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Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase footprinting

Nature Protocols volume 16, pages 5673–5706 (2021)Cite this article

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Abstract

Precise control of gene expression requires the coordinated action of multiple factors at cis-regulatory elements. We recently developed single-molecule footprinting to simultaneously resolve the occupancy of multiple proteins including transcription factors, RNA polymerase II and nucleosomes on single DNA molecules genome-wide. The technique combines the use of cytosine methyltransferases to footprint the genome with bisulfite sequencing to resolve transcription factor binding patterns at cis-regulatory elements. DNA footprinting is performed by incubating permeabilized nuclei with recombinant methyltransferases. Upon DNA extraction, whole-genome or targeted bisulfite libraries are prepared and loaded on Illumina sequencers. The protocol can be completed in 4–5 d in any laboratory with access to high-throughput sequencing. Analysis can be performed in 2 d using a dedicated R package and requires access to a high-performance computing system. Our method can be used to analyze how transcription factors cooperate and antagonize to regulate transcription.

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Fig. 1: Overview of the experimental workflow.
Fig. 2: Number of TFBSs that can be studied by SMF.
Fig. 3: Methylation efficiency of M.SssI and M.CvIPI is not affected by the sequence context when saturating conditions are used.
Fig. 4: QCs during the preparation of bait-captured SMF samples.
Fig. 5: Overview of the computational workflow.
Fig. 6: Controlling footprinting efficiency with low-coverage sequencing data.
Fig. 7: SMF data visualization.
Fig. 8: Single-molecule sorting.
Fig. 9: QCs during the preparation of amplicon SMF samples.

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Data availability

The data used to produce Fig. 6 and Fig. 7 were produced within the scope of ref. 8 and are available at ArrayExpress: E-MTAB-9123 and E-MTAB-9033. The data used to produce Fig. 3 are available at ArrayExpress: E-MTAB-10815.

Code availability

The SingleMoleculeFootprinting25 R package has been released and is available through Bioconductor. The code used to produce the figures for this paper is available at https://github.com/KrebsLab/Kleinendorst_et_al31.

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Acknowledgements

The authors are grateful to the members of the Krebs laboratory for helpful discussions, comments on the manuscript and feedback during the development of the R package. The authors would like to acknowledge C. Sönmezer for sharing data and E. Kreibich for sharing amplicon QC gels. The authors are thankful to L. Villacorta, V. Benes and the members of the Genomics Core facility for sequencing the libraries and technical assistance. The salary of G.B. is supported by the Deutsche Forschungsgemeinschaft (KR 5247/1-1). The authors thank W. Huber for supporting the development of the R package. The authors would like to thank C. Girardot and the Genome Biology Computational Support. Research in the laboratory of A.R.K is supported by core funding of the European Molecular Biology Laboratory, Deutsche Forschungsgemeinschaft (KR 5247/1-1 and KR 5247/2-1). M.L.S. is funded by The German Network for Bioinformatics Infrastructure (de.NBI) Förderkennzeichen Nr. 031A537B.

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Author notes

  1. These authors contributed equally: Rozemarijn W. D. Kleinendorst, Guido Barzaghi.

Authors and Affiliations

  1. European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany

    Rozemarijn W. D. Kleinendorst, Guido Barzaghi, Mike L. Smith & Arnaud R. Krebs

  2. Faculty of Biosciences, Collaboration for Joint PhD Degree between EMBL and Heidelberg University, Heidelberg, Germany

    Guido Barzaghi

  3. European Molecular Biology Laboratory (EMBL), Structural and Computational Biology Unit, Heidelberg, Germany

    Judith B. Zaugg

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  1. Rozemarijn W. D. Kleinendorst
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Contributions

A.R.K designed the study. R.W.D.K., G.B. and A.R.K wrote the manuscript. R.W.D.K performed the experiments. G.B. developed the package for data analysis with support from M.L.S. A.R.K supervised the conduction of the experiments and the data analysis. All authors discussed the results and commented on the manuscript.

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Correspondence to Arnaud R. Krebs.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Simon Bourdareau, Julia Zeitlinger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Sönmezer, C. et al. Mol. Cell 81, 255–267 (2021): https://doi.org/10.1016/j.molcel.2020.11.015

Krebs, A. et al. Mol. Cell 67, 411–422 (2017): https://doi.org/10.1016/j.molcel.2017.06.027

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Kleinendorst, R.W.D., Barzaghi, G., Smith, M.L. et al. Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase footprinting. Nat Protoc 16, 5673–5706 (2021). https://doi.org/10.1038/s41596-021-00630-1

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