Automated protein-DNA interaction screening of Drosophila regulatory elements

Journal name:
Nature Methods
Volume:
8,
Pages:
1065–1070
Year published:
DOI:
doi:10.1038/nmeth.1763
Received
Accepted
Published online

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.

At a glance

Figures

  1. Workflow underlying the generation of the Drosophila transcription factor (TF) ORF clone resource and the Drosophila Y1H AD transcription factor library.
    Figure 1: Workflow underlying the generation of the Drosophila transcription factor (TF) ORF clone resource and the Drosophila Y1H AD transcription factor library.

    Of 755 predicted Drosophila transcription factors, 501 were available as cDNA clones from the Berkeley Drosophila Genome Project (BDGP). The remaining transcription factors were targeted for de novo cloning. Transcription factor ORFs were PCR-amplified and cloned into the pDONR221 Entry vector. The resulting Entry clones were sequence-verified by high-throughput sequencing and categorized according to the quality and the coverage of the sequencing into three classes: gold for fully sequence–verified clones, silver for 5′ and 3′ end-sequenced clones, and bronze for partially sequenced clones. All nonrejected clones were transferred into the Y1H-compatible AD vectors pAD-DEST-ARS/CEN and pAD-DEST-2μ by Gateway cloning.

  2. Drosophila high-throughput Y1H platform.
    Figure 2: Drosophila high-throughput Y1H platform.

    A yeast DNA-bait strain was distributed over a 384-well plate. Each well of this plate was then transformed with a different AD transcription factor clone from the Drosophila Y1H AD transcription factor library by a robotic yeast transformation platform, which additionally spotted the 384 individually transformed yeast strains on a permissive agar plate. A colony-pinning robot then transferred the yeast colonies onto a permissive and a selective plate, quadruplicating each colony in a square pattern in the process. Transcription factor–DNA bait interactions were identified based on growth on a selective, 3-amino-1,2,4-triazole–containing yeast plate.

  3. Overview of the TIDY program.
    Figure 3: Overview of the TIDY program.

    (a) Flowchart of TIDY program steps. (b) Screenshot of the TIDY output upon image analysis of a selective plate from a Y1H screen. In this example, five interactions were observed (green circles). A different threshold was used for plate-interior and plate-exterior yeast colonies.

  4. DNA occupancy analysis of Y1H-identified transcription factors by MARE.
    Figure 4: DNA occupancy analysis of Y1H-identified transcription factors by MARE.

    (af) Analysis of the so10 element for binding of EY (a), TOY (b), CG9797 (c) and TTK (d) and of the yp1-1 element for binding of DSX (e) and TJ (f). Bound DNA levels normalized over surface-immobilized protein amounts are plotted for each 12-nucleotide stretch and as an interpolated curve. Peaks are indicated with a red line, peak maxima are indicated with a red dot. Peaks found in both replicates are indicated with an asterisk. Where available, DNase I footprinting data and PWM-based binding site predictions are indicated. Overlapping DNase I footprinting data and PWM-based binding site predictions are indicated. Note that as DNA occupancy is plotted as a relative signal normalized for the protein level in the microfluidics chamber, the scale of the y axis may vary between replicates.

  5. In vivo effects of RNAi-mediated knockdown of Y1H-identified transcription factors binding the so10 element.
    Figure 5: In vivo effects of RNAi-mediated knockdown of Y1H-identified transcription factors binding the so10 element.

    (a,b) Bright-field microscopy images of adult eyes, lateral view of OK107>CG9797-RNAiTRiP (a) and OK107>UAS-mCD8-GFP (b) flies. (c,d) Bright-field microscopy images of adult eyes, frontal view of OK107>ttk-RNAiVDRC (c) and OK107>UAS-mCD8-GFP (d) flies. Scale bars, 100 μm. (e,f) Quantitative real-time PCR analysis of so expression in third instar eye-antennal discs of OK107>CG9797-RNAiVDRC and OK107>CG9797-RNAiTRiP flies (e) and in the indicated tissues of OK107>ttk-RNAiVDRC flies (f). Values are relative to the corresponding controls. Error bars, s.e.m. (n = 3). *P < 0.05.

