Two Hybrid Technologies pp 189-208

Part of the Methods in Molecular Biology book series (MIMB, volume 812)

Gene-Centered Yeast One-Hybrid Assays

Protocol

Abstract

Transcription is regulated by sequence-specific transcription factors (TFs) that bind to short genomic DNA elements that can be located in promoters, enhancers and other cis-regulatory modules. Determining which TFs bind where requires techniques that enable the ab initio identification of TF–DNA interactions. These techniques can either be “TF-centered” (protein-to-DNA), where regions of DNA bound by a TF of interest are identified, or “gene-centered” (DNA-to-protein), where TFs that bind a DNA sequence of interest are identified. Here, we describe gene-centered yeast one-hybrid (Y1H) assays. Briefly, in Y1H assays, a DNA fragment is cloned upstream of two different reporters, and these reporter constructs are integrated into the genome of a yeast strain. Next, plasmids expressing TFs as hybrid proteins (hence the name of the assay) fused with the strong transcriptional activation domain (AD) of the yeast TF Gal4 are introduced into the yeast strain. When a TF interacts with the DNA fragment of interest, the AD moiety activates reporter expression in yeast regardless of whether the TF is an activator or repressor in vivo. Sequencing the plasmid in the colonies that exhibit reporter activation reveals the identity of the TFs that can bind the DNA fragment. We have shown Y1H to be a robust method for detecting interactions between a variety of DNA elements and multiple families of TFs.

