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.

A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors

Abstract

The DNA-binding specificities of transcription factors can be used to computationally predict cis-regulatory modules (CRMs) that regulate gene expression1. However, the absence of specificity data for the majority of transcription factors limits the widespread implementation of this approach. We have developed a bacterial one-hybrid system that provides a simple and rapid method to determine the DNA-binding specificity of a transcription factor. Using this technology, we successfully determined the DNA-binding specificity of seven previously characterized transcription factors and one novel transcription factor, the Drosophila melanogaster factor Odd-skipped. Regulatory targets of Odd-skipped were successfully predicted using this information, demonstrating that the data produced by the bacterial one-hybrid system are relevant to in vivo function.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Overview of the bacterial one-hybrid system.
Figure 2: Binding site motifs for seven proteins determined using the B1H system.
Figure 3: Analysis of the DNA-binding specificity of Odd determined using the B1H system.
Figure 4: Altered gene expression after ectopic expression of D. melanogaster Odd.

References

  1. Berman, B.P. et al. Exploiting transcription factor binding site clustering to identify cis-regulatory modules involved in pattern formation in the Drosophila genome. Proc. Natl. Acad. Sci. USA 99, 757–762 (2002).

    Article  CAS  Google Scholar 

  2. Roulet, E. et al. High-throughput SELEX–SAGE method for quantitative modeling of transcription-factor binding sites. Nat. Biotechnol. 20, 831–835 (2002).

    Article  CAS  Google Scholar 

  3. Bulyk, M.L., Huang, X., Choo, Y. & Church, G.M. Exploring the DNA-binding specificities of zinc fingers with DNA microarrays. Proc. Natl. Acad. Sci. USA 98, 7158–7163 (2001).

    Article  CAS  Google Scholar 

  4. Mukherjee, S. et al. Rapid analysis of the DNA-binding specificities of transcription factors with DNA microarrays. Nat. Genet. 36, 1331–1339 (2004).

    Article  CAS  Google Scholar 

  5. Lee, T.I. et al. Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 298, 799–804 (2002).

    Article  CAS  Google Scholar 

  6. Liu, X., Noll, D.M., Lieb, J.D. & Clarke, N.D. DIP-chip: rapid and accurate determination of DNA-binding specificity. Genome Res. 15, 421–427 (2005).

    Article  CAS  Google Scholar 

  7. Wilson, T.E., Fahrner, T.J., Johnston, M. & Milbrandt, J. Identification of the DNA binding site for NGFI-B by genetic selection in yeast. Science 252, 1296–1300 (1991).

    Article  CAS  Google Scholar 

  8. Deplancke, B., Dupuy, D., Vidal, M. & Walhout, A.J. A gateway-compatible yeast one-hybrid system. Genome Res. 14, 2093–2101 (2004).

    Article  CAS  Google Scholar 

  9. 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 

  10. Dove, S.L., Joung, J.K. & Hochschild, A. Activation of prokaryotic transcription through arbitrary protein-protein contacts. Nature 386, 627–630 (1997).

    Article  CAS  Google Scholar 

  11. Bailey, T.L. & Elkan, C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 2, 28–36 (1994).

    CAS  PubMed  Google Scholar 

  12. Wolfe, S.A., Greisman, H.A., Ramm, E.I. & Pabo, C.O. Analysis of zinc fingers optimized via phage display: evaluating the utility of a recognition code. J. Mol. Biol. 285, 1917–1934 (1999).

    Article  CAS  Google Scholar 

  13. Voz, M.L., Agten, N.S., Van de Ven, W.J. & Kas, K. PLAG1, the main translocation target in pleomorphic adenoma of the salivary glands, is a positive regulator of IGF-II. Cancer Res. 60, 106–113 (2000).

    CAS  PubMed  Google Scholar 

  14. Walhout, A.J. & Vidal, M. A genetic strategy to eliminate self-activator baits prior to high-throughput yeast two-hybrid screens. Genome Res. 9, 1128–1134 (1999).

    Article  CAS  Google Scholar 

  15. 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 

  16. Senger, K. et al. Immunity regulatory DNAs share common organizational features in Drosophila. Mol. Cell 13, 19–32 (2004).

    Article  CAS  Google Scholar 

  17. Kovall, R.A. & Hendrickson, W.A. Crystal structure of the nuclear effector of Notch signaling, CSL, bound to DNA. EMBO J. 23, 3441–3451 (2004).

    Article  CAS  Google Scholar 

  18. Tun, T. et al. Recognition sequence of a highly conserved DNA binding protein RBP-J kappa. Nucleic Acids Res. 22, 965–971 (1994).

    Article  CAS  Google Scholar 

  19. Jun, S. & Desplan, C. Cooperative interactions between paired domain and homeodomain. Development 122, 2639–2650 (1996).

