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.

Automated protein-DNA interaction screening of Drosophila regulatory elements

Nature Methods volume 8, pages 1065–1070 (2011)Cite this article

Subjects

Abstract

Drosophila melanogaster has one of the best characterized metazoan genomes in terms of functionally annotated regulatory elements. To explore how these elements contribute to gene regulation, we need convenient tools to identify the proteins that bind to them. Here we describe the development and validation of a high-throughput yeast one-hybrid platform, which enables screening of DNA elements versus an array of full-length, sequence-verified clones containing over 85% of predicted Drosophila transcription factors. Using six well-characterized regulatory elements, we identified 33 transcription factor–DNA interactions of which 27 were previously unidentified. To simultaneously validate these interactions and locate the binding sites of involved transcription factors, we implemented a powerful microfluidics-based approach that enabled us to retrieve DNA-occupancy data for each transcription factor throughout the respective target DNA elements. Finally, we biologically validated several interactions and identified two new regulators of sine oculis gene expression and hence eye development.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: Workflow underlying the generation of the Drosophila transcription factor (TF) ORF clone resource and the Drosophila Y1H AD transcription factor library.
Figure 2: Drosophila high-throughput Y1H platform.
Figure 3: Overview of the TIDY program.
Figure 4: DNA occupancy analysis of Y1H-identified transcription factors by MARE.
Figure 5: In vivo effects of RNAi-mediated knockdown of Y1H-identified transcription factors binding the so10 element.

References

  1. Adams, M.D. et al. The genome sequence of Drosophila melanogaster. Science 287, 2185–2195 (2000).

    Article  Google Scholar 

  2. O'Kane, C.J. & Gehring, W.J. Detection in situ of genomic regulatory elements in Drosophila. Proc. Natl. Acad. Sci. USA 84, 9123–9127 (1987).

    Article  CAS  Google Scholar 

  3. Zinzen, R.P., Girardot, C., Gagneur, J., Braun, M. & Furlong, E.E.M. Combinatorial binding predicts spatio-temporal cis-regulatory activity. Nature 462, 65–70 (2009).

    Article  CAS  Google Scholar 

  4. Filion, G.J. et al. Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 143, 212–224 (2010).

    Article  CAS  Google Scholar 

  5. Bischof, J., Maeda, R.K., Hediger, M., Karch, F. & Basler, K. An optimized transgenesis system for Drosophila using germ-line-specific phic31 integrases. Proc. Natl. Acad. Sci. USA 104, 3312–3317 (2007).

    Article  CAS  Google Scholar 

  6. Stark, A. et al. Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures. Nature 450, 219–232 (2007).

    Article  CAS  Google Scholar 

  7. Simicevic, J. & Deplancke, B. DNA-centered approaches to characterize regulatory protein-DNA interaction complexes. Mol. Biosyst. 6, 462–468 (2010).

    Article  CAS  Google Scholar 

  8. Deplancke, B. et al. A gene-centered C. elegans protein-DNA interaction network. Cell 125, 1193–1205 (2006).

    Article  CAS  Google Scholar 

  9. Adryan, B. & Teichmann, S.A. Flytf: A systematic review of site-specific transcription factors in the fruit fly Drosophila melanogaster. Bioinformatics 22, 1532–1533 (2006).

    Article  CAS  Google Scholar 

  10. Gallo, S.M. et al. Redfly v3.0: Toward a comprehensive database of transcriptional regulatory elements in Drosophila. Nucleic Acids Res. 39, D118–D123 (2011).

    Article  CAS  Google Scholar 

  11. Massouras, A., Decouttere, F., Hens, K. & Deplancke, B. Webprinses: Automated full-length clone sequence identification and verification using high-throughput sequencing data. Nucleic Acids Res. 38 (suppl.), W378–W384 (2010).

    Article  CAS  Google Scholar 

  12. Vermeirssen, V. et al. Matrix and steiner-triple-system smart pooling assays for high-performance transcription regulatory network mapping. Nat. Methods 4, 659–664 (2007).

    Article  CAS  Google Scholar 

  13. Reece-Hoyes, J.S. et al. Yeast one-hybrid assays for gene-centered human gene regulatory network mapping. Nat. Methods doi.10.1038/nmeth.1764 (30 October 2011).

