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Direct measurement of DNA affinity landscapes on a high-throughput sequencing instrument

Abstract

Several methods for characterizing DNA-protein interactions are available1,2,3,4,5,6, but none have demonstrated both high throughput and quantitative measurement of affinity. Here we describe 'high-throughput sequencing'-'fluorescent ligand interaction profiling' (HiTS-FLIP), a technique for measuring quantitative protein-DNA binding affinity at unprecedented depth. In this approach, the optics built into a high-throughput sequencer are used to visualize in vitro binding of a protein to sequenced DNA in a flow cell. Application of HiTS-FLIP to the protein Gcn4 (Gcn4p), the master regulator of the yeast amino acid starvation response7, yielded 440 million binding measurements, enabling determination of dissociation constants for all 12-mer sequences having submicromolar affinity. These data revealed a complex interdependency between motif positions, allowed improved discrimination of in vivo Gcn4p binding sites and regulatory targets relative to previous methods and showed that sets of genes with different promoter affinities to Gcn4p have distinct functions and expression kinetics. Broad application of this approach should increase understanding of the interactions that drive transcription.

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Figure 1: HiTS-FLIP method.
Figure 2: Positional interdependencies in the DNA binding affinity of Gcn4p.
Figure 3: HiTS-FLIP accurately predicts in vivo binding and regulation and reveals impact of lower-affinity binding sites.

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Acknowledgements

The authors thank G. Braus for providing the GCN4 containing plasmids, N. Clarke for sharing amino acid starvation microarray time course data, A. Keating and A. Reinke for advice on EMSA experiments, T. Theara, S. Levine and the Biomicro Center at Massachusetts Institute of Technology for help with Illumina sequencing technology and initial experiments, and L. Boyer, N. Clarke, W. Gilbert, P. Sharp and members of the Burge laboratory for helpful discussion and comments on the manuscript. This work was supported by a Human Frontiers Science Program long-term fellowship (R.N.), a Computational Science Graduate Fellowship from the Office of Science in the Department of Energy under contract DE-FG02-97ER25308 (R.C.F.), by a major equipment grant from the National Science Foundation (no. 0821391) and by grants from the US National Institutes of Health (C.B.B.).

Author information

Authors and Affiliations

Authors

Contributions

R.N., initial idea, experimental design and initial implementation, Gcn4p purification and EMSA experiments; R.C.F., experimental design and initial implementation, computational analysis of image data, all bioinformatic analyses; S.L. and G.P.S., experimental design and method optimization; I.K., D.S., R.L., L.Z., method optimization; C.B.B., initial idea, coordination of project, contributions to bioinformatics analyses.

Corresponding author

Correspondence to Christopher B Burge.

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Competing interests

S.L., I.K., D.S., R.L., L.Z. and G.P.S. are employees of Illumina.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 6, 8, Supplementary Methods, Discussion and Supplementary Figures 1–11 (PDF 1463 kb)

Supplementary Table 1

HiTS-FLIP dissociation constants for 8mers. (TXT 9 kb)

Supplementary Table 2

HiTS-FLIP dissociation constants for 9mers. (TXT 48 kb)

Supplementary Table 3

HiTS-FLIP dissociation constants for 10mers. (TXT 203 kb)

Supplementary Table 4

HiTS-FLIP dissociation constants for 11mers. (TXT 881 kb)

Supplementary Table 5

HiTS-FLIP dissociation constants for 12mers. (TXT 3887 kb)

Supplementary Table 7

Predicted promoter occupancies using 8mer Kd values or PBM data. (TXT 235 kb)

Supplementary Data (ZIP 17 kb)

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Nutiu, R., Friedman, R., Luo, S. et al. Direct measurement of DNA affinity landscapes on a high-throughput sequencing instrument. Nat Biotechnol 29, 659–664 (2011). https://doi.org/10.1038/nbt.1882

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