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:

Regulation of endogenous gene expression with a small-molecule dimerizer

Nature Biotechnology volume 20, pages 729–733 (2002)Cite this article

Abstract

Artificial transcription factors containing designer zinc-finger DNA-binding domains (DBDs) have been used to activate or repress expression of a growing number of endogenous genes. We have combined targeted zinc-finger DBD technology with a dimerizer-regulated gene expression system to permit the small-molecule control of endogenous gene transcription. We constructed a dimerizer-responsive transcription factor that incorporates an artificial zinc-finger DBD targeted to the promoter of the human VEGF gene. Introduction of this activator into human cells allowed expression of the chromosomal VEGF gene to be induced by a small-molecule dimerizer compound consisting of a nonimmunosuppressive rapamycin analog. We found that by directly regulating zinc-finger protein (ZFP) activity, we could circumvent difficulties encountered in the generation of cell lines stably expressing conventional unregulated activators. Dimerizer-dependent VEGF induction was rapid, tight, and dose dependent, and resulted in VEGF protein expression levels several-fold greater than those produced by the natural hypoxic response.

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: Strategy and constructs for dimerizer-regulated transcription.
Figure 2: Dimerizer-dependent induction of the chromosomal VEGF gene in transient transfection assays.
Figure 3: Dimerizer-dependent activation of the chromosomal VEGF gene in stable cell lines.
Figure 4: VEGF induction in the RS3H-VZ-8F3 stable pool.
Figure 5: Kinetics of VEGF mRNA and protein induction.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Pabo, C.O., Peisach, E. & Grant, R.A. Design and selection of novel Cys2His2 zinc finger proteins. Annu. Rev. Biochem. 70, 313–340 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Zhang, L. et al. Synthetic zinc finger transcription factor action at an endogenous chromosomal site. J. Biol. Chem. 275, 33850–33860 (2000).

    Article  CAS  Google Scholar 

  6. Bartsevich, V.V. & Juliano, R.L. Regulation of the MDR1 gene by transcriptional repressors selected using peptide combinatorial libraries. Mol. Pharmacol. 58, 1–10 (2000).

    Article  CAS  Google Scholar 

  7. 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 

  8. Dreier, B., Beerli, R.R., Segal, D.J., Flippin, J.D. & Barbas, C.F. 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 

  9. Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551 (1992).

    Article  CAS  Google Scholar 

  10. Wang, Y., O'Malley, B.W. Jr., Tsai, S.Y. & O'Malley, B.W. A regulatory system for use in gene transfer. Proc. Natl. Acad. Sci. USA 91, 8180–8184 (1994).

    Article  CAS  Google Scholar 

  11. No, D., Yao, T.P. & Evans, R.M. Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. USA 93, 3346–3351 (1996).

    Article  CAS  Google Scholar 

  12. Beerli, R.R., Schopfer, U., Dreier, B. & Barbas, C.F. Chemically regulated zinc finger transcription factors. J. Biol. Chem. 275, 32617–32627 (2000).

    Article  CAS  Google Scholar 

  13. Xu, L. et al. A versatile framework for the design of ligand-dependent, transgene-specific transcription factors. Mol. Ther. 3, 262–273 (2001).

    Article  CAS  Google Scholar 

  14. Rivera, V.M. et al. A humanized system for pharmacologic control of gene expression. Nat. Med. 2, 1028–1032 (1996).

    Article  CAS  Google Scholar 

  15. Ho, S.N., Biggar, S.R., Spencer, D.M., Schreiber, S.L. & Crabtree, G.R. Dimeric ligands define a role for transcriptional activation domains in reinitiation. Nature 382, 822–826 (1996).

    Article  CAS  Google Scholar 

  16. Pollock, R. et al. Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector. Proc. Natl. Acad. Sci. USA 97, 13221–13226 (2000).

    Article  CAS  Google Scholar 

  17. Pomerantz, J.L., Sharp, P.A. & Pabo, C.O. Structure-based design of transcription factors. Science 267, 93–96 (1995).

    Article  CAS  Google Scholar 

  18. Berger, S.L. et al. Genetic isolation of ADA2: a potential transcriptional adaptor required for function of certain acidic activation domains. Cell 70, 251–265 (1992).

    Article  CAS  Google Scholar 

  19. Gilbert, D.M., Heery, D.M., Losson, R., Chambon, P. & Lemoine, Y. Estradiol-inducible squelching and cell growth arrest by a chimeric VP16-estrogen receptor expressed in Saccharomyces cerevisiae: suppression by an allele of PDR1. Mol. Cell Biol. 13, 462–472 (1993).

    Article  CAS  Google Scholar 

  20. Shockett, P., Difilippantonio, M., Hellman, N. & Schatz, D.G. A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice. Proc. Natl. Acad. Sci. USA 92, 6522–6526 (1995).

    Article  CAS  Google Scholar 

  21. Baron, U., Gossen, M. & Bujard, H. Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential. Nucleic Acids Res. 25, 2723–2729 (1997).

    Article  CAS  Google Scholar 

  22. Molinari, E., Gilman, M. & Natesan, S. Proteasome-mediated degradation of transcriptional activators correlates with activation domain potency in vivo. EMBO J. 18, 6439–6447 (1999).

    Article  CAS  Google Scholar 

  23. Salghetti, S.E., Muratani, M., Wijnen, H., Futcher, B. & Tansey, W.P. Functional overlap of sequences that activate transcription and signal ubiquitin-mediated proteolysis. Proc. Natl. Acad. Sci. USA 97, 3118–3123 (2000).

    Article  CAS  Google Scholar 

  24. Guyer, D. et al. Activation of latent transgenes in Arabidopsis using a hybrid transcription factor. Genetics 149, 633–639 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Brand, A.H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993).

    CAS  Google Scholar 

  26. Carmeliet, P. VEGF gene therapy: stimulating angiogenesis or angioma-genesis? Nat. Med. 6, 1102–1103 (2000).

    Article  CAS  Google Scholar 

  27. 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 

  28. Kim, J.S. & Pabo, C.O. Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants. Proc. Natl. Acad. Sci. USA 95, 2812–2817 (1998).

    Article  CAS  Google Scholar 

  29. 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 98, 1437–1441 (2001).

    Article  CAS  Google Scholar 

  30. Vincent, K.A. et al. Angiogenesis is induced in a rabbit model of hindlimb ischemia by naked DNA encoding an HIF-1α/VP16 hybrid transcription factor. Circulation 102, 2255–2261 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Vic Rivera for helpful comments on the manuscript.

Author information

Authors and Affiliations

  1. ARIAD Gene Therapeutics, Inc., 26 Landsdowne Street, Cambridge, 02139, MA

    Roy Pollock, Maryann Giel, Katja Linher & Tim Clackson

Authors
  1. Roy Pollock
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maryann Giel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Katja Linher
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tim Clackson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Roy Pollock.

Ethics declarations

Competing interests

R.P., K.L., and T.C. are employed by and own equity in ARIAD Pharmaceuticals, Inc.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pollock, R., Giel, M., Linher, K. et al. Regulation of endogenous gene expression with a small-molecule dimerizer. Nat Biotechnol 20, 729–733 (2002). https://doi.org/10.1038/nbt0702-729

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0702-729

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