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ChIP-nexus enables improved detection of in vivo transcription factor binding footprints

Nature Biotechnology volume 33, pages 395–401 (2015)Cite this article

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Abstract

Understanding how eukaryotic enhancers are bound and regulated by specific combinations of transcription factors is still a major challenge. To better map transcription factor binding genome-wide at nucleotide resolution in vivo, we have developed a robust ChIP-exo protocol called ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode and single ligation), which utilizes an efficient DNA self-circularization step during library preparation. Application of ChIP-nexus to four proteins—human TBP and Drosophila NFkB, Twist and Max—shows that it outperforms existing ChIP protocols in resolution and specificity, pinpoints relevant binding sites within enhancers containing multiple binding motifs, and allows for the analysis of in vivo binding specificities. Notably, we show that Max frequently interacts with DNA sequences next to its motif, and that this binding pattern correlates with local DNA-sequence features such as DNA shape. ChIP-nexus will be broadly applicable to the study of in vivo transcription factor binding specificity and its relationship to cis-regulatory changes in humans and model organisms.

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Figure 1: Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors.
Figure 2: High reproducibility, resolution and specificity of ChIP-nexus as compared to ChIP-seq.
Figure 3: Analysis of Dorsal, Twist and Max in vivo footprints.
Figure 4: Favored interaction side of Max at E-box motifs correlates with DNA features in the flanking sequences.

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Gene Expression Omnibus

Referenced accessions

Sequence Read Archive

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Acknowledgements

We thank M. Blanchette and A. Stark for discussions and R. Krumlauf and R. Mohan for critical comments on the manuscript. This work was funded by the US National Institutes of Health (New Innovator Award 1DP2 OD004561 to J.Z.) and the Stowers Institute for Medical Research.

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Author notes

  1. Qiye He

    Present address: Present address: Institute of Neurosciences, Chinese Academy of Sciences, Shanghai, P.R. China.,

  2. Qiye He and Jeff Johnston: These authors contributed equally to this work.

Authors and Affiliations

  1. Stowers Institute for Medical Research, Kansas City, Missouri, USA

    Qiye He, Jeff Johnston & Julia Zeitlinger

  2. Department of Pathology, Kansas University Medical Center, Kansas City, Kansas, USA

    Julia Zeitlinger

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  1. Qiye He
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  2. Jeff Johnston
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Contributions

Q.H. and J.Z. conceived and designed the ChIP-nexus protocol. Q.H. performed all experiments. J.J. developed all computational analysis tools. Q.H., J.J. and J.Z. analyzed and interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Julia Zeitlinger.

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The authors have applied for a patent for ChIP-nexus.

Supplementary information

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Supplementary Figure 1 and Supplementary Protocol 1 (PDF 2694 kb)

Supplementary Table 1 (XLSX 57 kb)

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He, Q., Johnston, J. & Zeitlinger, J. ChIP-nexus enables improved detection of in vivo transcription factor binding footprints. Nat Biotechnol 33, 395–401 (2015). https://doi.org/10.1038/nbt.3121

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