Small-molecule-regulated gene expression offers the promise of titrating the dose and duration of action of DNA-based therapies. To this end, we show that engineered zinc-finger protein transcription factors (ZFP TFs) can be coupled with a drug-inducible regulatory domain to permit small-molecule control of endogenous gene transcription. We constructed a drug-responsive ZFP TF via the fusion of a ZFP DNA-binding domain (DBD) targeting the human VEGF-A gene and an effector domain containing a truncated progesterone receptor ligand-binding domain linked to the NFκB p65 activation domain. Introduction of this engineered ZFP TF into human or murine cells allowed expression of the chromosomal VEGF-A gene to be induced upon addition of mifepristone, a synthetic steroid analog. Mifepristone-dependent VEGF-A induction was rapid, dose-dependent and reversible. Moreover, stable lines expressing the drug-responsive ZFP TF could be maintained in a state of continuous induction for at least 30 days without loss of viability. Potent VEGF-A induction was demonstrated using different engineered ZFP DBDs, thus this approach may represent a general solution to small-molecule regulation of targeted endogenous genes.
The ability to place the expression of an investigator-specified endogenous gene under small-molecule control would be of significant value to basic research, biotechnology and genetic medicine. To this end, we and others have employed engineered zinc-finger protein transcription factors (ZFP TFs), which employ natural transcriptional control mechanisms, to bring about the up- or downregulation of a variety of endogenous genes (reviewed in references1, 2, 3, 4, 5, 6). These factors can function with single gene specificity,7, 8 modulate stem cell fate,9 and have shown promise in multiple pre-clinical animal studies, including models of angiogenesis10, 11, 12, 13 and experimental diabetic neuropathy,14 as well as in multiple clinical trials.
Small-molecule-driven regulation of such factors would permit the finer control of the dose and duration of ZFP TF action, which in therapeutic settings may prove critical for achieving both efficacy and/or safety. Several systems for small-molecule control of gene expression have been developed including the tetracycline repressor,15 steroid hormone receptor,16 ecdysone receptor17, 18 and dimerizer protein-based systems.19 Importantly, steroid hormone receptor zinc-finger fusions including those involving the mifepristone-regulated progesterone receptor have shown promise in earlier studies on reporter genes,20 offering the prospect of regulated endogenous gene expression via a single delivered transgene. Thus, to construct a drug-inducible ZFP TF regulator, we chose to evaluate the mifepristone-inducible progesterone receptor (PR) system.21, 22, 23 The PR system (see model in Figure 1b) creates a single fusion peptide that confers endogenous gene-specific targeting through the engineered ZFP DNA-binding domain (DBD), and small-molecule inducible gene activation via the truncated PR ligand-binding domain (PR-LBDdel19)21, 24, 25 and NFκB p65 activation domain.16, 26, 27 Here we demonstrate that this combination provides an effective means for achieving small-molecule-regulated control over targeted endogenous mammalian genes in vitro.
The engineered ZFP TF VZ+434–p65 described by Liu et al.,28 has been shown to drive the activation of the endogenous VEGF-A gene in human,28 mouse11, 28 and rat14, 28 cells in vitro as well as in various animal models of disease.10, 11, 12, 13, 14 To generate a small-molecule inducible engineered transcription factor, we were guided by the earlier work of Beerli et al.20 on steroid hormone receptor zinc-finger fusions, demonstrating that an engineered ZFP, PR, activation domain fusion could function as an autonomous and mifepristone-dependent transcription factor on an artificial reporter plasmid. For the current study, the DBD from VZ+434–p65 was fused to the truncated PR ligand-binding domain (PR-LBDdel19) and p65 activation domains (Figure 1a) from the pSwitch plasmid21 (Invitrogen, Carlsbad, CA, USA). The resulting ZFP TF fusion protein was designated VZ+434–PR–p65. The truncated human PR-LBDdel19 (containing amino acids 640–914 of the native protein) was selected for its inability to transactivate in response to progestins,24 the natural agonists of PR and yet activate transcription when bound by antiprogestins (such as the synthetic steroid mifepristone). Mifepristone thus acts as an agonist rather than as antagonist with this truncated domain (see references Nordstrom29, 30 and references therein). A model for the drug-inducible activity of the resultant ZFP–PR–p65 fusion is shown in Figure 1b. In the absence of inducer, constitutively produced VZ+434–PR–p65 should remain in an inactive form (Figure 1b, Uninduced) that likely resides in a cytoplasmic complex with heat-shock proteins and other chaperones.31, 32 Addition of the antiprogestin inducer triggers a conformational change that releases the ZFP–PR–p65 protein from the heat-shock proteins, permitting its nuclear translocation, binding to the target site in the VEGF-A gene and the stimulation of transcription at the targeted endogenous gene (Figure 1b, Induced). While speculative, this model is consistent with the results from other groups employing the truncated PR–p65 regulatory system (reviewed by Nordstrom29).
