De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis

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Abstract

Gene expression is regulated in part by protein transcription factors that bind target regulatory DNA sequences. Predicting DNA binding sites and affinities from transcription factor sequence or structure is difficult; therefore, experimental data are required to link transcription factors to target sequences. We present a microfluidics-based approach for de novo discovery and quantitative biophysical characterization of DNA target sequences. We validated our technique by measuring sequence preferences for 28 Saccharomyces cerevisiae transcription factors with a variety of DNA-binding domains, including several that have proven difficult to study by other techniques. For each transcription factor, we measured relative binding affinities to oligonucleotides covering all possible 8-bp DNA sequences to create a comprehensive map of sequence preferences; for four transcription factors, we also determined absolute affinities. We expect that these data and future use of this technique will provide information essential for understanding transcription factor specificity, improving identification of regulatory sites and reconstructing regulatory interactions.

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Figure 1: Overall experimental design and procedure.
Figure 2: Detailed analysis of measured Cy5 intensities and fluorescence intensity ratios (Cy5/BODIPY-FL) for rabbit reticulocyte lysate alone, Reb1p, Cin5p and Cup9p.
Figure 3: Comparison between Kd values derived from direct measurements of concentration-dependent binding and Kd values calculated from ratio measurements at a single concentration.
Figure 4: Comparison between motifs found for all 28 S. cerevisiae transcription factors and previous literature results (SWISS, SwissRegulon30; ChIP-chip, Harbison library22; PBM1, protein binding microarray14; PBM2, protein binding microarray15).

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Acknowledgements

P.M.F. was supported by a Howard Hughes Medical Institute/Helen Hay Whitney Foundation Postdoctoral Fellowship. J.L.D., S.R.Q. and this work were supported by the Howard Hughes Medical Institute. We thank A. Potanina for assistance with fabrication of microfluidic devices, O. Homann for implementation of PSAM functionality with MochiView and D. Breslow, F. Caro, S. Churchman, M. Dimon, T. Kiers, A. Kistler, C. Nelson, K. Sorber, E. Yeh and I. Zuleta for careful reading of the manuscript.

Author information

P.M.F. designed experiments, designed, created and printed the DNA library, made linear expression templates, fabricated microfluidic devices, performed microfluidic experiments assessing concentration-dependent binding and binding to the 8-mer library, analyzed data and wrote the manuscript. D.G. designed experiments, designed and fabricated microfluidic devices and performed microfluidic experiments assessing binding to the 8-mer library. D.T. fabricated microfluidic devices and performed microfluidic experiments assessing binding to the 8-mer library. J.Z. and H.L. analyzed data. S.R.Q. designed experiments, analyzed data and wrote the manuscript. J.L.D. designed experiments, assisted with printing the DNA library, analyzed data and wrote the manuscript.

Correspondence to Joseph L DeRisi or Stephen R Quake.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Tables 1–8, Supplementary Figs. 1–19 and Supplementary Methods (PDF 4961 kb)

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Fordyce, P., Gerber, D., Tran, D. et al. De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis. Nat Biotechnol 28, 970–975 (2010) doi:10.1038/nbt.1675

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