  14. Reece-Hoyes, J.S. et al. Enhanced yeast one-hybrid assays for high-throughput gene-centered regulatory network mapping. Nat. Methods doi:10.1038/nmeth.1748 (30 October 2011).

  15. Koegl, M. & Uetz, P. Improving yeast two-hybrid screening systems. Brief. Funct. Genomics Proteomics 6, 302–312 (2007).

    Article  CAS  Google Scholar 

  16. Vermeirssen, V. et al. Matrix and steiner-triple-system smart pooling assays for high-performance transcription regulatory network mapping. Nat. Methods 4, 659–664 (2007).

    Article  CAS  Google Scholar 

  17. Braun, P. et al. An experimentally derived confidence score for binary protein-protein interactions. Nat. Methods 6, 91–97 (2009).

    Article  CAS  Google Scholar 

  18. Deplancke, B., Dupuy, D., Vidal, M. & Walhout, A.J. A gateway-compatible yeast one-hybrid system. Genome Res. 14 10B, 2093–2101 (2004).

    Article  CAS  Google Scholar 

  19. Maerkl, S.J. & Quake, S.R. A systems approach to measuring the binding energy landscapes of transcription factors. Science 315, 233–237 (2007).

    Article  CAS  Google Scholar 

  20. Punzo, C., Seimiya, M., Flister, S., Gehring, W.J. & Plaza, S. Differential interactions of eyeless and twin of eyeless with the sine oculis enhancer. Development 129, 625–634 (2002).

    CAS  PubMed  Google Scholar 

  21. Czerny, T. et al. Twin of eyeless, a second pax-6 gene of Drosophila, acts upstream of eyeless in the control of eye development. Mol. Cell 3, 297–307 (1999).

    Article  CAS  Google Scholar 

  22. Burtis, K.C., Coschigano, K.T., Baker, B.S. & Wensink, P.C. The doublesex proteins of Drosophila melanogaster bind directly to a sex-specific yolk protein gene enhancer. EMBO J. 10, 2577–2582 (1991).

    Article  CAS  Google Scholar 

  23. Serikaku, M.A. & Otousa, J.E. Sine oculis is a homeobox gene required for Drosophila visual-system development. Genetics 138, 1137–1150 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Cheyette, B.N.R. et al. The Drosophila sine oculis locus encodes a homeodomain-containing protein required for the development of the entire visual-system. Neuron 12, 977–996 (1994).

    Article  CAS  Google Scholar 

  25. Callaerts, P. et al. Drosophila pax-6/eyeless is essential for normal adult brain structure and function. J. Neurobiol. 46, 73–88 (2001).

    Article  CAS  Google Scholar 

  26. Lai, Z.C. & Li, Y. Tramtrack69 is positively and autonomously required for Drosophila photoreceptor development. Genetics 152, 299–305 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Chen, Y.C., Rajagopala, S.V., Stellberger, T. & Uetz, P. Exhaustive benchmarking of the yeast two-hybrid system. Nat. Methods 7, 667–668 (2010).

    Article  CAS  Google Scholar 

  28. Mito, T. et al. Divergent and conserved roles of extradenticle in body segmentation and appendage formation, respectively, in the cricket Gryllus bimaculatus. Dev. Biol. 313, 67–79 (2008).

    Article  CAS  Google Scholar 

  29. Hutson, S.F. & Bownes, M. The regulation of yp3 expression in the Drosophila melanogaster fat body. Dev. Genes Evol. 213, 1–8 (2003).

    CAS  PubMed  Google Scholar 

  30. Li, M.A., Alls, J.D., Avancini, R.M., Koo, K. & Godt, D. The large maf factor traffic jam controls gonad morphogenesis in Drosophila. Nat. Cell Biol. 5, 994–1000 (2003).

    Article  CAS  Google Scholar 

  31. Turatsinze, J.V., Thomas-Chollier, M., Defrance, M. & van Helden, J. Using rsat to scan genome sequences for transcription factor binding sites and cis-regulatory modules. Nat. Protoc. 3, 1578–1588 (2008).

