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A novel RU486 inducible system for the activation and repression of genes
Advanced Drug Delivery Reviews 30 (1998) 23–31
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A novel RU486 inducible system for the activation and repression of genes Steven S. Chua, Yaolin Wang, Franco J. DeMayo, Bert W. O’Malley, Sophia Y. Tsai* Department of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA Received 1 July 1997; accepted 30 July 1997
Abstract We have developed an inducible system that consists of a transactivator and a target gene. The transactivator encodes a chimeric regulator that is responsive to RU486 (mifepristone, a progesterone receptor antagonist) but not to progestins and other hormones or endogenous ligands for activation. The target gene can be any gene under the control of Gal4 DNA binding sites. When the regulator is activated by RU486, it induces target gene expression by binding to the Gal4 recognition sequences upstream of the target. To verify this concept, we have successfully demonstrated the functionality of this system in tissue culture and in transgenic mice. Furthermore, for applications that require higher levels of a target gene, we also have generated regulators that can induce greater target gene expression. In addition, we also have constructed a modified regulator which can repress gene expression. The versatility of our system should prove useful for many applications in biology and gene therapy. 1998 Elsevier Science B.V. Keywords: Regulatable system; Regulator; Target; Activation; Repression; Temporal expression
Contents 1. 2. 3. 4.
Introduction ............................................................................................................................................................................ The need for more inducible gene systems ................................................................................................................................ Genesis of the RU486 inducible system .................................................................................................................................... Principle of the RU486 inducible system................................................................................................................................... 4.1. Regulator design .............................................................................................................................................................. 4.2. Components of the system ................................................................................................................................................ 4.3. Requirements of our inducible system ............................................................................................................................... 4.4. Activation process............................................................................................................................................................ 5. Results ................................................................................................................................................................................... 5.1. In vitro functionality ........................................................................................................................................................ 5.2. Growth hormone induction in vivo .................................................................................................................................... 5.3. Improvements to our system .............................................................................................................................................
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Abbreviations: CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; E1b, adenovirus E1b; Gal4, a yeast transcription factor; Gal4DBD, DNA binding factor of the Gal4 protein; GR, glucocorticoid receptor; GRE, glucocorticoid response element; hGH, human ¨ growth hormone; Kid-1, a kidney-specific transcription factor with a KRAB domain; KRAB, Kruppel-associated box; PR-891, a progesterone receptor mutant activated by RU486; PRE, progesterone response element; PRLBD, progesterone receptor ligand binding domain; RSV, Rous sarcoma virus; RU27987, a progestin agonist; RU486, mifepristone, a progesterone receptor antagonist; R5020, a progestin agonist; RXR, retinoid X receptor; TK, thymidine kinase; VP16AD, activation domain of the Herpes simplex viral protein VP16 *Corresponding author. Tel.: 1 1 713 7986251; fax: 1 1 713 7988227
0169-409X / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0169-409X( 97 )00104-X
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5.4. Inducible repressor system ................................................................................................................................................ 6. Conclusion ............................................................................................................................................................................. 7. Future directions ..................................................................................................................................................................... References ..................................................................................................................................................................................
1. Introduction The tight and precise expression of genes in mammalian systems represents a highly organized and complex paradigm of gene regulation. In the past decade, investigators have sought to mimic this principle in the form of inducible systems. These attempts have met with some success. Ideally, these systems should allow one to reversibly control the expression of a gene(s). Studies aimed at discerning the role of temporal expression of a gene product is only one of the numerous reasons inducible systems were developed. This is particularly important in developmental and cancer studies because the phenotype observed is usually a consequence of the stage in development when the causative gene is first expressed [1–3]. An additional impetus is to circumvent lethality-associated issues in transgenic mice studies resulting from the constitutive expression of a certain gene(s) [4–6]. In the important ultimate goal of applying gene therapy to cure certain debilitating diseases in humans, a regulated system of gene expression is critical to ensure that no unpleasant side-effects result from the uncontrolled expression of a therapeutic target gene. The multitude of these applications underscores the crucial need to develop a reliable and efficient gene regulatory system.
2. The need for more inducible gene systems A number of strategies have been developed over the years to regulate the expression of transferred genes. The earlier inducible systems (i.e. heat-shock [7], metallothionein [8], steroid receptor-responsive [9,10] and lac repressor–operator [11]) suffer from one or more of the following problems: leakiness of basal expression, toxicity of the inducer, pleiotropic effects in host cells and insufficient induction potency. While these systems are not perfect, they still find general use in select instances. Since an extensive discussion of all the systems available is
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beyond the scope of this mini-review, we will only briefly mention some of the newer systems available. A second generation of gene regulatory systems resulted from the need for better control of gene expression [12]; these have shown more promise than their predecessors and they utilize foreign regulatory elements and exogenous inducers to improve the specificity of induction of target genes. They include the tetracycline system [13], our RU486 (mifepristone, a progesterone receptor antagonist) system [14] and the ecdysone system [15]. The tetracycline-based system has seen many applications in tissue culture [13,16–21] and also in transgenic studies [22–27]. Nevertheless, chronic administration of tetracycline and the slow rate of clearance of this drug from bone and other tissues are issues of concern. In the ecdysone system, the induction of target genes requires the activation of the ecdysone receptor by muristerone and the participation of the retinoid X receptor (RXR) [15,28]. The addition of RXR plus the ecdysone receptor to transactivate the target gene has made this system more complex and not suitable for human use due to the potential interference of the retinoid signal transduction pathway. While no single system is perfect at present, it is the ultimate goal of researchers working in this field to achieve just that. In the following section, we will present merits of the current RU486 inducible system developed in our laboratory and describe its potential for additional modifications.
