Cited by 197Genomics and Chromatin DynamicsDiffusion and Search DynamicsGenomics and Phylogenetic Studies

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

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Nutiu, Razvan, Friedman, Robin C, Luo, Shujun, Khrebtukova, Irina, Silva, David, Li, Robin, Zhang, Lu, Schroth, Gary P +1 more·2011·Nature Biotechnology

Abstract

Several methods for characterizing DNA-protein interactions are available, 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 response, 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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  • Normalized fluorescence intensity is proportional to poly(A)-tract length after accounting for cluster density.

    “To account for the density of each cluster, this raw intensity is normalized to that of the fluorescent bases added during sequencing by synthesis12, thereby yielding a normalized fluorescence-intensity for the poly(A) tail of each transcript, paired with a sequencing read that identifies its poly(A) site and thus the gene of origin.”

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Authors
Nutiu, R., Friedman, R. C., Luo, S., Khrebtukova, I., Silva, D., Li, R., Zhang, L., Schroth, G. P., & Burge, C. B.
Affiliations
  • Massachusetts Institute of Technology (US)
  • Illumina (United States) (US)
Corresponding author
Burge, Christopher B
Venue
Nature Biotechnology
Published
2011-06-26
Volume
29
Issue
7
Pages
659-664
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
OpenAlex
W2069985409
Cited by
194
References
32
Citations by year
2026: 7 · 2025: 5 · 2024: 8 · 2023: 6 · 2022: 8 · 2021: 7 · 2020: 9 · 2019: 17 · 2018: 14 · 2017: 21 · 2016: 14 · 2015: 15 · 2014: 20 · 2013: 18 · 2012: 22
FWCI
8.30
Type
journal article
Language
en
Open access
Locations
  • Nature Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
Funders
  • National Human Genome Research Institute (R01HG002439)
  • National Institute of General Medical Sciences (R01GM085319)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Diffusion and Search Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
Keywords
protein-DNA binding affinity, GCN4, DNA-binding motifs, dissociation constant, promoter binding sites, gene expression prediction, transcriptional regulation, yeast
MeSH terms
Chromatography, Affinity, DNA, DNA-Binding Proteins, Equipment Design, Spectrometry, Fluorescence, Sequence Analysis, DNA, Equipment Failure Analysis, Protein Interaction Mapping

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