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Stem Cells 2003;21:632-637 www.StemCells.com
© 2003 AlphaMed Press

Rapid Communication

Engineered Zinc Finger Proteins for Controlling Stem Cell Fate

Victor V. Bartsevich, Jeffrey C. Miller, Casey C. Case, Carl O. Pabo

Sangamo BioSciences, Inc., Point Richmond Tech Center, Richmond, California, USA

Key Words. Zinc finger proteins • Embryonic stem cells • Differentiation

Victor V. Bartsevich, Ph.D., Sangamo BioSciences, Inc., Point Richmond Tech Center, 501 Canal Boulevard, Suite A100, Richmond, California 94804, USA. Telephone: 510-970-6000, ext. 253; Fax: 510-236-8951; e-mail: victorb{at}sangamo.com


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
 References
 
Stem cells are functionally defined as progenitor cells that can self-renew and differentiate. Critical transitions in these cells are controlled via signaling pathways and subsequent transcriptional regulation. Technologies capable of modulating the levels of gene expression, especially those of transcription factors, represent powerful tools for research and could potentially be used in therapeutic applications. In this study, we evaluated the ability of synthetic zinc finger protein transcription factors (ZFP-TFs) to cause the differentiation of embryonic stem (ES) cells. We constructed ZFP-TFs that target the mouse Oct-4 gene (which is a major regulator of ES cell pluripotency and self-renewal). These designed transcription factors were able to regulate the transcription of Oct-4, affecting the expression of downstream genes and thus regulating ES cell differentiation.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Conclusion
 References
 
Stem cells are defined functionally as progenitor cells that can self-renew and differentiate into specialized cell types. Embryonic stem (ES) cells isolated from the inner cell mass of the blastocyst have the capacity to generate all cell types of the body and, thus, are considered to be the most pluripotent cell type known [1]. The ability of stem cells to differentiate into a wide range of cell types should make them suitable for cell-based transplantation therapies of various diseases. However, a number of technical problems still need to be solved, especially those involving isolation of stem cells, propagation, and directed differentiation (in a way that is robust enough and practical enough for commercial application).

Transcription factors control many critical transitions in stem cell fate, and it has been shown that regulation of a single factor can either trigger stem cell differentiation or enhance stem cell longevity. For example, in mouse ES cells, activation or repression of Oct-4 [2] resulted in cell differentiation. Overexpression of another factor, HOXB4, did not trigger cell differentiation but rather resulted in extension of the lifespan of hematopoietic stem cells [3, 4]. Regulating the expression of such critical genes should provide a powerful approach for stem cell manipulation.

A number of techniques are available for modulating gene expression, but we have focused on the use of engineered zinc finger protein transcription factors (ZFP-TFs) to control stem cell fate. ZFP-TF design and selection have been comprehensively reviewed [5–7]. These proteins contain two main modules: a DNA-binding domain and a transcription regulation domain. The synthetic DNA-binding module typically consists of three to six Cys2-His2 zinc finger domains.

Each finger in such a protein contacts three to four base pairs (bp) of DNA, and the full proteins bind extended sequences (typically 9–20 bp) with high affinity and specificity. The attached regulatory module in a typical ZFP-TF can be either an activation or a repression domain (making the use of ZFP-TFs more versatile than other approaches). An additional level of regulation can be achieved when domains are added that allow nuclear localization of the ZFP-TF to be controlled by a small molecular switch [8]. ZFP-TFs have been tested in many settings [9–13], and recent work, for example, has shown how they can induce vascular endothelial growth factor A expression and thus stimulate the growth of new blood vessels in mice [14].