    CAS  PubMed  Google Scholar 

  20. Melnikova, I.N., Crute, B.E., Wang, S. & Speck, N.A. Sequence specificity of the core-binding factor. J. Virol. 67, 2408–2411 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Golling, G., Li, L., Pepling, M., Stebbins, M. & Gergen, J.P. Drosophila homologs of the proto-oncogene product PEBP2/CBF beta regulate the DNA-binding properties of Runt. Mol. Cell. Biol. 16, 932–942 (1996).

    Article  CAS  Google Scholar 

  22. Sosinsky, A., Bonin, C.P., Mann, R.S. & Honig, B. Target Explorer: an automated tool for the identification of new target genes for a specified set of transcription factors. Nucleic Acids Res. 31, 3589–3592 (2003).

    Article  CAS  Google Scholar 

  23. Riddihough, G. & Ish-Horowicz, D. Individual stripe regulatory elements in the Drosophila hairy promoter respond to maternal, gap, and pair-rule genes. Genes Dev. 5, 840–854 (1991).

    Article  CAS  Google Scholar 

  24. Saulier-Le Drean, B., Nasiadka, A., Dong, J. & Krause, H.M. Dynamic changes in the functions of Odd-skipped during early Drosophila embryogenesis. Development 125, 4851–4861 (1998).

    CAS  PubMed  Google Scholar 

  25. Serebriiskii, I. & Joung, J. in Protein-Protein Interactions: A Molecular Cloning Manual (ed. E. Golemis) 93–142, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

    Google Scholar 

  26. Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1–I2 regulatory elements. Nucleic Acids Res. 25, 1203–1210 (1997).

    Article  CAS  Google Scholar 

  27. Liu, X., Brutlag, D.L. & Liu, J.S. BioProspector: discovering conserved DNA motifs in upstream regulatory regions of co-expressed genes. Pac. Symp. Biocomput. 6, 127–138 (2001).

    Google Scholar 

  28. Schneider, T.D. & Stephens, R.M. Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 18, 6097–6100 (1990).

    Article  CAS  Google Scholar 

  29. Crooks, G.E., Hon, G., Chandonia, J.M. & Brenner, S.E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    Article  CAS  Google Scholar 

  30. Tautz, D. & Pfeifle, C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81–85 (1989).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank Keith Joung, Jessica Hurt, Carl Pabo and Hermann Bujard for precursor plasmids and strains. Henry Krause for providing the HSodd2 flies. Lucio Castilla, Sean Landrette, Marian Walhout, Marc Freeman, Tony Ip and the Drosophila Genomic Resource Center for various cDNAs. Nadine McGinnis and Robin Smith for technical support. Marian Walhout and Keith Joung for very helpful discussions. We thank the UCSC genome bioinformatics site and the Institute for Genomic Research, the Genome Sequencing Center at Washington University, Agencourt Bioscience Corporation and HGSC at Baylor College of Medicine for access to and analysis of unpublished Drosophila genome data. S.A.W. and X.M. were supported in part by the Concern Foundation and National Institutes of Health (NIH) grant 1R01GM068110; M.H.B. was supported in part by a Basil O'Connor Starter Research Award from the March of Dimes Birth Defects Foundation and by the American Cancer Society grant RSG-05-026-01-CCG. This work was supported by NIH grant 1R01GM068110 (S.A.W.). This work was supported by NIH grant 1R01GM068110 (S.A.W.) and ACS grant RSG-05-026-01-CCG (M.H.B.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Scot A Wolfe.

Ethics declarations

Competing interests

The authors have a pending patent application on related subject matter.

Supplementary information

Supplementary Fig. 1

Comparison of the number of self-activating clones in the original and purified library. (PDF 418 kb)

Supplementary Fig. 2

The Runt/Bgb heterodimer is required for reporter activation. (PDF 450 kb)

Supplementary Fig. 3

Growth rates of cells containing different Odd bait-prey combinations. (PDF 754 kb)

Supplementary Fig. 4

Schematic representation of the conserved Odd binding sites near hairy, paired, gooseberry and Goosecoid. (PDF 153 kb)

Supplementary Table 1

Details of the selections performed with each bait using the B1H system. (PDF 20 kb)

Supplementary Table 2

Unique insert sequences from the prey isolated using the B1H system with each bait. (PDF 61 kb)

Supplementary Table 3

Search results using a PWM for Odd built in Target Explorer. (PDF 82 kb)

Supplementary Methods (PDF 44 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Meng, X., Brodsky, M. & Wolfe, S. A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors. Nat Biotechnol 23, 988–994 (2005). https://doi.org/10.1038/nbt1120

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1120

This article is cited by

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