Abstract
Despite the critical importance of transcription factors in mediating gene regulation, there exists no general, genome-wide tool that uses transcription factors to induce or silence a target gene or select for a particular phenotype. In the strategy described here, we prepared large combinatorial libraries of artificial transcription factors comprising three or six zinc-finger domains, and selected transcription factor–DNA interactions able to upregulate several genes in human cells. Selected transcription factors either induced the expression of an endothelial-specific differentiation marker, VE-cadherin, in non-endothelial cell lines or, when combined with a repression domain, knocked down expression. Potential binding sites for a number of these transcription factors were mapped along the promoter of CDH5, the gene encoding VE-cadherin. Transcription factor libraries represent a useful approach for studying and modulating gene function in cells and potentially in whole organisms.
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References
Beerli, R.R. & Barbas, C.F. III. Engineering polydactyl zinc-finger transcription factors. Nat. Biotechnol. 12, 632–641 (2002).
Brummelkamp, T.R., Benards, R. & Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553 (2002).
Hiroaki, K., Onuki, R., Suyama, E. & Taira, K. Identification of genes that function in the TNF-α-mediated apoptotic pathway using randomized hybrid ribozyme libraries. Nat. Biotechnol. 20, 376–380 (2002).
Walden, R. et al. Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development. Plant Mol. Biol. 26, 1521–1528 (1994).
Beerli, R.R., Dreier, B. & Barbas, C.F. III. Positive and negative regulation of endogenous genes by designed transcription factors. Proc. Natl. Acad. USA 97, 1495–1500 (2000).
Zhang, L. et al. Synthetic zinc finger transcription factor action at an endogenous chromosomal site. Activation of the human erythropoietin gene. J. Biol. Chem. 275, 33850–33860 (2000).
Liu, P.Q. 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. 276, 11323–11334 (2001).
Dreier, B., Beerli, R.R., Segal, D.J., Flippin, J.D. & Barbas, C.F. III. Development of zinc finger domains for recognition of the 5′-ANN-3′ family of DNA sequences and their use in the construction of artificial transcription factors. J. Biol. Chem. 276, 29466–29478 (2001).
Jamieson, A.C., Kim, S.H. & Wells, J.A. In vitro selection of zinc fingers with altered DNA-binding specificity. Biochemistry 33, 5689–5695 (1994).
Segal, D.J., Dreier, B., Beerli, R.R & Barbas, C.F. III. Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences. Proc. Natl. Acad. USA 96, 2758–2763 (1999).
Beerli, R.R., Segal, D.J., Dreier, B. & Barbas, C.F. III. Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc. Natl. Acad. USA 95, 14628–14633 (1998).
Dreier, B., Segal, D.J. & Barbas, C.F III. Insights into the molecular recognition of the 5′-GNN-3′ family of DNA sequences by zinc finger domains. J. Mol. Biol. 303, 489–502 (2000).
Liu, Q., Xia, Z. & Case, C.C. Validated zinc finger protein designs for all 16 GNN DNA triplet targets. J. Biol. Chem. 277, 3850–3856 (2002).
Venter, J.C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001).
Liu, X., Sun, Y., Constantinescu, S.N, Karam, E., Weinberg, R.A. & Lodish, H.F. Transforming growth factor β-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells. Proc. Natl. Acad. Sci. USA, 94, 10669–10674 (1997).
Dejana, E., Bazzoni, G. & Lampugnani, M.G. Vascular endothelial (VE)–cadherin: only an intercellular glue? Exp. Cell Res. 252, 13–19 (1999).
Vittet, D., Buchou, T., Schweitzer, A., Dejana, E. & Hubert, P. Targeted null-mutation in the vascular endothelial–cadherin gene impairs the organization of vascular-like structures in embryoid bodies. Proc. Natl. Acad. USA 94, 6273–6278 (1997).
Carmeliet, P. et al. Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell 98, 147–157 (1999).
