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Automated protein-DNA interaction screening of Drosophila regulatory elements


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.

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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.


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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, 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.

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Authors and Affiliations



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.

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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)

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Hens, K., Feuz, JD., Isakova, A. et al. Automated protein-DNA interaction screening of Drosophila regulatory elements. Nat Methods 8, 1065–1070 (2011).

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