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

Nat Protoc. 2021 Dec;16(12):5673-5706. doi: 10.1038/s41596-021-00630-1. Epub 2021 Nov 12.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Animals
  • Cell Nucleus / metabolism
  • DNA / genetics
  • DNA / metabolism*
  • DNA Footprinting / methods*
  • DNA Modification Methylases / genetics
  • DNA Modification Methylases / metabolism*
  • Gene Expression Regulation
  • Gene Library
  • Genome*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Mice
  • Mouse Embryonic Stem Cells / cytology
  • Mouse Embryonic Stem Cells / metabolism
  • Nucleosomes / chemistry
  • Nucleosomes / metabolism
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism
  • Sequence Analysis, DNA / statistics & numerical data
  • Single Molecule Imaging / methods*
  • Software
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*


  • Nucleosomes
  • Transcription Factors
  • DNA
  • DNA Modification Methylases
  • RNA Polymerase II