3. Genesis of the RU486 inducible system In the course of studies to understand the mechanism of progesterone receptor antagonist activity, we found that truncating a wild-type progesterone receptor at position 891 renders this mutant, PR-891, incapable of binding to the progestin agonist R5020 [29]. Surprisingly, PR-891 was able to bind to RU486 [30] and transactivate progesterone receptor-
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mediated genes only in the presence of this antihormone. Since PR-891 is 42 amino acids shorter than the wild-type receptor, we deduced that the progesterone binding domain lies in this latter stretch of amino acids while the RU486 binding domain lies further upstream of this region [29]. A useful application for this mutant was evident. Activation of target genes by RU486 would be preferable to using progestin agonists since the activation of endogenous progesterone receptors can induce other cellular target genes that may have pleiotropic effects on cells. Moreover, we do not want to turn on progesterone receptor-mediated genes by using the PR-891 mutant which could bind to progesterone response elements (PREs) or glucocorticoid response elements (GREs) when activated by RU486. To circumvent these problems, modifications to this mutant were made and are discussed below.
4. Principle of the RU486 inducible system
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Fig. 1. Regulator construct. The transactivator gene encodes a chimeric regulator (GLVP) consisting of a VP16 activation domain (VP16AD), a Gal4 DNA binding domain (Gal4DBD) and a truncated progesterone ligand binding domain PRLBD-891 responsive to RU486 [30] but not to R5020 for activation. Any promoter can be used to target the expression of the regulator to any particular cell or tissue of interest.
minimal promoter [i.e. TATA box or thymidine kinase (TK) promoter] and four high-affinity Gal4 DNA binding sites (Fig. 2). The regulator binds only to the Gal4 DNA binding sites of the target gene and induces target gene expression when RU486 is administered.
4.3. Requirements of our inducible system Before a gene regulator system can find general application, the following criteria have to be met.
4.1. Regulator design Based on the modular nature of transcription factors [31], we created an inducible chimeric transcription factor (regulator) [14], utilizing a DNA binding domain derived from the yeast transcription factor Gal4 (Gal4DBD) [32], an activation domain from the Herpes simplex viral protein VP16 (VP16AD) [33] and a modified C-terminal ligand binding domain from the PR-891 mutant (PRLBD891) [29,14]. This chimeric regulator (GLVP) no longer binds to PREs or GREs and also will not interfere with progesterone receptor-mediated transcription. Instead the chimeric regulator can now bind to and only activate target genes with Gal4 DNA binding sites when RU486 is administered. A schematic representation of the regulator is depicted in Fig. 1.
1. The transactivator (regulator) should not activate target gene expression in the absence of the inducer. 2. The expression of the regulator should be nontoxic and impart no phenotype. 3. The target gene should be silent until induced by the activated regulator. 4. In the absence of induction, the target gene should impart no phenotype. 5. Activation of the regulator should occur via an exogenous compound. 6. The inducer compound should have no adverse effects on cells or tissues. In the case of RU486, it must be administered at levels that can activate the regulator but do not inhibit endogenous
4.2. Components of the system Our inducible system consists of two components: a transactivator (regulator) and a target. The regulator can be placed under the control of any promoter. The target can be any gene placed under the control of a
Fig. 2. Target construct. The target gene can be any gene with an SV40 polyadenylation signal (SV40pA) placed under the control of a minimal promoter (i.e. TATA box) and four Gal4 DNA binding sites (17 3 4). Upon activation, the regulator can then bind to the 17 3 4 Gal4 sites and induce target gene expression.
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progesterone receptor or glucocorticoid receptor (GR) function. 7. The induction process should be reversible. 8. This system should allow for high levels of target gene expression.
(Fig. 3). Modulation of not only the level but also the duration of target gene expression can occur by adjusting the dose and duration of administration of RU486.
4.4. Activation process
5. Results
The activation process of our chimeric regulator is similar to that of known steroid receptors [34,35] by their cognate ligands and will be discussed only briefly. The regulator can be expressed in any cells or tissues by utilizing the appropriate tissue-specific promoter. Though it is expressed constitutively, it is inactive in the absence of the RU486 [30] and target gene expression will not result. When RU486 is added, a conformational change is induced in the regulator rendering it active. The activated regulator can now bind to the Gal4 DNA binding sites located upstream of the target gene to induce its expression
To substantiate the above principles, we tested the functionality of our system in vitro in tissue culture and in vivo using a bitransgenic mice model.
Fig. 3. Activation process of our inducible system. Though our regulator is expressed constitutively, it is inactive in the absence of RU486 and the target gene is not expressed. When RU486 is added, the regulator is activated and undergoes a conformational change. The activated regulator then dimerizes and binds to the Gal4 DNA binding sites of the target gene and then induces target gene expression.