Liao, F. et al. Monoclonal antibody to vascular endothelial–cadherin is a potent inhibitor of angiogenesis, tumor growth, and metastasis. Cancer Res. 60, 6805–6810 (2000).
Liao, F. et al. Selective targeting of angiogenic tumor vasculature by vascular endothelial–cadherin antibody inhibits tumor growth without affecting vascular permeability. Cancer Res. 62, 2567–2575 (2002).
Gory, S., Vernet, M., Laurent, M., Dejana, E., Dalmon, J. & Huber, P. The vascular endothelial-cadherin promoter directs endothelial-specific expression in transgenic mice. Blood 93, 184–192 (1999).
Gory, S. et al. Requirement of a GT box (Sp1 site) and two Ets binding sites for vascular endothelial cadherin gene transcription. J. Biol. Chem. 273, 6750–6755 (1998).
Lelievre, E., Mattot, V., Huber, P., Vandenbunder, B. & Soncin, F. ETS1 lowers capillary endothelial cell density at confluence and induces the expression of VE-cadherin. Oncogene 19, 2438–2446 (2000).
Lelievre, E., Lionneton, F., Mattot, V., Spruyt, N. & Soncin, F. Ets-1 regulates fli-1 expression in endothelial cells. Identification of ETS binding sites in the fli-1 gene promoter. J. Biol. Chem. 277, 25143–25151 (2002).
Elrod-Erickson, M., Rould, M.A., Nekludova, L. & Pabo, C.O. Zif268 protein-DNA complex refined at 1.6 A: a model system for understanding zinc finger-DNA interactions. Structure 4, 1171–1180 (1996).
Hendrix, M.J.C. et al. Expression and functional significance of VE-cadherin in aggressive human melanoma cells: role in vasculogenic mimicry. Proc. Natl. Acad. USA 94, 8018–8023 (2001).
Ordiz, M.I., Barbas, C.F III & Beachy, R.N. Regulation of transgene expression in plants with polydactyl zinc finger transcription factors. Proc. Natl. Acad. USA 99, 13290–13295 (2002).
Guan, X. et al. Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors. Proc. Natl. Acad. Sci. USA 99, 13296–13301 (2002).
Barbas, C.F III, Burton, D.R., Scott, J.K., Silverman, G.J. Phage-display vectors. in Phage Display: A Laboratory Manual 2.1–2.19 (CSH, New York, 2001).
Segal, D.J. et al. Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins. Biochemistry (in press).
Acknowledgements
The authors thank D. Valente and N. Niederberger for technical support, and D.J. Segal and X. Li for the critical reading of the manuscript. This work was supported by the US National Institutes of Health CA86258 and DK61803. L. Magnenat was the recipient of postdoctoral fellowships from the Swiss National Science Foundation.
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Supplementary information
Supplementary Figure 1.
Analysis of A431 cells infected with some of the selected pMX-TFsZF pools by flow cytometry from the 3ZF selections (A) or 6ZF selections (B). Blue represents A431 cells infected with the selected pMX-TFsZF pools and stained with the corresponding antibody. Orange represents A431 cells infected with the 3ZF or 6ZF unselected libraries. Green represents mockinfected cells. The stippled line indicates control staining without primary antibody. (PDF 410 kb)
Supplementary Figure 2.
Specificity of isolated 3ZF TFsZF clones VE-5, VE-8, VE-13 and VE-18 (A-D) and 6ZF TFsZF 144-4, 144-5 and 144-13 (E-G) activating VE-cadherin determined by FACS using a panel of 10 cell surface markers. Legend is as described in figure 1. (PDF 1787 kb)
Supplementary Figure 3.
Luciferase transactivation assay of all selected TFsZF with the VE-cadherin promoter. The amount of effector TFsZF construct used in the assay is indicated. (PDF 70 kb)
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Blancafort, P., Magnenat, L. & Barbas, C. Scanning the human genome with combinatorial transcription factor libraries. Nat Biotechnol 21, 269–274 (2003). https://doi.org/10.1038/nbt794
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DOI: https://doi.org/10.1038/nbt794
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