Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Phenotypic alteration of eukaryotic cells using randomized libraries of artificial transcription factors

Nature Biotechnology volume 21, pages 1208–1214 (2003)Cite this article

A Corrigendum to this article was published on 01 April 2004

Abstract

We have developed a method in which randomized libraries of zinc finger–containing artificial transcription factors are used to induce phenotypic variations in yeast and mammalian cells. By linking multiple zinc-finger domains together, we constructed more than 100,000 zinc-finger proteins with diverse DNA-binding specificities and fused each of them to either a transcription activation or repression domain. The resulting transcriptional regulatory proteins were expressed individually in cells, and the transfected cells were screened for various phenotypic changes, such as drug resistance, thermotolerance or osmotolerance in yeast, and differentiation in mammalian cells. Genes associated with the selected phenotypes were also identified. Our results show that randomized libraries of artificial transcription factors are useful tools for functional genomics and phenotypic engineering.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic representation of the ZFP library approach.
Figure 2: Various phenotypic changes in yeast induced by artificial ZFP-TFs.
Figure 3: Characterization of ketoconazole-resistant transformants.
Figure 4: Identification of target gene associated with ketoconazole resistance.
Figure 5: Phenotypic changes induced by ZFP-TFs in mammalian cell culture systems.

Similar content being viewed by others

Article 17 June 2019

Anna J. Simon, Simon d’Oelsnitz & Andrew D. Ellington

References

  1. Pavletich, N.P. & Pabo, C.O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1Å. Science 252, 809–817 (1991).

    Article  CAS  Google Scholar 

  2. Wolfe, S.A., Kekludova, L. & Pabo, C.O. DNA recognition by Cys2His2 zinc finger proteins. Annu. Rev. Biophys. Biomol. Struct. 29, 183–212 (2000).

    Article  CAS  Google Scholar 

  3. Lee, D.K., Seol, W. & Kim, J.-S. Custom DNA-binding proteins and artificial transcription factors. Curr. Top. Med. Chem. 3, 645–657 (2003).

    Article  CAS  Google Scholar 

  4. Rebar, E.J. & Pabo, C.O. Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science 263, 671–673 (1994).

    Article  CAS  Google Scholar 

  5. 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).

    Article  CAS  Google Scholar 

  6. Choo, Y. & Klug, A. Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage. Proc. Natl. Acad. Sci. USA 91, 11163–11167 (1994).

    Article  CAS  Google Scholar 

  7. Wu, H., Yang, W.P. & Barbas, C.F. III. Building zinc fingers by selection: toward a therapeutic application. Proc. Natl. Acad. Sci. USA 92, 344–348 (1995).

    Article  CAS  Google Scholar 

  8. 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 

  9. Desjarlais, J.R. & Berg, J.M. Redesigning the DNA-binding specificity of a zinc finger protein: a data base-guided approach. Proteins. Struct. Funct. Genet. 12, 101–104 (1992).

    Article  CAS  Google Scholar 

  10. Nardelli, J., Gibson, T. & Charnay, P. Zinc finger-DNA recognition: analysis of base specificity by site-directed mutagenesis. Nucl. Acids Res. 20, 4137–4144 (1992).

    Article  CAS  Google Scholar 

  11. Taylor, W.E. et al. Designing zinc-finger ADR1 mutants with altered specificity of DNA binding to T in UAS1 sequences. Biochemistry 34, 3222–3230 (1995).

    Article  CAS  Google Scholar 

  12. Kim, J.-S. & Pabo, C.O. Transcriptional repression by zinc finger peptides: exploring the potential for applications in gene therapy. J. Biol. Chem. 272, 29795–29800 (1997).

    Article  CAS  Google Scholar 

  13. Kang, J.S. & Kim, J.-S. Zinc finger proteins as designer transcription factors. J. Biol. Chem. 275, 8742–8748 (2000).

    Article  CAS  Google Scholar 

  14. Bae, K.H. et al. Human zinc fingers as building blocks in the construction of artificial transcription factors. Nat. Biotechnol. 21, 275–280 (2003).

    Article  CAS  Google Scholar 

  15. Gogos, J.A., Jin, J., Wan, H., Kokkinidis, M. & Kafatos, F.C. Recognition of diverse sequences by class I zinc fingers: asymmetries and indirect effects on specificity in the interaction between CF2II and A+T-rich elements. Proc. Natl. Acad. Sci. USA 93, 2159–2164 (1996).

    Article  CAS  Google Scholar 

  16. Hsu, T., Gogos, J.A., Kirsh, S.A. & Kafatos, F.C. Multiple zinc finger forms resulting from developmentally regulated alternative splicing of a transcription factor gene. Science 257, 1946–1950 (1992).

    Article  CAS  Google Scholar 

  17. 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 

  18. 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 

  19. 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 

  20. Liu, P.Q. et al. Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. J. Biol. Chem. 276, 11323–11334 (2001).

    Article  CAS  Google Scholar 

  21. Lee, J.H., Van Montagu, M. & Verbruggen, N. A highly conserved kinase is an essential component for stress tolerance in yeast and plant cells. Proc. Natl. Acad. Sci. USA 96, 5873–5877 (1999).

    Article  CAS  Google Scholar 

  22. Vanden Bossche, H. et al. Antifungal drug resistance in pathogenic fungi. Med. Mycol. 36, 119–128 (1998).

    CAS  PubMed  Google Scholar 

  23. Kadosh, D. & Struhl, K. Repression by Ume6 involves recruitment of a complex containing Sin3 corepressor and Rpd3 histone deacetylase to target promoters. Cell 89, 365–371 (1997).