5.1. In vitro functionality We first assessed the transactivation potential of our regulator on a chloramphenicol acetyltransferase (CAT) reporter in transient transfections. The constructs used for the transfection studies are shown in Fig. 4. The GLVP regulator is driven by the Rous sarcoma virus (RSV) promoter and the CAT reporter is under the control of four Gal4 DNA binding sites and a minimum TK promoter. As depicted in Fig. 5, little or no CAT activity is seen in the absence of RU486 administration [30]. When RU486 is added, a significant transactivation of the CAT gene is observed. In results already published [14], we have demonstrated that other progestin antagonists such as the ZK [36,37] and Org [38] series of compounds have similar efficacies in the induction of target gene expression. It is noteworthy that no induction of CAT gene is seen in the presence of RU27987, a potent progestin agonist. The potential of our system to be induced not only by RU486 but by other synthetic antagonists of progesterone such as the ZK [36,37] and Org [38] compounds, further underscores its versatility.
Fig. 4. Diagram of regulator and target reporter. The GLVP regulator is driven by the RSV promoter and the CAT reporter is under the control of four Gal4 DNA binding sites (17 3 4) and the minimum TK promoter.
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Fig. 5. Transactivation potential of the GLVP regulator. To assess the transactivation potential of the GLVP regulator on the CAT reporter, transient transfections in monkey kidney (CV1) cells were performed. As shown in the autoradiogram, the GLVP regulator is only able to transactivate the 17 3 4 TK CAT significantly in the presence of RU486 but not in the presence of RU27987. In the absence of RU486, there is hardly any CAT activity.
5.2. Growth hormone induction in vivo We next generated a bitransgenic mice model similar to those of others [39–41] to examine the induction of a growth hormone reporter in vivo. Our bitransgenic mice system contains both a transactivator and a target gene. We generated liver-specific transactivator mice by targeting the expression of our GLVP regulator to the liver using the transthyretin promoter and we used the human growth hormone (hGH) gene as a reporter in our target mice. Chromosomal insulator fragments derived from the hypersensitive site IV (HS4) of the chicken b-globin gene were used to shield the regulator from the positional effects of integration [42]. When these two heterozygote lines are crossed, bitransgenic, wild type and monogenic target and transactivator mice can result. We would expect that only the bitransgenic mice given RU486 would show induction of growth hormone. Fig. 6 shows the transgenic constructs used to generate the mice. To assess growth hormone induction, blood collected from seven bitransgenic mice before and after
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Fig. 6. Schematic representation of transactivator and target constructs. The GLVP regulator is cloned into a transgenic vector with the SV40 small t splicing and polyadenylation signal under the control of the transthyretin (TTR) promoter. This transcription unit is flanked by chromosomal insulator fragments derived from the hypersensitive site 4 (HS4) of the chicken b-globin gene to insure position-independent expression of the regulator [42]. The target construct contains the human growth hormone gene (hGH) placed under the control of four Gal4 DNA binding sites and a minimal TK promoter. Prior to microinjection into fertilized onecelled embryos, these constructs were linearized.
a day of an oral dose of 250 mg / kg bodyweight of RU486 were subjected to radioimmunoassay. As shown in Fig. 7, an average concentration of 288.1 ng / ml of hGH was measured in bitransgenic mice given RU486 compared to an almost undetectable level of hGH in the absence of RU486. This is a huge induction of approximately 1500-fold. These results once again demonstrated the specificity of our inducible system. More importantly, these results show that our system was silent until activated by RU486 administration. To understand the induction potential of RU486 activation, we titrated the amount of RU486 given to the bitransgenic mice and analyzed hGH levels in the blood. There was a dose-dependent increase in serum hGH levels. A dose of RU486 at 500 mg / kg bodyweight gave a greater induction of serum hGH levels than a dose of 250 mg / kg bodyweight (unpublished data). The potential to modulate the rate of expression of a target gene is important in studies aimed at discerning the physiological role of a target gene by allowing for variation in levels of the target gene product [12]. In kinetic studies (unpublished data), we showed that the growth hormone level is maximum at 12 h
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Fig. 7. Induction of hGH in bitransgenic mice. To verify our inducible system in vivo, we examined hGH induction in bitransgenic mice generated from a cross between the monogenic liverspecific transactivator and hGH target lines. Blood collected from seven bitransgenic mice before (2RU486) and after a day ( 1 RU486) of an oral dose of 250 mg / kg bodyweight of RU486 injection were subjected to radioimmunoassay. As depicted in the graph, an approximate 1500-fold induction of hGH expression is only seen in bitransgenic mice given RU486.
following administration of a dose of 250 mg / kg bodyweight of RU486. By 25 h, the growth hormone levels in blood have been reduced to half maximal levels. By 100 h, most of the RU486 has been metabolized and cleared from the blood and target gene expression has ceased (unpublished data). To assess the effects of RU486 levels on mouse development, we injected plugged female mice over their course of pregnancy with varying doses of RU486 intraperitoneally on alternate days from day 1 of mating. Our results indicated that a dose of 100 mg / kg bodyweight of RU486 did not cause pregnant mice to abort. Our results imply that this level of RU486 does not impair endogenous progesterone receptor and GR function.
5.3. Improvements to our system While we have successfully demonstrated the functionality of our system, we also wished to improve the transactivation potential of our regulator
without sacrificing a low basal activity in the absence of RU486 [30]. Though the GLVP regulator is sufficiently potent to transactivate target genes in the presence of a TK promoter, it is weaker in the transactivation of genes containing a TATA box (unpublished results). Depending on the ultimate application of our system, higher levels of a target gene may be needed. Towards this goal, we have generated a number of new constructs to improve the transactivation potential of our regulator. We relocated the VP16AD to the C-terminal part of the regulator protein (GL 891 VPc9), downstream from the ligand binding domain (Fig. 8). As depicted in Table 1, a stronger transactivation potential is observed with this regulator. Based on the finding that extending the length of our mutated progesterone ligand binding domain actually increases the affinity of the receptor to RU486, we also have generated a new regulator (GL 914 VPc9) whereby 23 additional amino acids have been added to this domain [43]. This modified regulator demonstrated enhanced transactivation potential when compared to earlier regulator forms without sacrificing low basal activity in the absence of RU486 (Table 1). The finding also that the GL 914 VPc9 regulator responds to a concentration of RU486 that is an order of a magnitude lower than the other regulators should further enhance the desirability of our system. We can now use even lower RU486 concentrations to
Fig. 8. Improved regulator constructs. To improve the transactivation potential of the regulator, modifications were made. GLVP represents the original regulator and GL 891 VPc9 and GL 914 VPc9 denote the modified regulators. GL 891 VPc9 is similar to GLVP with the exception that the VP16AD is placed at the C terminus of the regulator. GL 914 VPc9 is similar to GL 891 VPc9 with the exception that 23 amino acids have been added to the PRLBD to increase its affinity to RU486. All regulators are driven by the CMV promoter.
S.S. Chua et al. / Advanced Drug Delivery Reviews 30 (1998) 23 – 31 Table 1 Transactivation potential of regulators % Conversion Regulator
2RU486
1RU486
Fold induction
GLVP GL 891 VPc9 GL 914 VPc9
0.560.1 0.860.1 1.160.3
11.661.9 46.766.0 79.7611.3
24 56 74
The three regulators shown in Fig. 8 were tested for their transactivation potential on the 17 3 4 E1b TATA CAT reporter in transient transfections in CV1 cells. As shown in the table, significant CAT activity is seen only in the presence of RU486. The new regulators GL 891 VPc9 and GL 914 VPc9 showed much higher conversion of the substrate into acetylated forms than the GLVP regulator. % Conversion denotes the average percent of substrate converted into the acetylated forms of chloramphenicol of three separate experiments. Fold induction denotes the ratio of substrate conversion in the presence and absence of RU486.
activate our target genes without worrying about compromising GR and progesterone receptor activities [43].
5.4. Inducible repressor system To expand the utility of our system, we investigated the potential of RU486-mediated repression. We replaced the VP16AD of our regulator with that ¨ of the Kruppel-associated box (KRAB) domain from the Kid-1 transcription factor, a kidney specific factor [44]. The KRAB domain is a stretch of about 75 amino acids found at the N-terminus of a number ¨ of the Kruppel-class transcription factors that contain two amphipathic helices believed to be involved in protein–protein interactions [45]. Fusion of this KRAB domain to DNA binding domains of heterologous proteins have rendered these fusions potent repressors of reporter gene expression [46]. In transfection studies using the CAT reporter placed under the control of the SV40 enhancer and five Gal4 DNA binding sites, our inducible repressor was able to repress CAT activity two to six-fold when RU486 was added [43], thereby demonstrating the potential to repress in addition to induce gene expression.
6. Conclusion The results of these studies have demonstrated the versatility and feasibility of our RU486 inducible
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system. Though our regulator is expressed constitutively, it is inactive in the absence of RU486. When RU486 is added, the regulator is activated and proceeds to induce target gene expression. This ensures specificity in that our regulator is only activated when experimentally dictated and only will induce specific target genes containing Gal4 DNA binding sites. The levels and duration of target gene expression can be modulated by altering the dose and duration of RU486 administration. Moreover, the ability to replace the VP16AD with a repressor domain further expands the utility of our system to now turn off target genes with the cognate Gal4 DNA binding sites. The qualities of specificity, nontoxicity and high induction potential coupled with low basal activity should make this inducible system useful for many applications.
7. Future directions Our RU486-mediated gene regulatory system can be applied to control the temporal expression of genes involved in development and cancer and to create transgenic mouse models for these important processes. This inducible system should see application also in the field of gene therapy whereby the regulated expression of a therapeutic gene is vital. With further improvements to reduce the dose of RU486 needed to activate our system, we should be able to make our inducible system even better. Ultimately, the possibility of our inducible system to be used in addition with those of others (i.e. the tetracycline [13], ecdysone [15], etc.) will further enhance the versatility of these systems by allowing us to control multiple levels of gene expression.
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[41] D.M. Ornitz, R.W. Moreadith, P. Leder, Binary system for the regulating transgene expression in mice: targeting int-2 gene expression with yeast Gal4 / UAS control elements, Proc. Natl. Acad. Sci. USA 88 (1991) 698–702. [42] J.H. Chung, M. Whiteley, G. Felsenfeld, A 59 element of the chicken b-globin domain serves as an insulator in human erythroid cells and protects against position effect in drosophila, Cell 74 (1993) 505–514. [43] Y. Wang, J. Xu, T. Pierson, B.W. O’Malley, S.Y. Tsai, Positive and negative regulation of gene expression in eukaryotic cells with an inducible transcriptional regulator. (1996) submitted. [44] R. Witzgall, E. Oleary, R. Gessner, A.J. Ouellette, J.V. Bonventre, Kid-1, a putative renal transcription factor: regulation during ontogeny and in response to ischemia and toxic injury, Mol. Cell. Biol. 13 (1993) 1933–1942. [45] E.J. Bellefroid, D.A. Poncelet, P.J. Lecocq, O. Revelant, J.A. ¨ Martial, The evolutionary conserved Kruppel-associated box domain defines a subfamily of eukaryotic multi-fingered proteins, Proc. Natl. Acad. Sci. USA 88 (1991) 3608–3612. [46] J.F. Margolin, J.R. Friedman, W.K.-H. Meyer, H. Vissing, ¨ H.-J. Thiesen, F.J. Rauscher III, Kruppel-associated boxes are potent transcriptional repression domains, Proc. Natl. Acad. Sci. USA 91 (1994) 4509–4513.