References

  1. Adams, M.D. et al. The genome sequence of Drosophila melanogaster. Science 287, 21852195 (2000).
  2. O'Kane, C.J. & Gehring, W.J. Detection in situ of genomic regulatory elements in Drosophila. Proc. Natl. Acad. Sci. USA 84, 91239127 (1987).
  3. Zinzen, R.P., Girardot, C., Gagneur, J., Braun, M. & Furlong, E.E.M. Combinatorial binding predicts spatio-temporal cis-regulatory activity. Nature 462, 6570 (2009).
  4. Filion, G.J. et al. Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 143, 212224 (2010).
  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, 33123317 (2007).
  6. Stark, A. et al. Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures. Nature 450, 219232 (2007).
  7. Simicevic, J. & Deplancke, B. DNA-centered approaches to characterize regulatory protein-DNA interaction complexes. Mol. Biosyst. 6, 462468 (2010).
  8. Deplancke, B. et al. A gene-centered C. elegans protein-DNA interaction network. Cell 125, 11931205 (2006).
  9. Adryan, B. & Teichmann, S.A. Flytf: A systematic review of site-specific transcription factors in the fruit fly Drosophila melanogaster. Bioinformatics 22, 15321533 (2006).
  10. Gallo, S.M. et al. Redfly v3.0: Toward a comprehensive database of transcriptional regulatory elements in Drosophila. Nucleic Acids Res. 39, D118D123 (2011).
  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.), W378W384 (2010).
  12. Vermeirssen, V. et al. Matrix and steiner-triple-system smart pooling assays for high-performance transcription regulatory network mapping. Nat. Methods 4, 659664 (2007).
  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, 302312 (2007).
  16. Vermeirssen, V. et al. Matrix and steiner-triple-system smart pooling assays for high-performance transcription regulatory network mapping. Nat. Methods 4, 659664 (2007).
  17. Braun, P. et al. An experimentally derived confidence score for binary protein-protein interactions. Nat. Methods 6, 9197 (2009).
  18. Deplancke, B., Dupuy, D., Vidal, M. & Walhout, A.J. A gateway-compatible yeast one-hybrid system. Genome Res. 14 10B, 20932101 (2004).
  19. Maerkl, S.J. & Quake, S.R. A systems approach to measuring the binding energy landscapes of transcription factors. Science 315, 233237 (2007).
  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, 625634 (2002).
  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, 297307 (1999).
  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, 25772582 (1991).
  23. Serikaku, M.A. & Otousa, J.E. Sine oculis is a homeobox gene required for Drosophila visual-system development. Genetics 138, 11371150 (1994).
  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, 977996 (1994).
  25. Callaerts, P. et al. Drosophila pax-6/eyeless is essential for normal adult brain structure and function. J. Neurobiol. 46, 7388 (2001).
  26. Lai, Z.C. & Li, Y. Tramtrack69 is positively and autonomously required for Drosophila photoreceptor development. Genetics 152, 299305 (1999).
  27. Chen, Y.C., Rajagopala, S.V., Stellberger, T. & Uetz, P. Exhaustive benchmarking of the yeast two-hybrid system. Nat. Methods 7, 667668 (2010).
  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, 6779 (2008).
  29. Hutson, S.F. & Bownes, M. The regulation of yp3 expression in the Drosophila melanogaster fat body. Dev. Genes Evol. 213, 18 (2003).
  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, 9941000 (2003).
  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, 15781588 (2008).
  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, D102D106 (2008).
  33. Matys, V. et al. Transfac and its module transcompel: transcriptional gene regulation in eukaryotes. Nucleic Acids Res. 34, D108D110 (2006).
  34. Dietzl, G. et al. A genome-wide transgenic rnai library for conditional gene inactivation in Drosophila. Nature 448, 151156 (2007).
  35. Ni, J.Q. et al. A Drosophila resource of transgenic rnai lines for neurogenetics. Genetics 182, 10891100 (2009).
  36. Gubelmann, C. et al. Getprime: A gene- or transcript-specific primer database for quantitative real-time PCR. Database doi:10.1093/database/bar040 (2011).

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Author information

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. Laboratory of Developmental Genetics, Department of Human 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

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.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (3M)

    Supplementary Figures 1–18, Supplementary Tables 2, 4–6, Supplementary Data

Excel files

  1. Supplementary Table 1 (1M)

    Predicted transcription factors in the Drosophila genome and their cloning status.

  2. Supplementary Table 3 (25K)

    CRMs used in this study.

Additional data