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Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production

Abstract

Through the shuffling of predefined modular zinc finger domains with predictable target site recognition in vitro, we have generated a large repertoire of artificial transcription factors with five zinc finger domains (TFZFs). Here we report an effective strategy for the selection of ATF libraries by coupling expression of transcriptional activators of the promoter of interest to the enhanced production of retroviral vector particles transferring the TFZF encoding gene. Using this strategy, we successfully selected specific TFZFs that upregulate the expression of the γ-globin promoter. Selected transcription factors induced the expression of γ-globin when coupled to an activation domain and reduced expression when linked to a repression domain. This new retroviral approach might be used to select other TFZFs but might also be generalized for the selection of other protein and small-molecule interactions.

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Figure 1: Schematic illustration of the selection strategy.
Figure 2: Selected TFZF libraries show increased GFP-positive transduced cells after each round of selection.
Figure 3: Selected TFZFs shows repression of γ-globin promoter expression when fused to the repressor domain KRAB.
Figure 4: Activation of human γ-globin in β-YAC bone marrow cells (BMCs) containing selected zinc fingers, as measured by RT-PCR.

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References

  1. Beerli, R.R. & Barbas, C.F. III. Engineering polydactyl zinc-finger transcription factors. Nat. Biotechnol. 20, 135–141 (2002).

    Article  CAS  Google Scholar 

  2. 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).

    Article  CAS  Google Scholar 

  3. Greisman, H.A. & Pabo, C.O. A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites. Science 275, 657–661 (1997).

    Article  CAS  Google Scholar 

  4. 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).

    Article  CAS  Google Scholar 

  5. 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. Sci. USA 96, 2758–2763 (1999).

    Article  CAS  Google Scholar 

  6. Beerli, R.R., Dreier, B. & Barbas, C.F. III. Positive and negative regulation of endogenous genes by designed transcription factors. Proc. Natl. Acad. Sci. USA 97, 1495–1500 (2000).

    Article  CAS  Google Scholar 

  7. Stege, J.T., Guan, X., Ho, T., Beachy, R.N. & Barbas, C.F. III. Controlling gene expression in plants using synthetic zinc finger transcription factors. Plant J. 32, 1077–1086 (2002).

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. Blancafort, P., Magnenat, L. & Barbas, C.F. III. Scanning the human genome with combinatorial transcription factor libraries. Nat. Biotechnol. 21, 269–274 (2003).

    Article  CAS  Google Scholar 

  10. Magnenat, L., Blancafort, P. & Barbas, C.F. III. In vivo selection of combinatorial libraries and designed affinity maturation of polydactyl zinc finger transcription factors for ICAM-1 provides new insights into gene regulation. J. Mol. Biol. 341, 635–649 (2004).

    Article  CAS  Google Scholar 

  11. Chada, K., Magram, J. & Costantini, F. An embryonic pattern of expression of a human fetal globin gene in transgenic mice. Nature 319, 685–689 (1986).

    Article  CAS  Google Scholar 

  12. Chada, K. et al. Specific expression of a foreign β-globin gene in erythroid cells of transgenic mice. Nature 314, 377–380 (1985).

    Article  CAS  Google Scholar 

  13. Kollias, G., Wrighton, N., Hurst, J. & Grosveld, F. Regulated expression of human Aγ-, β-, and hybrid γβ-globin genes in transgenic mice: manipulation of the developmental expression patterns. Cell 46, 89–94 (1986).

    Article  CAS  Google Scholar 

  14. Peterson, K.R. Hemoglobin switching: new insights. Curr. Opin. Hematol. 10, 123–129 (2003).

    Article  CAS  Google Scholar 

  15. Rutherford, T. & Nienhuis, A.W. Human globin gene promoter sequences are sufficient for specific expression of a hybrid gene transfected into tissue culture cells. Mol. Cell. Biol. 7, 398–402 (1987).

    Article  CAS  Google Scholar 

  16. Li, Q., Peterson, K.R., Fang, X. & Stamatoyannopoulos, G. Locus control regions. Blood 100, 3077–3086 (2002).

    Article  CAS  Google Scholar 

  17. Wijgerde, M., Grosveld, F. & Fraser, P. Transcription complex stability and chromatin dynamics in vivo. Nature 377, 209–213 (1995).

    Article  CAS  Google Scholar 

  18. Murray, N., Serjeant, B.E. & Serjeant, G.R. Sickle cell-hereditary persistence of fetal haemoglobin and its differentiation from other sickle cell syndromes. Br. J. Haematol. 69, 89–92 (1988).