To test whether VZ+434–PR–p65 would permit mifepristone-inducible activation of the endogenous VEGF-A gene, plasmids expressing either VZ+434–PR–p65, Gal4 DBD–PR–p65 (pSwitch), the constitutively active VEGF-A activator VZ+434–p65 or green fluorescent protein (pGFP) were transiently transfected into HEK293 cells and the cells either induced for 48 h with 10 nM mifepristone or mock induced as control. Figure 1c demonstrates that expression of VZ+434–PR–p65 in the presence of mifepristone resulted in a ∼14-fold increase in VEGF-A levels secreted into the medium as determined by enzyme-linked immunosorbent assay (ELISA), and concomitant increase in VEGF-A mRNA (Figure 1d). Activation via the mifepristone-inducible VZ+434–PR–p65 construct was ∼50% of that generated by expression of the constitutively active VZ+434–p65 construct (Figure 1c). The VZ+434 DBD was necessary for VEGF-A gene activation since transfection of plasmid pSwitch (which contains the same PR–p65 regulatory/activation module fused to the Gal4 DBD) resulted in VEGF-A levels similar to uninduced cells or those obtained via transfection with the eGFP expressing control plasmid.
Western blot analysis of whole-cell protein extracts using an antibody against the NFκB p65 domain confirmed the expression of each ZFP TF construct (Figure 1c). Cross reactivity of this antibody with the endogenous p65 protein served as an internal loading control. ZFP TF constructs showed a measurable decrease in protein level upon addition of mifepristone consistent with ligand-dependent increased turnover observed with the endogenous steroid receptors33 (see Figures 1c and 2). This supports the proposed model for regulated gene activation (Figure 1b), namely, that the presence or absence of the inducer regulates the activity of the ZFP–PR–p65 chimera rather than the levels of the engineered factor itself. These data suggest that VZ+434–PR–p65 is a mifepristone-regulated activator of the endogenous VEGF-A gene.
To demonstrate that the results obtained above were not unique to the VZ+434 DBD and/or its target site, a second drug-inducible ZFP TF was constructed incorporating a second previously characterized ZFP DBD that binds the VEGF-A gene at position −573 relative to the transcription start site, that is ∼1 kb upstream of VZ+434. An inducible version of this transcription factor, named VZ−573–PR–p65, was generated as described for VZ+434-PR–p65 (Figure 1a) except that the DBD was derived from the constitutive activator protein VZ−573–p65.28 Plasmid DNA encoding VZ−573–PR–p65 was transiently transfected into HEK293 cells and induced with 10 nM mifepristone or mock induced as described above. VZ+434–PR–p65 was included for comparison, as were the constitutively active engineered ZFP TF constructs VZ−573–p65 and VZ+434–p65, and the negative control plasmids pGFP and pSwitch. Figure 1e shows that VZ−573–PR–p65 also demonstrated mifepristone-dependent activation of VEGF-A with both induced and uninduced levels matching closely those obtained with VZ+434–PR–p65. Both constitutive activators VZ+434–p65 and VZ−573–p65 provided mifepristone-independent activation, as expected. Uninduced levels of VEGF-A expression were comparable to the levels obtained with the negative control plasmids pSwitch, pGFP and GFP–PR–p65 demonstrating the tight control over the uninduced state. Small-molecule inducible activation of VEGF-A gene expression can, therefore, be obtained through two different transactivator proteins (VZ−573–PR–p65 and VZ+434–PR–p65) that target distinct sites upstream and downstream of the transcription start site, respectively. Thus, the combination of engineered ZFP TFs targeting specific endogenous genes and the PR–p65 regulatory/activation domain may represent a general solution to small-molecule regulation of targeted endogenous genes.
To confirm the generality of this observation, we next tested a panel of cell lines representing different tissue types and species for inducible expression of VEGF-A via VZ+434-PR–p65 (Figures 2a–c) including, U2OS human osteosarcoma, HER911 human embryonic retinoblasts, Neuro2A mouse neuroblastoma. As shown above for HEK293 cells (Figure 1c), VZ+434–PR–p65 was found to be a mifepristone-dependent activator of the chromosomal VEGF-A gene (Figures 2a–c). Secreted VEGF-A, induced by VZ+434–p65, has been shown previously to be biologically active in both in vitro and in vivo assays of function.14 Thus, VZ+434–PR–p65 is a cell type- and species-independent inducible activator of the endogenous VEGF-A gene.