Here we have constructed ZFP-TFs that target the mouse Oct-4 gene. This is an important target, since Oct-4 is expressed in ES and germ cells [15, 16], and this transcription factor is important for the maintenance of pluripotency and self-renewal [17]. Cells that lose Oct-4 during embryonic development differentiate into somatic lineages. In mouse ES cells, the transcriptional responses and morphological changes that result from altered levels of Oct-4 expression have been well characterized [2]. We show here that appropriately designed ZFP-TFs can regulate Oct-4 levels and thus control stem cell fate.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Conclusion
 References
 
Cell Culture and Transfection
Mouse ES cells, ES-D3, were received from American Type Culture Collection (http://www.atcc.org) and propagated on gelatin-coated dishes in knockout Dulbecco’s modified Eagle’s medium (GIBCO/BRL; Carlsbad, CA; http://www.invitrogen.com) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 10 ng/ml murine leukemia inhibitory factor (LIF).

For transfection in 12-well plates, we plated 2 x 105 cells per well 1 day before transfection. In these experiments, 1.7 µg DNA were diluted into 180 µl serum-free OPTI-MEM I medium (GIBCO/BRL), mixed with 4 µl LipofectAMINE 2000 (Invitrogen; Carlsbad, CA; http://www.invitrogen.com) diluted into 180 µl OPTI-MEM I, incubated for 20 minutes, and added to ES cells. After incubation for 5 hours at 37°C in a CO2 incubator, the transfection mixture was replaced with regular growth medium.

For stable transfection and for analysis of differentiation, cells were replated (at a 1:14 dilution) into fresh growth medium after 48 hours and G418 antibiotic was added for selection at 400 µg/ml. Cells were analyzed after an additional incubation of 4–6 days.

ZFP Design, Analysis, and Plasmid Construction
Cys2-His2 ZFPs with the desired DNA sequence specificity were assembled by joining three two-finger units, where each two-finger unit was chosen from a pool of premade ZFP libraries selected via phage display [18]. Within each unit, zinc finger domains were connected with the canonical TGEKP linker; the two-finger units were connected with extended linkers [19] to enhance DNA-binding specificity. These designed six-finger proteins targeted 18–19 bp binding sites.

The in vitro DNA-binding activity of the ZFPs was verified with an enzyme-linked immunosorbent assay (ELISA). In this assay, ZFPs with an HA tag YPYDVPDYA were mixed with biotin-tagged double-stranded DNA and an anti-HA peroxidase-fused antibody. After equilibration, this mixture was applied to a streptavidin-coated plate; unbound material was washed away, and the peroxidase activity was then measured using the QuantaBlu system (Pierce Chemical Co.; Rockford, IL; http://www.piercenet.com).

To construct the expression plasmids, the ZFPs were subcloned into the pcDNA3.1 mammalian expression vector (Invitrogen) under control of a human cytomegalovirus (CMV) immediate-early promoter. In some constructs, the CMV promoter was replaced by a human elongation factor 1{alpha} (EF-1{alpha}) promoter cloned from pTracer-CMV/Bsd vector (Invitrogen). All ZFP constructs contained an N-terminal nuclear localization signal from the simian virus 40 large T antigen and a C-terminal FLAG peptide DYKDDDDK. The herpes simplex virus VP16 activation domain (amino acids 413–490) or the repression domain from the human KOX1 protein (amino acids 1–97) was inserted between the ZFP and FLAG domains to generate the desired ZFP-TFs.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Quantitative RT-PCR
Samples were analyzed 48 hours after transfection. Total RNA was isolated with a High Pure RNA Isolation Kit (Roche Diagnostics Corporation; Basel, Switzerland; http://www.roche.com) and analyzed by quantitative RT-PCR on an ABI PRISM 7700 Sequence Detector (Applied Biosystems; Foster City, CA; http://www.appliedbiosystems.com) using the Taqman assay according to the manufacturer’s recommendations. Primers and probes for the Taqman assay are shown in Table 1Go. All experiments were repeated three to five times. General RT-PCR was performed using a MasterAmp High Fidelity RT-PCR Kit (Epicentre Technologies; Madison, WI; http://www.epicentre.com).


