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.
Laboratory of Systems Biology and Genetics, Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Korneel
K
Hens
Jean-Daniel
JD
Feuz
Alina
A
Isakova
Antonina
A
Iagovitina
Andreas
A
Massouras
Julien
J
Bryois
Patrick
P
Callaerts
Susan E
SE
Celniker
Bart
B
Deplancke
D000818:Animals; D001331:Automation; D001665:Binding Sites; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D044085:Microfluidics; D016366:Open Reading Frames; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D015203:Reproducibility of Results; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D014157:Transcription Factors; D004247:DNA