The data shown in Figure 1c and Figure 2 also provide four independent experimental settings in which to evaluate how tightly this regulatable system restricts the activity of the engineered ZFP in the uninduced or off state. In all four cases, the uninduced VZ+434–PR–p65 VEGF-A levels were similar to those generated with the parallel samples transfected with the control pSwitch plasmid DNA. Thus, under transient transfection conditions, the truncated PR-LBDdel19-p65 domain appears to restrict the activity of VZ+434–PR–p65 effectively, resulting in low/negligible basal activity. Taken together, these data demonstrate that VZ+434–PR–p65 is a tightly regulated mifepristone-dependent activator of the endogenous VEGF-A gene across different cell types of both human and murine origin.
Next, we wished to determine whether the ZFP–PR–p65 system would permit the reversible regulation of endogenous gene expression. To achieve this, we constructed a HEK293 cell line that stably expressed VZ+434–PR–p65. Single-cell-derived clones expressing VZ+434–PR–p65 were selected and screened for VEGF-A production in the absence and presence of mifepristone. A clone, referred to throughout the remainder of this work as HEK293 (VZ+434–PR–p65), was selected for further studies based upon both the degree of VEGF-A activation in the presence of the inducer and low background expression in its absence. First, we examined the dose responsiveness of the HEK293 (VZ+434–PR–p65) stable line by incubating these cells for 48 h with increasing concentrations of mifepristone (from 0.4 pM to 100 nM) and assaying for secreted VEGF-A in the culture medium. Induction of the endogenous VEGF-A gene was dose-dependent with a clearly detectable increase in VEGF-A secretion at 10 pM and maximal induction obtained at 4 nM (Figure 3a). This experiment demonstrates that the induction through mifepristone provides a ‘rheostat’ for gradual, dose-responsive induction over a broad concentration range of inducer. Moreover, activation of the endogenous VEGF-A is observed at low doses of mifepristone (∼10 pM).
To address the reversibility of VEGF-A induction via VZ+434–PR–p65, we induced the stable line HEK293 (VZ+434–PR–p65) in the presence of mifepristone for 72 h followed by its removal and growth in the absence of inducer for a further 48 h (Figure 3b). As a control, the same cell line was cultured in parallel in the absence or presence of inducer for the entire period of the study. Analysis of VEGF-A level in the medium showed robust induction after 48 h in each of the mifepristone-treated samples. Importantly, when mifepristone was withdrawn after 72 h of culture, a marked decrease in VEGF-A secretion was observed 24 h later, with near complete return to basal VEGF-A levels occurring 48 h after the removal of inducer (Figure 3b). In contrast, continued exposure to mifepristone resulted in maintenance of high levels of VEGF-A secretion through day 5 of the experiment. Thus, withdrawal of the inducer rapidly reverses the induction of the ZFP-targeted endogenous gene.
Finally, to determine whether mifepristone-induced endogenous gene regulation could be achieved for extended periods, as might be needed in a gene therapy approach to chronic conditions, we maintained the stable line HEK293 (VZ+434–PR–p65) in the presence of 10 nM mifepristone for a period of 1 month. During this induction period, the cells continued to expand and were split twice weekly to prevent over-growth. Culture medium was removed from the cells and analyzed by ELISA on the indicated days. Cells were also observed visually for any changes in growth rate or morphology resulting from continuous induction.
As shown in Figure 3c, HEK293 (VZ+434–PR–p65) could be maintained under constant induction for at least 1 month, as demonstrated by the ability of the cells to continuously secrete high levels of VEGF-A. No reduction in the level of gene expression was observed throughout the induction period. Moreover, no differences in the rate of growth or morphology of the cells were observed when compared directly to uninduced cultures grown in parallel. These data demonstrate that small-molecule-driven induction of endogenous gene activation, mediated via a suitably engineered ZFP TF, was well tolerated while supporting long-term induction of the target gene product.