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  		Table 1. Nucleotide sequences of the primers and probes used for Taqman and RT-PCR analyses
 

   RESULTS AND DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Conclusion
 References
 
Zinc Finger Protein Design
In an initial screen, we designed 10 DNA-binding modules that targeted the promoter of the Oct-4 gene. Each module contained six Cys2-His2 zinc finger domains and was assembled by joining three two-finger units (as described in Materials and Methods). These DNA-binding domains were then linked to the herpes simplex virus VP16 activation domain or to the human KOX1 repression domain. In preliminary studies, these 10 ZFP-TFs were analyzed with ELISA and Taqman assays to see which bound DNA the best and which were most effective in regulating the expression of the chromosomal Oct-4 gene. Here, we present data for the one protein that demonstrated the best results in both tests. This ZFP-TF targets a 19-bp site (AGGGTGGGATTGGGGAGGG), which is located between bp -25 and -7 relative to the first transcription initiation start in the Oct-4 promoter [20].

Regulation of Oct-4 with ZFP-TFs
The chosen ZFP was carefully retested for its ability to regulate the Oct-4 gene in mouse ES cells. Cells were transiently transfected with plasmids expressing the DNA-binding domain alone or as a fusion with the VP16 activation or the KOX1 repression domains. The transfection efficiency was at least 80%, as detected by fluorescence microscopy in cells transfected with the green fluorescent protein (GFP). Because of a controversy in the literature about the functional efficiency of a human CMV immediate-early promoter in ES cells [21, 22], we constructed plasmids driving ZFP expression via CMV and also via a human EF-1{alpha} promoter (EF-labeled constructs, Fig. 1Go). After 2 days, relative levels of Oct-4 expression were analyzed by quantitative RT-PCR (Fig. 1AGo). Transfection with the DNA-binding domain alone did not affect Oct-4 transcription. In contrast, activators constructed with this ZFP induced overexpression of Oct-4 up to 1.6 fold, and the corresponding repressors gave a threefold reduction in transcription. Both promoters, CMV and EF-1{alpha}, were functional in the ES cells, but the EF-1{alpha} promoter was consistently more efficient.



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  		Figure 1. Quantitative RT-PCR analysis. A) Modulation of Oct-4 expression by ZFP-TFs and subsequent changes in transcription of OTX1 and HAND1. B) Relative levels of ZFP expression. RT-PCR was performed on total RNA isolated from ES cells transiently transfected for 48 hours. Data were normalized against GAPDH expression. Gene expression was driven by human CMV immediate-early promoter or by human EF-1{alpha} promoter (indicated as EF). Designed ZFP was linked to the VP16 activation or KOX1 (KOX) repression domains (as indicated). As a control, we used RNA from untransfected cells (NT) or from cells transfected with plasmid expressing GFP.

 
We also monitored expression of OTX1, which is a target gene for Oct-4 [23], and HAND1, which is a transcription factor involved in trophoblast differentiation [24]. It had previously been shown that changes in Oct-4 levels lead to changes (in the same direction) in OTX1 levels [2]. In contrast, it had been shown that lowering Oct-4 levels increased HAND1 levels. In our experiments, activation or repression of Oct-4 expression (caused by transient ZFP-TF overexpression) gave the expected downstream effects on OTX1 and HAND1 levels (Fig. 1AGo). We also observed that HAND1 was repressed when Oct-4 was activated.

Modulation of the expression of these downstream "marker genes" was consistently stronger when the EF-1{alpha} promoter was used to drive ZFP-TF expression. Quantitative RT-PCR analysis revealed that this correlated with enhanced ZFP-TF overexpression from the EF-1{alpha} promoter (relative to the CMV promoter, Fig. 1BGo).

In the case of Oct-4 repression by EF-ZFP-KOX, RT-PCR analysis revealed the induction of expression of two additional trophoblast markers, CDX2 and MASH2 [25], in transiently transfected cells (Fig. 2Go).



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  		Figure 2. RT-PCR analysis. RT-PCR was performed on total RNA isolated from ES cells transiently transfected for 48 hours with GFP or EF-ZFP-KOX.