    Article  CAS  Google Scholar 

  24. Mendez-Vidal, C., Wilhelm, M.T., Hellborg, F., Qian, W. & Wiman, K.G. The p53-induced mouse zinc finger protein wig-1 binds double-stranded RNA with high affinity. Nucl. Acids Res. 30, 1991–1996 (2002).

    Article  CAS  Google Scholar 

  25. Morii, E., Oboli, K., Kataoka, T.R., Iagarashi, K. & Kitamura, Y. Interaction and cooperation of mi transcription factor (MITF) and myc-associated zinc-finger protein-related factor (MAZR) for transcription of mouse mast cell protease 6 gene. J. Biol. Chem. 277, 8566–8571 (2002).

    Article  CAS  Google Scholar 

  26. Sanglard, D. et al. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob. Agents Chemother. 39, 2378–2386 (1998).

    Article  Google Scholar 

  27. Moore, P.A., Ruben, S.M. & Rosen, C.A. Conservation of transcriptional activation functions of the NF-kappa B p50 and p65 subunits in mammalian cells and Saccharomyces cerevisiae. Mol. Cell Biol. 13, 1666–1674 (1993).

    Article  CAS  Google Scholar 

  28. Witzgall, R., O'Leary, E., Leaf, A., Onaldi, D. & Bonventre, J.V. The Kruppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. Proc. Natl. Acad. Sci. USA 91, 4514–4518 (1994).

    Article  CAS  Google Scholar 

  29. Beerli, R.R., Segal, D.J., Drier, 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. Sci. USA 95, 14628–14633 (1998).

    Article  CAS  Google Scholar 

  30. Wainwright, L.J., Lasorella, A. & Lavarone, A. Distinct mechanisms of cell cycle arrest control the decision between differentiation and senescence in human neuroblastoma cells. Proc. Natl. Acad. Sci. USA 98, 9396–9400 (2001).

    Article  CAS  Google Scholar 

  31. Katagiri, T. et al. Bone morphogenic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J. Cell. Biol. 127, 1755–1766 (1994).

    Article  CAS  Google Scholar 

  32. Campling, B.G., Pym, J., Galbraith, P.R. & Cole, S.P. Use of the MTT assay for rapid determination of chemosensitivity of human leukemic blast cells. Leuk. Res. 12, 823–831 (1988).

    Article  CAS  Google Scholar 

  33. Mayer, T.U. et al. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286, 971–974 (1999).

    Article  CAS  Google Scholar 

  34. De Backer, M.D. et al. An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans. Nat. Biotechnol. 19, 235–241 (2001).

    Article  CAS  Google Scholar 

  35. Welch, P.J. et al. Identification and validation of a gene involved in anchorage-independent cell growth control using a library of randomized hairpin ribozymes. Genomics 66, 274–283 (2000).

    Article  CAS  Google Scholar 

  36. Kruger, M. et al. Identification of eIF2Bgamma and eIF2gamma as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach. Proc. Natl. Acad. Sci. USA 97, 8566–8571 (2000).

    Article  CAS  Google Scholar 

  37. Pierce, M.L. & Ruffner, D.E. Construction of a directed hammerhead ribozyme library; toward the identification of optimal target sites for antisense-mediated gene inhibition. Nucl. Acids Res. 26, 5093–5101 (1998).

    Article  CAS  Google Scholar 

  38. Ashrafi, K. et al. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421, 268–272 (2003).

    Article  CAS  Google Scholar 

  39. 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 

  40. Sanches, J.P., Ullman, C., Moore, M., Choo, Y. & Chua, N.H. Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol. 43, 1465–1472 (2002).

    Article  Google Scholar 

  41. Joung, J.K., Ramm, E.I. & Pabo, C.O. A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions. Proc. Natl. Acad. Sci. USA 97, 7382–7387 (2000).

    Article  CAS  Google Scholar 

  42. Gordon, C.L. et al. Glucoamylase: green fluorescent protein fusions to monitor protein secretion in Aspergillus niger. Microbiology 146, 415–426 (2000).

    Article  CAS  Google Scholar 

  43. 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 

  44. Tupler, R., Perini, G. & Green, M.R. Expressing the human genome. Nature 409, 832–833 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K.H. Bae, H.C. Shin and J.W. Park for helpful discussions. We also thank Hyun-Mo Ryoo for providing materials, Jae-Ran Lee and Eunjoon Kim for help with immunofluorescence microscopy and K. LaMarco for carefully reading our manuscript. This work was partially supported by the National Research Laboratory Program (M1-0104-00-0048) and by the 21C Frontier Microbial Genomics and Applications Program (MG02-0302-007-2-1-0) of the Korean Ministry of Science and Technology.

Author information

Author notes

  1. Kyung-Soon Park and Dong-ki Lee: These authors contributed equally to this work.

Authors and Affiliations

  1. ToolGen, Inc., 461-6 Jeonmin-Dong, Yuseong-Gu, Daejeon, 305-390, South Korea

    Kyung-Soon Park, Dong-ki Lee, Horim Lee, Yangsoon Lee, Young-Soon Jang, Yong Ha Kim, Hyo-Young Yang, Sung-Il Lee, Wongi Seol & Jin-Soo Kim

Authors
  1. Kyung-Soon Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dong-ki Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Horim Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yangsoon Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Young-Soon Jang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yong Ha Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hyo-Young Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sung-Il Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Wongi Seol
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jin-Soo Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jin-Soo Kim.

Ethics declarations

Competing interests

The research described in the paper was partially funded by ToolGen, Inc., a privately-held company, and most of the authors are employees. J.-S.K. was one of the founders of the company and is a major shareholder.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Park, KS., Lee, Dk., Lee, H. et al. Phenotypic alteration of eukaryotic cells using randomized libraries of artificial transcription factors. Nat Biotechnol 21, 1208–1214 (2003). https://doi.org/10.1038/nbt868

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt868

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing