A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system
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Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others.
In this report, we describe the combination of dimerizer-regulated two-hybrid and tetracycline regulatory elements in an ordered cascade, placing expression of endpoint reporters under the control of two distinct drugs. In this Dual Drug Control (DDC) system, a first plasmid expresses fusion proteins to DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid encodes a reporter gene transcriptionally responsive to binding by Tet-OFF. We evaluate the dynamic range and specificity of this system in comparison to other available systems.
This study demonstrates the feasibility of combining two discrete drug-regulated expression systems in a temporally sequential cascade, without loss of dynamic range of signal induction. The efficient layering of control levels allowed by this combination of elements provides the potential for the generation of complex control circuitry that may advance ability to regulate gene expression in vivo.
KeywordsInternal Ribosomal Entry Site Gene Expression System Split System Internal Ribosomal Entry Site Element SEAP Activity
To achieve effective control of gene expression in vivo, higher eukaryotes generally utilize extensive promoter/enhancer/locus control regions spanning many kilobases of DNA, and encompass multiple discrete binding sites for transcription factors that combinatorially encode specificity of gene transcription. However, for studies of gene function requiring the introduction of heterologous protein into cultured cells, there is a practical limit governing the size of promoter that can be utilized, corresponding to the 2–3 kb of enhancer/promoter sequence that can be accommodated on a plasmid that also must contain other required sequence elements. Currently, many of the gene expression systems commonly utilized for studies of protein function utilize small, virally derived enhancers (for example, from cytomegalovirus, CMV) that typically do not provide many options for calibrating the expression of encoded proteins, but rather produce constitutive high levels of protein product following introduction into cells. Although some inducible gene expression systems have been developed using natural promoter elements, disadvantages of these systems prevented them from gaining broad use. As one example, although use of the metallothionein promoter  allows induction of gene expression through addition of metals to cell culture, the attendant cellular stress response arising from exposure to metals can cause secondary effects complicating the interpretation of resulting data.
As an alternative approach, a number of groups have explored the possibilities of developing small artificial promoters or transcriptional regulatory systems with desirable properties for gene expression control. It has long been appreciated that it is possible to generate novel transcriptional control systems by fusing separable DNA binding domains (DBDs) and transcriptional activation domains (ADs) to achieve a desired transcriptional activation specificity . The fact that it is possible to create functional transcriptional regulatory control systems even in situations where the DBD and AD are not covalently attached is the guiding principle behind technologies such as the yeast two hybrid system . In adapting these concepts to the design of artificial gene expression systems, previous work by some groups has demonstrated the possibility of constructing elegant combinatorial systems incorporating feedback loops, based on the integration of two-hybrid system paradigms with diverse tissue specific promoters (e.g. [4, 5, 6]). Separately, others have shown the efficient regulation of gene expression through artificial promoters dependent on the action of small molecule modulators, including tetracycline and derivatives (reviewed in ); streptogramin and macrolide antibiotics, [8, 9, 10]; combined coumermycin and novobiocin systems ; ecdysone ; dimerizer molecules such as FK1012 and other rapamycin-related derivatives [13, 14]. Further afield, derivatives of the "quorum-sensing" circuits of bacteria, that assess cell density to regulate interconversion between biofilms and other growth forms , and components of the plant phytochrome system, in which promoter activity can be regulated by light , have been adapted for artificial promoter construction.
Each of these systems offers specific advantages for some applications. Notably, in order to generate flexible tools for general usage in achieving fine control of gene expression, it would be useful to be able to combine elements of these different systems. However, it has not to date been clear whether these artificial transcriptional regulatory reagents are sufficiently robust to be merged into more complex regulatory cascades, particularly under the conditions of transient transfection generally preferred for rapid experimentation. This is potentially a non-trivial problem, as in increasing the number of cascade elements, more control points are introduced that might contribute to loss of efficiency, specificity, or dynamic range. Nevertheless, sequential cascades combining different groups of transcription factors, with localization and activity regulated by multiple inputs from modifying factors such as kinases, phosphatases, acetylases, and other enzymes represents a standard means of gene regulation in naturally occurring living organisms. It is likely that the complexity arising from the multiple inputs leads to more fine-tuning of output, and enables a more complex biological response. Our goals in undertaking the present study were two-fold: first, to evaluate the results of arraying two discrete, artificial transcriptional control systems in contrast to the results obtained with each system in isolation; and second, optimally, to develop a reagent system that is practically useful in achieving more fine control of gene expression.