 
Morphological Changes in ES Cells
It is known that changes in Oct-4 levels are correlated with morphological changes in ES cells. Previous studies demonstrated that, for self-renewal and maintenance of ES cell pluripotency, Oct-4 should be expressed at ~50%-150% of the endogenous amount [2]. A greater than 50% increase in Oct-4 expression causes ES cells to differentiate into primitive endoderm and mesoderm. Twofold repression induces ES cells to differentiate into large, flattened trophoblast cells.

Because our ZFP-TFs modulated Oct-4 expression beyond these threshold levels, we expected that these ZFP-TFs could induce morphological changes. To test this, ES cells were transfected with ZFPs and then treated with an antibiotic to select for transfected cells. Under our conditions, the mouse ES cells grew undifferentiated in the presence of murine LIF and formed three-dimensional colonies of tightly packed uniform cells (Fig. 3AGo). The absence of LIF triggered the differentiation and formation of a variety of different cell types (Fig. 3BGo). Almost all cells that were transfected with the EF-ZFP-KOX repressor and selected via antibiotic resistance differentiated and formed large, flattened cells (Fig. 3CGo) in the presence of LIF. Using a similar protocol with the ZFP activator showed that only a small percentage of the cells changed their morphology (a cluster of differentiated cells is shown in Fig. 3DGo). Control experiments with the DNA-binding domain alone or with the regulatory domains fused only to GFP caused no such effects in stably transfected ES cells (data not shown). We conclude that both our ZFP repressor and our ZFP activator were able to induce ES cell differentiation via modulating Oct-4 levels. The activator was less efficient, presumably because of the relatively low activation of Oct-4 (around the critical level indicated in [2]). It is also possible that different cell lines and growth conditions might reveal differences in Oct-4 expression levels that are permissive for the undifferentiated growth of ES cells.



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  		Figure 3. Mouse ES cell differentiation. A) Undifferentiated cells cultured in the presence of LIF. B) Differentiated cells cultured in the absence of LIF for 10 days. C and D) Differentiation of cells in the presence of LIF induced by repression or activation of the Oct-4 gene for 6–8 days using the ZFP with the KOX1 (KOX) repression or VP16 activation domains. In the case of Oct-4 activation, only a cluster of differentiated cells is shown (the majority of cells did not differentiate).

 

   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
References
 
In this study, we constructed ZFP-TFs that regulate the mouse Oct-4 gene and affect ES cell fate. Using the ZFP to induce or repress the transcription of Oct-4 resulted in corresponding downstream transcriptional responses and led to cell differentiation. Our data clearly demonstrate that engineered ZFPs can control ES cell phenotypes.

There are many potential applications of ZFP-TFs in controlling stem cell fate. In cases where the regulatory pathways are still being analyzed, ZFP technology can be used for the functional evaluation of candidate genes that may be involved in mammalian lineage determination and in cellular specialization. When the critical genes are known, ZFP-TFs might be used to help to maintain stem cells indefinitely in the undifferentiated state or to control stem cell differentiation as needed for cell-based therapies.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
 References
 
We thank Dr. Fyodor D. Urnov, Dr. Andreas Reik, Dr. Michael C. Holmes, Dr. Trevor N. Collingwood, and Dr. Simon P. Chandler for helpful discussions and advice on this manuscript.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusion
 References
 

  1. Smith AG. Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol 2001;17:435–462.[CrossRef][Medline]

  2. Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24:372–376.[CrossRef][Medline]

  3. Helgason CD, Sauvageau G, Lawrence HJ et al. Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro. Blood 1996;87:2740–2749.[Abstract/Free Full Text]

  4. Antonchuk J, Sauvageau G, Humphries RK. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 2002;109:39–45.[CrossRef][Medline]

  5. Pabo CO, Peisach E, Grant RA. Design and selection of novel Cys2His2 zinc finger proteins. Annu Rev Biochem 2001;70:313–340.[CrossRef][Medline]