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A novel RU486 inducible system for the activation and repression of genes Steven S. Chua, Yaolin Wang, Franco J. DeMayo, Bert W. O’Malley, Sophia Y. Tsai* Department of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA Received 1 July 1997; accepted 30 July 1997
Abstract We have developed an inducible system that consists of a transactivator and a target gene. The transactivator encodes a chimeric regulator that is responsive to RU486 (mifepristone, a progesterone receptor antagonist) but not to progestins and other hormones or endogenous ligands for activation. The target gene can be any gene under the control of Gal4 DNA binding sites. When the regulator is activated by RU486, it induces target gene expression by binding to the Gal4 recognition sequences upstream of the target. To verify this concept, we have successfully demonstrated the functionality of this system in tissue culture and in transgenic mice. Furthermore, for applications that require higher levels of a target gene, we also have generated regulators that can induce greater target gene expression. In addition, we also have constructed a modified regulator which can repress gene expression. The versatility of our system should prove useful for many applications in biology and gene therapy. 1998 Elsevier Science B.V. Keywords: Regulatable system; Regulator; Target; Activation; Repression; Temporal expression
Contents 1. 2. 3. 4.
Introduction ............................................................................................................................................................................ The need for more inducible gene systems ................................................................................................................................ Genesis of the RU486 inducible system .................................................................................................................................... Principle of the RU486 inducible system................................................................................................................................... 4.1. Regulator design .............................................................................................................................................................. 4.2. Components of the system ................................................................................................................................................ 4.3. Requirements of our inducible system ............................................................................................................................... 4.4. Activation process............................................................................................................................................................ 5. Results ................................................................................................................................................................................... 5.1. In vitro functionality ........................................................................................................................................................ 5.2. Growth hormone induction in vivo .................................................................................................................................... 5.3. Improvements to our system .............................................................................................................................................
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Abbreviations: CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; E1b, adenovirus E1b; Gal4, a yeast transcription factor; Gal4DBD, DNA binding factor of the Gal4 protein; GR, glucocorticoid receptor; GRE, glucocorticoid response element; hGH, human ¨ growth hormone; Kid-1, a kidney-specific transcription factor with a KRAB domain; KRAB, Kruppel-associated box; PR-891, a progesterone receptor mutant activated by RU486; PRE, progesterone response element; PRLBD, progesterone receptor ligand binding domain; RSV, Rous sarcoma virus; RU27987, a progestin agonist; RU486, mifepristone, a progesterone receptor antagonist; R5020, a progestin agonist; RXR, retinoid X receptor; TK, thymidine kinase; VP16AD, activation domain of the Herpes simplex viral protein VP16 *Corresponding author. Tel.: 1 1 713 7986251; fax: 1 1 713 7988227
0169-409X / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0169-409X( 97 )00104-X
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5.4. Inducible repressor system ................................................................................................................................................ 6. Conclusion ............................................................................................................................................................................. 7. Future directions ..................................................................................................................................................................... References ..................................................................................................................................................................................
1. Introduction The tight and precise expression of genes in mammalian systems represents a highly organized and complex paradigm of gene regulation. In the past decade, investigators have sought to mimic this principle in the form of inducible systems. These attempts have met with some success. Ideally, these systems should allow one to reversibly control the expression of a gene(s). Studies aimed at discerning the role of temporal expression of a gene product is only one of the numerous reasons inducible systems were developed. This is particularly important in developmental and cancer studies because the phenotype observed is usually a consequence of the stage in development when the causative gene is first expressed [1–3]. An additional impetus is to circumvent lethality-associated issues in transgenic mice studies resulting from the constitutive expression of a certain gene(s) [4–6]. In the important ultimate goal of applying gene therapy to cure certain debilitating diseases in humans, a regulated system of gene expression is critical to ensure that no unpleasant side-effects result from the uncontrolled expression of a therapeutic target gene. The multitude of these applications underscores the crucial need to develop a reliable and efficient gene regulatory system.