Key words

Transcription factor Yeast one-hybrid Gene expression Transcription 

References

  1. 1.
    Wang, M. M., and Reed, R. R. (1993) Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast Nature 364, 121–126.PubMedCrossRefGoogle Scholar
  2. 2.
    Li, J. J., and Herskowitz, I. (1993) Isolation of the ORC6, a component of the yeast origin recognition complex by a one-hybrid system Science 262, 1870–1874.Google Scholar
  3. 3.
    Fields, S., and Song, O. (1989) A novel genetic system to detect protein-protein interactions Nature 340, 245–246.PubMedCrossRefGoogle Scholar
  4. 4.
    Walhout, A. J. M., Sordella, R., Lu, X., Hartley, J. L., Temple, G. F., Brasch, M. A., Thierry-Mieg, N., and Vidal, M. (2000) Protein interaction mapping in C. elegans using proteins involved in vulval development Science 287, 116–122.Google Scholar
  5. 5.
    Rual, J. F., Venkatesan, K., Hao, T., Hirozane-Kishikawa, T., Dricot, A., Li, N., Berriz, G. F., Gibbons, F. D., Dreze, M., Ayivi-Guedehoussou, N., Klitgord, N., Simon, C., Boxem, M., Milstein, S., Rosenberg, J., Goldberg, D. S., Zhang, L. V., Wong, S. L., Franklin, G., Li, S., Albala, J. S., Lim, J., Fraughton, C., Llamosas, E., Cevik, S., Bex, C., Lamesch, P., Sikorski, R. S., Vandenhaute, J., Zoghbi, H. Y., Smolyar, A., Bosak, S., Sequerra, R., Doucette-Stamm, L., Cusick, M. E., Hill, D. E., Roth, F. P., and Vidal, M. (2005) Towards a proteome-scale map of the human protein-protein interaction network Nature 437, 1173–1178.PubMedCrossRefGoogle Scholar
  6. 6.
    Ren, B., Robert, F., Wyrick, J. J., Aparicio, O., Jennings, E. G., Simon, I., Zeitlinger, J., Schreiber, J., Hannett, N., Kanin, E., Volkert, T. L., Wilson, C. J., Bell, S. P., and Young, R. A. (2000) Genome-wide location and function of DNA binding proteins Science 290, 2306–2309.PubMedCrossRefGoogle Scholar
  7. 7.
    van Steensel, B., Delrow, J., and Henikoff, S. (2001) Chromatin profiling using targeted DNA adenine methyltransferase Nat Genet 27, 304–8.PubMedCrossRefGoogle Scholar
  8. 8.
    Berger, M. F., Philippakis, A. A., Qureshi, A. M., He, F. S., Estep, P. W., 3rd, and Bulyk, M. L. (2006) Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities Nat Biotechnol 24, 1429–35.PubMedCrossRefGoogle Scholar
  9. 9.
    Walhout, A. J. M. (2006) Unraveling transcription regulatory networks by protein-DNA and protein-protein interaction mapping Genome Res 16, 1445–1454.PubMedCrossRefGoogle Scholar
  10. 10.
    Deplancke, B., Dupuy, D., Vidal, M., and Walhout, A. J. M. (2004) A Gateway-compatible yeast one-hybrid system Genome Res 14, 2093–2101.PubMedCrossRefGoogle Scholar
  11. 11.
    Deplancke, B., Mukhopadhyay, A., Ao, W., Elewa, A. M., Grove, C. A., Martinez, N. J., Sequerra, R., Doucette-Stam, L., Reece-Hoyes, J. S., Hope, I. A., Tissenbaum, H. A., Mango, S. E., and Walhout, A. J. M. (2006) A gene-centered C. elegans protein-DNA interaction network Cell 125, 1193–1205.PubMedCrossRefGoogle Scholar
  12. 12.
    Vermeirssen, V., Barrasa, M. I., Hidalgo, C., Babon, J. A. B., Sequerra, R., Doucette-Stam, L., Barabasi, A. L., and Walhout, A. J. M. (2007) Transcription factor modularity in a gene-centered C. elegans core neuronal protein-DNA interaction network Genome Res 17, 1061–1071.PubMedCrossRefGoogle Scholar
  13. 13.
    Martinez, N. J., Ow, M. C., Barrasa, M. I., Hammell, M., Sequerra, R., Doucette-Stamm, L., Roth, F. P., Ambros, V., and Walhout, A. J. M. (2008) A C. elegans genome-scale microRNA network contains composite feedback motifs with high flux capacity Genes Dev 22, 2535–2549.PubMedCrossRefGoogle Scholar
  14. 14.
    Arda, H. E., Taubert, S., Conine, C., Tsuda, B., Van Gilst, M. R., Sequerra, R., Doucette-Stam, L., Yamamoto, K. R., and Walhout, A. J. M. (2010) Functional modularity of nuclear hormone receptors in a C. elegans gene regulatory network Molecular Systems Biology in press.Google Scholar
  15. 15.
    Reboul, J., Vaglio, P., Rual, J. F., Lamesch, P., Martinez, M., Armstrong, C. M., Li, S., Jacotot, L., Bertin, N., Janky, R., Moore, T., Hudson, J. R., Jr., Hartley, J. L., Brasch, M. A., Vandenhaute, J., Boulton, S., Endress, G. A., Jenna, S., Chevet, E., Papasotiropoulos, V., Tolias, P. P., Ptacek, J., Snyder, M., Huang, R., Chance, M. R., Lee, H., Doucette-Stamm, L., Hill, D. E., and Vidal, M. (2003) C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression Nat. Genet. 34, 35–41.PubMedCrossRefGoogle Scholar
  16. 16.
    Rual, J.-F., Hirozane-Kishikawa, T., Hao, T., Bertin, N., Li, S., Dricot, A., Li, N., Rosenberg, J., Lamesch, P., Vidalain, P.-O., Clingingsmith, T. R., Hartley, J. L., Esposito, D., Cheo, D., Moore, T., Simmons, B., Sequerra, R., Bosak, S., Doucette-Stam, L., Le Peuch, C., Vandenhaute, J., Cusick, M. E., Albala, J. S., Hill, D. E., and Vidal, M. Human ORFeome version 1.1: a platform for reverse proteomics (2004) Genome Res 14, 2128–2135.Google Scholar
  17. 17.
    Vermeirssen, V., Deplancke, B., Barrasa, M. I., Reece-Hoyes, J. S., Arda, H. E., Grove, C. A., Martinez, N. J., Sequerra, R., Doucette-Stamm, L., Brent, M., and Walhout, A. J. M. (2007) Matrix and Steiner-triple-system smart pooling assays for high-performance transcription regulatory network mapping Nat Methods 4, 659–664.PubMedCrossRefGoogle Scholar
  18. 18.
    Walhout, A. J. M., Temple, G. F., Brasch, M. A., Hartley, J. L., Lorson, M. A., van den Heuvel, S., and Vidal, M. (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes Methods in enzymo-logy: “Chimeric genes and proteins” 328, 575–592.Google Scholar
  19. 19.
    Walhout, A. J. M., and Vidal, M. (1999) A genetic strategy to eliminate self-activator baits prior to high-throughput yeast two-hybrid screens Genome Res. 9, 1128–1134.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • John S. Reece-Hoyes
    • 1
  • Albertha J. M. Walhout
    • 1
  1. 1.University of Massachusetts Medical SchoolWorcesterUSA

Personalised recommendations