  6. Jamieson AC, Miller JC, Pabo CO. Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov 2003;2:361–368.[CrossRef][Medline]

  7. Beerli RR, Barbas CF 3rd. Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol 2002;20:135–141.[CrossRef][Medline]

  8. Habeck M. Regulatable gene therapy. Drug Discov Today 2002;7:888–889.[CrossRef][Medline]

  9. Bartsevich VV, Juliano RL. Regulation of the MDR1 gene by transcriptional repressors selected using peptide combinatorial libraries. Mol Pharmacol 2000;58:1–10.[Abstract/Free Full Text]

  10. Zhang L, Spratt SK, Liu Q et al. Synthetic zinc finger transcription factor action at an endogenous chromosomal site. Activation of the human erythropoietin gene. J Biol Chem 2000;275:33850–33860.[Abstract/Free Full Text]

  11. Beerli RR, Dreier B, Barbas CF 3rd. Positive and negative regulation of endogenous genes by designed transcription factors. Proc Natl Acad Sci USA 2000;97:1495–1500.[Abstract/Free Full Text]

  12. Liu PQ, Rebar EJ, Zhang L et al. Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. J Biol Chem 2001;276:11323–11334.[Abstract/Free Full Text]

  13. Ren D, Collingwood TN, Rebar EJ et al. PPARgamma knockdown by engineered transcription factors: exogenous PPARgamma2 but not PPARgamma1 reactivates adipogenesis. Genes Dev 2002;16:27–32.[Abstract/Free Full Text]

  14. Rebar EJ, Huang Y, Hickey R et al. Induction of angiogenesis in a mouse model using engineered transcription factors. Nat Med 2002;8:1427–1432.[CrossRef][Medline]

  15. Scholer HR, Ruppert S, Suzuki N et al. New type of POU domain in germ line-specific protein Oct-4. Nature 1990;344:435–439.[CrossRef][Medline]

  16. Rosner MH, Vigano MA, Ozato K et al. A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 1990;345:686–692.[CrossRef][Medline]

  17. Pesce M, Scholer HR. Oct-4: gatekeeper in the beginnings of mammalian development. STEM CELLS 2001;19:271–278.[Abstract/Free Full Text]

  18. Isalan M, Klug A, Choo Y. A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nat Biotechnol 2001;19:656–660.[CrossRef][Medline]

  19. Moore M, Klug A, Choo Y. Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units. Proc Natl Acad Sci USA 2001;98:1437–1441.[Abstract/Free Full Text]

  20. Okazawa HK, Okamoto K, Ishino F et al. The oct3 gene, a gene for embryonic transcription factor, is controlled by a retinoic acid repressible enhancer. EMBO J 1991;10:2997–3005.[Medline]

  21. Chung S, Andersson T, Sonntag KC et al. Analysis of different promoter systems for efficient transgene expression in mouse embryonic stem cell lines. STEM CELLS 2002;20:139–145.[Abstract/Free Full Text]

  22. Ward CM, Stern PL. The human cytomegalovirus immediate-early promoter is transcriptionally active in undifferentiated mouse embryonic stem cells. STEM CELLS 2002;20:472–475.[Abstract/Free Full Text]

  23. Saijoh Y, Fujii H, Meno C et al. Identification of putative downstream genes of Oct-3, a pluripotent cell-specific transcription factor. Genes Cells 1996;1:239–252.[Abstract]

  24. Cserjesi P, Brown D, Lyons GE et al. Expression of the novel basic helix-loop-helix gene eHAND in neural crest derivatives and extraembryonic membranes during mouse development. Dev Biol 1995;170:664–678.[CrossRef][Medline]

  25. Tanaka S, Kunath T, Hadjantonakis AK et al. Promotion of trophoblast stem cell proliferation by FGF-4. Science 1998;282:2072–2075.[Abstract/Free Full Text]

Received February 10, 2003; accepted for publication August 4, 2003.



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