2. The need for more inducible gene systems A number of strategies have been developed over the years to regulate the expression of transferred genes. The earlier inducible systems (i.e. heat-shock [7], metallothionein [8], steroid receptor-responsive [9,10] and lac repressor–operator [11]) suffer from one or more of the following problems: leakiness of basal expression, toxicity of the inducer, pleiotropic effects in host cells and insufficient induction potency. While these systems are not perfect, they still find general use in select instances. Since an extensive discussion of all the systems available is
29 29 29 29
beyond the scope of this mini-review, we will only briefly mention some of the newer systems available. A second generation of gene regulatory systems resulted from the need for better control of gene expression [12]; these have shown more promise than their predecessors and they utilize foreign regulatory elements and exogenous inducers to improve the specificity of induction of target genes. They include the tetracycline system [13], our RU486 (mifepristone, a progesterone receptor antagonist) system [14] and the ecdysone system [15]. The tetracycline-based system has seen many applications in tissue culture [13,16–21] and also in transgenic studies [22–27]. Nevertheless, chronic administration of tetracycline and the slow rate of clearance of this drug from bone and other tissues are issues of concern. In the ecdysone system, the induction of target genes requires the activation of the ecdysone receptor by muristerone and the participation of the retinoid X receptor (RXR) [15,28]. The addition of RXR plus the ecdysone receptor to transactivate the target gene has made this system more complex and not suitable for human use due to the potential interference of the retinoid signal transduction pathway. While no single system is perfect at present, it is the ultimate goal of researchers working in this field to achieve just that. In the following section, we will present merits of the current RU486 inducible system developed in our laboratory and describe its potential for additional modifications.
3. Genesis of the RU486 inducible system In the course of studies to understand the mechanism of progesterone receptor antagonist activity, we found that truncating a wild-type progesterone receptor at position 891 renders this mutant, PR-891, incapable of binding to the progestin agonist R5020 [29]. Surprisingly, PR-891 was able to bind to RU486 [30] and transactivate progesterone receptor-
S.S. Chua et al. / Advanced Drug Delivery Reviews 30 (1998) 23 – 31
mediated genes only in the presence of this antihormone. Since PR-891 is 42 amino acids shorter than the wild-type receptor, we deduced that the progesterone binding domain lies in this latter stretch of amino acids while the RU486 binding domain lies further upstream of this region [29]. A useful application for this mutant was evident. Activation of target genes by RU486 would be preferable to using progestin agonists since the activation of endogenous progesterone receptors can induce other cellular target genes that may have pleiotropic effects on cells. Moreover, we do not want to turn on progesterone receptor-mediated genes by using the PR-891 mutant which could bind to progesterone response elements (PREs) or glucocorticoid response elements (GREs) when activated by RU486. To circumvent these problems, modifications to this mutant were made and are discussed below.
4. Principle of the RU486 inducible system
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Fig. 1. Regulator construct. The transactivator gene encodes a chimeric regulator (GLVP) consisting of a VP16 activation domain (VP16AD), a Gal4 DNA binding domain (Gal4DBD) and a truncated progesterone ligand binding domain PRLBD-891 responsive to RU486 [30] but not to R5020 for activation. Any promoter can be used to target the expression of the regulator to any particular cell or tissue of interest.
minimal promoter [i.e. TATA box or thymidine kinase (TK) promoter] and four high-affinity Gal4 DNA binding sites (Fig. 2). The regulator binds only to the Gal4 DNA binding sites of the target gene and induces target gene expression when RU486 is administered.
4.3. Requirements of our inducible system Before a gene regulator system can find general application, the following criteria have to be met.
4.1. Regulator design Based on the modular nature of transcription factors [31], we created an inducible chimeric transcription factor (regulator) [14], utilizing a DNA binding domain derived from the yeast transcription factor Gal4 (Gal4DBD) [32], an activation domain from the Herpes simplex viral protein VP16 (VP16AD) [33] and a modified C-terminal ligand binding domain from the PR-891 mutant (PRLBD891) [29,14]. This chimeric regulator (GLVP) no longer binds to PREs or GREs and also will not interfere with progesterone receptor-mediated transcription. Instead the chimeric regulator can now bind to and only activate target genes with Gal4 DNA binding sites when RU486 is administered. A schematic representation of the regulator is depicted in Fig. 1.
1. The transactivator (regulator) should not activate target gene expression in the absence of the inducer. 2. The expression of the regulator should be nontoxic and impart no phenotype. 3. The target gene should be silent until induced by the activated regulator. 4. In the absence of induction, the target gene should impart no phenotype. 5. Activation of the regulator should occur via an exogenous compound. 6. The inducer compound should have no adverse effects on cells or tissues. In the case of RU486, it must be administered at levels that can activate the regulator but do not inhibit endogenous
4.2. Components of the system Our inducible system consists of two components: a transactivator (regulator) and a target. The regulator can be placed under the control of any promoter. The target can be any gene placed under the control of a
Fig. 2. Target construct. The target gene can be any gene with an SV40 polyadenylation signal (SV40pA) placed under the control of a minimal promoter (i.e. TATA box) and four Gal4 DNA binding sites (17 3 4). Upon activation, the regulator can then bind to the 17 3 4 Gal4 sites and induce target gene expression.
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progesterone receptor or glucocorticoid receptor (GR) function. 7. The induction process should be reversible. 8. This system should allow for high levels of target gene expression.
(Fig. 3). Modulation of not only the level but also the duration of target gene expression can occur by adjusting the dose and duration of administration of RU486.
4.4. Activation process
5. Results
The activation process of our chimeric regulator is similar to that of known steroid receptors [34,35] by their cognate ligands and will be discussed only briefly. The regulator can be expressed in any cells or tissues by utilizing the appropriate tissue-specific promoter. Though it is expressed constitutively, it is inactive in the absence of the RU486 [30] and target gene expression will not result. When RU486 is added, a conformational change is induced in the regulator rendering it active. The activated regulator can now bind to the Gal4 DNA binding sites located upstream of the target gene to induce its expression
To substantiate the above principles, we tested the functionality of our system in vitro in tissue culture and in vivo using a bitransgenic mice model.