    Article  CAS  Google Scholar 

  32. Bryne, J.C. et al. Jaspar, the open access database of transcription factor-binding profiles: new content and tools in the 2008 update. Nucleic Acids Res. 36, D102–D106 (2008).

    Article  CAS  Google Scholar 

  33. Matys, V. et al. Transfac and its module transcompel: transcriptional gene regulation in eukaryotes. Nucleic Acids Res. 34, D108–D110 (2006).

    Article  CAS  Google Scholar 

  34. Dietzl, G. et al. A genome-wide transgenic rnai library for conditional gene inactivation in Drosophila. Nature 448, 151–156 (2007).

    Article  CAS  Google Scholar 

  35. Ni, J.Q. et al. A Drosophila resource of transgenic rnai lines for neurogenetics. Genetics 182, 1089–1100 (2009).

    Article  CAS  Google Scholar 

  36. Gubelmann, C. et al. Getprime: A gene- or transcript-specific primer database for quantitative real-time PCR. Database 10.1093/database/bar040 (2011).

Download references

Acknowledgements

We thank the members of the Lausanne genomic technologies facility for performing the Illumina sequencing, K.H. Wan for managing cDNA sequencing and transcription factor cDNA clone production, J. Reece-Hoyes and M. Walhout (University of Massachusetts Medical School, Worcester) for discussions of this work and for providing the Y1H-aS2 strain, N. Gheldof for making figures, N.W. Kelley (Biozentrum, University of Basel) for providing PWMs, S. Waszak for MARE data analysis, S. Plaza (Centre de Biologie du Développement, Université de Toulouse) for providing so10-GAL4 flies, and members of the TRiP at Harvard Medical School (US National Institutes of Health National Institute of General Medical Sciences R01-GM084947) and the Vienna Drosophila RNAi Center for providing transgenic RNAi fly stocks used in this study. This work was supported by funds from the Swiss National Science Foundation and SystemsX.ch, by a Marie Curie International Reintegration grant (BD ) from the Seventh Research Framework Programme, by the Frontiers in Genetics National Centres of Competence in Research Program and by Institutional support from the Ecole Polytechnique Fédérale de Lausanne.

Author information

Authors and Affiliations

  1. Laboratory of Systems Biology and Genetics, Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    Korneel Hens, Jean-Daniel Feuz, Alina Isakova, Antonina Iagovitina, Andreas Massouras, Julien Bryois & Bart Deplancke

  2. Laboratory of Developmental Genetics, Vlaams Instituut voor Biotechnologie, Leuven, Belgium

    Patrick Callaerts

  3. Department of Human Genetics, Laboratory of Developmental Genetics, Catholic University of Leuven, Leuven, Belgium

    Patrick Callaerts

  4. Department of Genome Dynamics, Berkeley Drosophila Genome Project, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Susan E Celniker

Authors
  1. Korneel Hens
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jean-Daniel Feuz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alina Isakova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Antonina Iagovitina
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andreas Massouras
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Julien Bryois
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Patrick Callaerts
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Susan E Celniker
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Bart Deplancke
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.D. supervised the study. K.H. and B.D. designed the study. K.H. and J.B. built the transcription factor clone collection. K.H. and J.-D.F. performed Y1H screens. K.H. performed in vivo validations. A. Iagovitina developed image analysis software. A. Isakova performed MARE analyses. A.M. analyzed high-throughput sequencing data. P.C. provided cDNA clones and financial support. S.E.C. identified transcription factors with sequence-specific DNA-binding domains used in this study and provided transcription factor cDNA clones. K.H. and B.D. provided the manuscript.

Corresponding author

Correspondence to Bart Deplancke.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18, Supplementary Tables 2, 4–6, Supplementary Data (PDF 3014 kb)

Supplementary Table 1

Predicted transcription factors in the Drosophila genome and their cloning status. (XLSX 1119 kb)

Supplementary Table 3

CRMs used in this study. (XLSX 21 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hens, K., Feuz, JD., Isakova, A. et al. Automated protein-DNA interaction screening of Drosophila regulatory elements. Nat Methods 8, 1065–1070 (2011). https://doi.org/10.1038/nmeth.1763

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1763

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research