Automated protein-DNA interaction screening of Drosophila regulatory elements

Journal name:
Nature Methods
Year published:
Published online


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


  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.


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Author information


  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


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.

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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.

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