    Article  CAS  Google Scholar 

  19. Swank, R.A. & Stamatoyannopoulos, G. Fetal gene reactivation. Curr. Opin. Genet. Dev. 8, 366–370 (1998).

    Article  CAS  Google Scholar 

  20. Blouin, M.J. et al. Genetic correction of sickle cell disease: insights using transgenic mouse models. Nat. Med. 6, 177–182 (2000).

    Article  CAS  Google Scholar 

  21. Gräslund, T., Li, X., Magnenat, L., Popkov, M. & Barbas, C.F. III. Exploring strategies for the design of artificial transcription factors: targeting sites proximal to known regulatory regions for the induction of γ-globin expression and the treatment of sickle cell disease. J. Biol. Chem. 280, 3707–3714 (2005).

    Article  Google Scholar 

  22. May, C. et al. Therapeutic haemoglobin synthesis in β-thalassaemic mice expressing lentivirus-encoded human β-globin. Nature 406, 82–86 (2000).

    Article  CAS  Google Scholar 

  23. Blau, C.A. et al. γ-Globin gene expression in chemical inducer of dimerization (CID)-dependent multipotential cells established from human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice. J. Biol. Chem. 280, 36642–36647 (2005).

    Article  CAS  Google Scholar 

  24. Collis, P., Antoniou, M. & Grosveld, F. Definition of the minimal requirements within the human β-globin gene and the dominant control region for high level expression. EMBO J. 9, 233–240 (1990).

    Article  CAS  Google Scholar 

  25. Powars, D.R., Chan, L. & Schroeder, W.A. The influence of fetal hemoglobin on the clinical expression of sickle cell anemia. Ann. NY Acad. Sci. 565, 262–278 (1989).

    Article  CAS  Google Scholar 

  26. Rebar, E.J. et al. Induction of angiogenesis in a mouse model using engineered transcription factors. Nat. Med. 8, 1427–1432 (2002).

    Article  CAS  Google Scholar 

  27. Maeder, M.L. et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol. Cell 31, 294–301 (2008).

    Article  CAS  Google Scholar 

  28. Lund, C.V., Blancafort, P., Popkov, M. & Barbas, C.F. III. Promoter-targeted phage display selections with preassembled synthetic zinc finger libraries for endogenous gene regulation. J. Mol. Biol. 340, 599–613 (2004).

    Article  CAS  Google Scholar 

  29. Gordley, R.M., Smith, J.D., Graslund, T. & Barbas, C.F. III. Evolution of programmable zinc finger-recombinases with activity in human cells. J. Mol. Biol. 367, 802–813 (2007).

    Article  CAS  Google Scholar 

  30. Miller, J.C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778–785 (2007).

    Article  CAS  Google Scholar 

  31. Nomura, W. & Barbas, C.F. III. In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase. J. Am. Chem. Soc. 129, 8676–8677 (2007).

    Article  CAS  Google Scholar 

  32. Szczepek, M. et al. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat. Biotechnol. 25, 786–793 (2007).

    Article  CAS  Google Scholar 

  33. Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).

    Article  CAS  Google Scholar 

  34. Xu, G.L. & Bestor, T.H. Cytosine methylation targeted to pre-determined sequences. Nat. Genet. 17, 376–378 (1997).

    Article  CAS  Google Scholar 

  35. Mandell, J.G. & Barbas, C.F. III. Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res. 34, W516–W523 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

Thanks to F.C. Costa and R. Neades for technical assistance with the BMC transfections and expression assays. This study was supported by the Skaggs Institute for Chemical Biology and in part by US National Institutes of Health Grants RO1 DK61803 and R01GM065059 (C.F.B.) and R01 DK061804 (K.R.P.).

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U.T., K.R.P. and C.F.B. designed the research; U.T., K.R.P., B.G. and H.F. performed the experiments; U.T., K.R.P. and C.F.B., wrote the manuscript, which all authors commented on.

Corresponding author

Correspondence to Carlos F Barbas III.

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Supplementary Table 1 and Supplementary Methods (PDF 408 kb)

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Tschulena, U., Peterson, K., Gonzalez, B. et al. Positive selection of DNA-protein interactions in mammalian cells through phenotypic coupling with retrovirus production. Nat Struct Mol Biol 16, 1195–1199 (2009). https://doi.org/10.1038/nsmb.1677

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