We show here effective small-molecule inducible activation of an endogenous target gene (VEGF-A) by engineered ZFP TFs. Induction is robust, dose-responsive, reversible, DBD dependent and can be sustained for extended periods of time. Furthermore, in agreement with studies of Gal4–PR–p65,34, 35 peak induction was obtained at low (4 nM) mifepristone relative to the 600 mg/day (∼20 μ M) dose of Mifeprex used clinically. Indeed, the Gal4–PR–p65 inducible expression system has been shown to be highly effective in animals via oral or intraperitoneal delivery30, 36, 37 and insects (Drosophila) with induction obtained by feeding or larval bathing.30, 37, 38 These results, together with the successful application of constitutive ZFP TFs in animal models,10, 11, 12, 13, 14 suggest that small-molecule regulation of engineered ZFP–PR–p65 transcription factors would also function in vivo.
Interestingly, the LBD domain of the PR–p65 can dimerize on addition of Mifepristone, a fact that was exploited directly in previous work on ZFP–PR fusions on reporter genes.20 In the present study, we observed activation of the chromosomal VEGF-A gene from each of two distinct single ZFP-binding sites in the promoter (see Figure 1a), although we do not know formally whether a dimer of the ZFP–PR–p65 is recruited to, or assembled at, each site. Such dimerization may explain the reduced activation observed with the ZFP–PR–p65 in comparison to its constitutive ZFP–p65 relative. Moreover, the genome-wide specificity of the ZFP–PR–p65 fusion may be altered (potentially adversely) via such dimerization, although it should be noted that experimentally we observed long-term induction was well tolerated (Figure 3).
In contrast to the majority of regulatory systems described (reviewed recently in Goverdhana et al.39), the approach described here requires only a single protein to be introduced into the cell. The modular nature of the ZFP DBD and regulatory domains allows both to function in the context of a fusion protein. This advantage is significant in two respects. First, the ‘payload’ for delivery to the target cell is reduced, since the need for expression of a multicomponent regulatable system is obviated. Indeed, a single ZFP–PR–p65 fusion protein is sufficiently compact to be combined with any vector system, including those with limited cargo capacity, such as self-complementary adeno-associated vectors (AAV).40 Secondly, all components of the ZFP–PR–p65 are human in origin, theoretically reducing the potential for an overt immunogenic response (though in vivo testing would of course be necessary). The ability of the PR–p65 regulatory/activation domain to function with alternative engineered ZFP DBDs suggests that this system may be a general solution to obtaining small-molecule regulation of endogenous gene expression. Note, however, that the effect of antagonists such as mifepristone on the activity of the wild-type PR can depend upon variables such as cell type, promoter context and general cellular environment.41 Such promoter and/or cell type-specific differences may also affect the ability of the PR-derived domain used in our constructs to control gene expression in response to mifepristone in specific contexts.
Reversible and sustainable gene expression demonstrated using this small-molecule-controlled system is of particular importance to therapeutic applications that require long-term expression of a corrective factor. Firstly, potent natural transcription factors containing powerful activation domains can result in cellular toxicity when expressed at constitutively high levels, potentially via a ‘squelching’ mechanism that may result from competition for essential components of the transcriptional regulation machinery.42 By demonstrating long-term induction of VEGF-A over a period of 1 month, we show that sustained expression and activation of the VZ+434–PR–p65 engineered transcription factor is both functional and well tolerated. We speculate that degradation of the activated transcription factor limits ‘squelching’ and thus permits long-term gene regulation. Note that numerous naturally occurring transcription factors, including the estrogen receptor and VP1643, 44, 45 employ ubiquitination and proteosome-mediated degradation that contribute also to the rapid turn-off of transcription upon cessation of the induction signal. In this regard, the reversibility of endogenous gene expression using ZFP–PR–p65 was found to be rapid (reverting to basal activation levels within 48 h), consistent with the rapid turn over of the active form of the ZFP TF. The rapid shut off of target gene expression in response to the withdrawal of inducer is an important potential safety feature of this system in applications of DNA-based therapy. In summary, the ZFP–PR–p65 system investigated here holds promise as a general method for placing endogenous gene expression under small-molecule control, and should find broad applications in basic research and gene therapy.
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We thank Drs Trevor Collingwood, Michael Holmes, Edward Rebar and Sean Brennan for careful reading of the manuscript. We are also grateful to Edward Lanphier for encouragement and support.
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Dent, C., Lau, G., Drake, E. et al. Regulation of endogenous gene expression using small molecule-controlled engineered zinc-finger protein transcription factors. Gene Ther 14, 1362–1369 (2007). https://doi.org/10.1038/sj.gt.3302985
- gene expression
- receptors, progesterone
- vascular endothelial growth factor
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