In this report we combine a set of elements developed by various research groups with new elements constructed herein to create a complex multi-component system intended for use in transient experiments. In this system, a first plasmid expresses fusion proteins to DBD and AD, which interact contingent on the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid encodes a reporter gene transcriptionally responsive to binding by Tet-OFF. In a series of tests, we evaluate the dynamic range and specificity of this Dual Drug Control (DDC) system in contrast to simpler systems, and note issues of particular importance for future tool development. Finally, we discuss several potential applications for a DDC system.
Results and discussion
IRES versus split expression of two-hybrid components. Units shown represent fold-induction, induced over uninduced conditions, of a ZFHD-dependent SEAP reporter, in three different cell lines.
In summary, this pilot study demonstrates the feasibility of combining two discrete drug-regulated expression systems in an ordered cascade. It further demonstrates that this sequential arrangement of systems does not result in unworkable reduction in dynamic range of signal induction, and shows that a comparable level of induction is obtained using simultaneous or sequential addition of regulatory drugs. As such, it may prove to be a useful addition to the toolbox of reagents for control of gene expression in mammalian cells. Other studies in this area (for example, the work of Kramer et al., ), have also begun to address the sequential combination of elements derived from different artificial regulatory systems, with the goal of "fine-tuning" gene expression. This is likely to represent an important future theme in promoter design, and will involve much trial and error of many system components. Ultimately, it may also provide an informative model for analysis of elements contributing to efficient propagation of information in signal transduction cascade, as systems biology evolves as an experimental science.
pSEAP2-Enhancer, pRetro-Off, pBI-EGFP, and pEGFP-N1 were acquired from Clontech Labs. pC4N2-RHS/ZF3, pZ12I-PL-2, pLH-Z12-I-S and pZ12I-hGH-2 were developed by Ariad Pharmaceuticals, and are described in (the ARGENT Regulated Transcription Plasmid Kit Version 2.0, Available on-line at http://www.ariad.com/). For this work, the Renilla luciferase gene (SRUC3) was recloned from the plasmid pBluescript/SRUC3  to replace the EGFP gene in pEGFP-N1, creating pSRUC3-N1, a reporter plasmid with expression of SRUC3 under the control of the CMV promoter. In pZBS-Tet-OFF, the pRetro-OFF vector has been modified to place the tTA (tetracycline repressor fused to viral VP16, ) protein under transcriptional control of 12 ZFHD1-binding sites in a minimal promoter context derived from IL2 (from pZ12I-PL-2). In pBI-EGFP-SEAP, the tetracycline responsive promoter was used to bidirectionally express pEGFP and the secreted alkaline phosphatase (SEAP) gene. The plasmids pAR-DBD and pAR-AD were constructed as derivatives of pC4N2-RHS/ZF3 by eliminating one of the two co-expressed components initially expressed, such that ZFHD-FKBP12 (pAR-DBD) or p65-FRAP (pAR-AD) is separately expressed under the control of the CMV promoter.
Cos7, HeLa, and A2780 cells were used as hosts for transfection experiments, and were cultured using standard conditions as recommended by the ATCC. Experiments for optimization of plasmid ratios were planned and analyzed using the software program STATISTICA http://www.statsoftinc.com. Transfections were performed using a TransIT-LT1 reagent from Mirus. The pSRUC3-N1 plasmid was transfected together with test combinations of other plasmids (as described in Results) as a control for transfection efficiency: typically, 10% of a transfection mixture would be pSRUC3-N1, and levels of Renilla luciferase assessed in collected medium conditioned by transfected cells, based on standard recommended conditions . In a standard experiment, transfected cells were grown in the presence or absence of 25 nM dimerizer AP21967 (Ariad Pharmaceuticals) and/or in the presence or absence of 2 μg/ml tetracycline (Sigma). Reporter activation was determined 42 – 96 hours after transfection. Comparable expression of p65 activation domain fusion proteins under induced conditions was confirmed using polyclonal antibodies directed against NFκB p65 (Geneka Biotechnology Inc.) in Western analysis.
Measurement of Renilla luciferase activity and SEAP activity was performed in a luminometer LKB 1250. Measurement of SEAP activity in culture medium was by standard means using the chemiluminescent Great EscAPe SEAP reporter System (Clontech Labs). Renilla luciferase activity was measured using the Renilla Luciferase Assay System (Promega). In each case, cells were lysed by incubation for 15 min with Renilla Luciferase Assay Lysis Buffer, and the lysed samples were cleared by centrifugation for 30 sec at 14000 min-1 in a microcentrifuge. Data presented for SEAP were corrected based on the calculated transfection efficiency. Human growth hormone levels were assayed using an ELISA kit from Roche (# 1 585 878).
This work was supported by the Ovarian SPORE program at Fox Chase Cancer Center, and by ACS Pilot Project Funding (to IS). We are very grateful to Tom Hamilton and Denise Connolly for useful discussions. We thank Vic Rivera for comments on the manuscript.
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