Fig. 3. Activation process of our inducible system. Though our regulator is expressed constitutively, it is inactive in the absence of RU486 and the target gene is not expressed. When RU486 is added, the regulator is activated and undergoes a conformational change. The activated regulator then dimerizes and binds to the Gal4 DNA binding sites of the target gene and then induces target gene expression.
5.1. In vitro functionality We first assessed the transactivation potential of our regulator on a chloramphenicol acetyltransferase (CAT) reporter in transient transfections. The constructs used for the transfection studies are shown in Fig. 4. The GLVP regulator is driven by the Rous sarcoma virus (RSV) promoter and the CAT reporter is under the control of four Gal4 DNA binding sites and a minimum TK promoter. As depicted in Fig. 5, little or no CAT activity is seen in the absence of RU486 administration [30]. When RU486 is added, a significant transactivation of the CAT gene is observed. In results already published [14], we have demonstrated that other progestin antagonists such as the ZK [36,37] and Org [38] series of compounds have similar efficacies in the induction of target gene expression. It is noteworthy that no induction of CAT gene is seen in the presence of RU27987, a potent progestin agonist. The potential of our system to be induced not only by RU486 but by other synthetic antagonists of progesterone such as the ZK [36,37] and Org [38] compounds, further underscores its versatility.
Fig. 4. Diagram of regulator and target reporter. The GLVP regulator is driven by the RSV promoter and the CAT reporter is under the control of four Gal4 DNA binding sites (17 3 4) and the minimum TK promoter.
S.S. Chua et al. / Advanced Drug Delivery Reviews 30 (1998) 23 – 31
Fig. 5. Transactivation potential of the GLVP regulator. To assess the transactivation potential of the GLVP regulator on the CAT reporter, transient transfections in monkey kidney (CV1) cells were performed. As shown in the autoradiogram, the GLVP regulator is only able to transactivate the 17 3 4 TK CAT significantly in the presence of RU486 but not in the presence of RU27987. In the absence of RU486, there is hardly any CAT activity.
5.2. Growth hormone induction in vivo We next generated a bitransgenic mice model similar to those of others [39–41] to examine the induction of a growth hormone reporter in vivo. Our bitransgenic mice system contains both a transactivator and a target gene. We generated liver-specific transactivator mice by targeting the expression of our GLVP regulator to the liver using the transthyretin promoter and we used the human growth hormone (hGH) gene as a reporter in our target mice. Chromosomal insulator fragments derived from the hypersensitive site IV (HS4) of the chicken b-globin gene were used to shield the regulator from the positional effects of integration [42]. When these two heterozygote lines are crossed, bitransgenic, wild type and monogenic target and transactivator mice can result. We would expect that only the bitransgenic mice given RU486 would show induction of growth hormone. Fig. 6 shows the transgenic constructs used to generate the mice. To assess growth hormone induction, blood collected from seven bitransgenic mice before and after
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Fig. 6. Schematic representation of transactivator and target constructs. The GLVP regulator is cloned into a transgenic vector with the SV40 small t splicing and polyadenylation signal under the control of the transthyretin (TTR) promoter. This transcription unit is flanked by chromosomal insulator fragments derived from the hypersensitive site 4 (HS4) of the chicken b-globin gene to insure position-independent expression of the regulator [42]. The target construct contains the human growth hormone gene (hGH) placed under the control of four Gal4 DNA binding sites and a minimal TK promoter. Prior to microinjection into fertilized onecelled embryos, these constructs were linearized.
a day of an oral dose of 250 mg / kg bodyweight of RU486 were subjected to radioimmunoassay. As shown in Fig. 7, an average concentration of 288.1 ng / ml of hGH was measured in bitransgenic mice given RU486 compared to an almost undetectable level of hGH in the absence of RU486. This is a huge induction of approximately 1500-fold. These results once again demonstrated the specificity of our inducible system. More importantly, these results show that our system was silent until activated by RU486 administration. To understand the induction potential of RU486 activation, we titrated the amount of RU486 given to the bitransgenic mice and analyzed hGH levels in the blood. There was a dose-dependent increase in serum hGH levels. A dose of RU486 at 500 mg / kg bodyweight gave a greater induction of serum hGH levels than a dose of 250 mg / kg bodyweight (unpublished data). The potential to modulate the rate of expression of a target gene is important in studies aimed at discerning the physiological role of a target gene by allowing for variation in levels of the target gene product [12]. In kinetic studies (unpublished data), we showed that the growth hormone level is maximum at 12 h
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Fig. 7. Induction of hGH in bitransgenic mice. To verify our inducible system in vivo, we examined hGH induction in bitransgenic mice generated from a cross between the monogenic liverspecific transactivator and hGH target lines. Blood collected from seven bitransgenic mice before (2RU486) and after a day ( 1 RU486) of an oral dose of 250 mg / kg bodyweight of RU486 injection were subjected to radioimmunoassay. As depicted in the graph, an approximate 1500-fold induction of hGH expression is only seen in bitransgenic mice given RU486.
following administration of a dose of 250 mg / kg bodyweight of RU486. By 25 h, the growth hormone levels in blood have been reduced to half maximal levels. By 100 h, most of the RU486 has been metabolized and cleared from the blood and target gene expression has ceased (unpublished data). To assess the effects of RU486 levels on mouse development, we injected plugged female mice over their course of pregnancy with varying doses of RU486 intraperitoneally on alternate days from day 1 of mating. Our results indicated that a dose of 100 mg / kg bodyweight of RU486 did not cause pregnant mice to abort. Our results imply that this level of RU486 does not impair endogenous progesterone receptor and GR function.
5.3. Improvements to our system While we have successfully demonstrated the functionality of our system, we also wished to improve the transactivation potential of our regulator
without sacrificing a low basal activity in the absence of RU486 [30]. Though the GLVP regulator is sufficiently potent to transactivate target genes in the presence of a TK promoter, it is weaker in the transactivation of genes containing a TATA box (unpublished results). Depending on the ultimate application of our system, higher levels of a target gene may be needed. Towards this goal, we have generated a number of new constructs to improve the transactivation potential of our regulator. We relocated the VP16AD to the C-terminal part of the regulator protein (GL 891 VPc9), downstream from the ligand binding domain (Fig. 8). As depicted in Table 1, a stronger transactivation potential is observed with this regulator. Based on the finding that extending the length of our mutated progesterone ligand binding domain actually increases the affinity of the receptor to RU486, we also have generated a new regulator (GL 914 VPc9) whereby 23 additional amino acids have been added to this domain [43]. This modified regulator demonstrated enhanced transactivation potential when compared to earlier regulator forms without sacrificing low basal activity in the absence of RU486 (Table 1). The finding also that the GL 914 VPc9 regulator responds to a concentration of RU486 that is an order of a magnitude lower than the other regulators should further enhance the desirability of our system. We can now use even lower RU486 concentrations to
Fig. 8. Improved regulator constructs. To improve the transactivation potential of the regulator, modifications were made. GLVP represents the original regulator and GL 891 VPc9 and GL 914 VPc9 denote the modified regulators. GL 891 VPc9 is similar to GLVP with the exception that the VP16AD is placed at the C terminus of the regulator. GL 914 VPc9 is similar to GL 891 VPc9 with the exception that 23 amino acids have been added to the PRLBD to increase its affinity to RU486. All regulators are driven by the CMV promoter.
S.S. Chua et al. / Advanced Drug Delivery Reviews 30 (1998) 23 – 31 Table 1 Transactivation potential of regulators % Conversion Regulator
2RU486
1RU486
Fold induction
GLVP GL 891 VPc9 GL 914 VPc9
0.560.1 0.860.1 1.160.3
11.661.9 46.766.0 79.7611.3
24 56 74
The three regulators shown in Fig. 8 were tested for their transactivation potential on the 17 3 4 E1b TATA CAT reporter in transient transfections in CV1 cells. As shown in the table, significant CAT activity is seen only in the presence of RU486. The new regulators GL 891 VPc9 and GL 914 VPc9 showed much higher conversion of the substrate into acetylated forms than the GLVP regulator. % Conversion denotes the average percent of substrate converted into the acetylated forms of chloramphenicol of three separate experiments. Fold induction denotes the ratio of substrate conversion in the presence and absence of RU486.
activate our target genes without worrying about compromising GR and progesterone receptor activities [43].
5.4. Inducible repressor system To expand the utility of our system, we investigated the potential of RU486-mediated repression. We replaced the VP16AD of our regulator with that ¨ of the Kruppel-associated box (KRAB) domain from the Kid-1 transcription factor, a kidney specific factor [44]. The KRAB domain is a stretch of about 75 amino acids found at the N-terminus of a number ¨ of the Kruppel-class transcription factors that contain two amphipathic helices believed to be involved in protein–protein interactions [45]. Fusion of this KRAB domain to DNA binding domains of heterologous proteins have rendered these fusions potent repressors of reporter gene expression [46]. In transfection studies using the CAT reporter placed under the control of the SV40 enhancer and five Gal4 DNA binding sites, our inducible repressor was able to repress CAT activity two to six-fold when RU486 was added [43], thereby demonstrating the potential to repress in addition to induce gene expression.
6. Conclusion The results of these studies have demonstrated the versatility and feasibility of our RU486 inducible
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system. Though our regulator is expressed constitutively, it is inactive in the absence of RU486. When RU486 is added, the regulator is activated and proceeds to induce target gene expression. This ensures specificity in that our regulator is only activated when experimentally dictated and only will induce specific target genes containing Gal4 DNA binding sites. The levels and duration of target gene expression can be modulated by altering the dose and duration of RU486 administration. Moreover, the ability to replace the VP16AD with a repressor domain further expands the utility of our system to now turn off target genes with the cognate Gal4 DNA binding sites. The qualities of specificity, nontoxicity and high induction potential coupled with low basal activity should make this inducible system useful for many applications.
7. Future directions Our RU486-mediated gene regulatory system can be applied to control the temporal expression of genes involved in development and cancer and to create transgenic mouse models for these important processes. This inducible system should see application also in the field of gene therapy whereby the regulated expression of a therapeutic gene is vital. With further improvements to reduce the dose of RU486 needed to activate our system, we should be able to make our inducible system even better. Ultimately, the possibility of our inducible system to be used in addition with those of others (i.e. the tetracycline [13], ecdysone [15], etc.) will further enhance the versatility of these systems by allowing us to control multiple levels of gene expression.
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