Measuring quantitative effects of methylation on transcription factor-DNA binding affinity

Sci Adv. 2017 Nov 17;3(11):eaao1799. doi: 10.1126/sciadv.aao1799. eCollection 2017 Nov.


Methylation of CpG (cytosine-phosphate-guanine) dinucleotides is a common epigenetic mark that influences gene expression. The effects of methylation on transcription factor (TF) binding are unknown for most TFs and, even when known, such knowledge is often only qualitative. In reality, methylation sensitivity is a quantitative effect, just as changes to the DNA sequence have quantitative effects on TF binding affinity. We describe Methyl-Spec-seq, an easy-to-use method that measures the effects of CpG methylation (mCPG) on binding affinity for hundreds to thousands of variants in parallel, allowing one to quantitatively assess the effects at every position in a binding site. We demonstrate its use on several important DNA binding proteins. We calibrate the accuracy of Methyl-Spec-seq using a novel two-color competitive fluorescence anisotropy method that can accurately determine the relative affinities of two sequences in solution. We also present software that extends standard methods for representing, visualizing, and searching for matches to binding site motifs to include the effects of methylation. These tools facilitate the study of the consequences for gene regulation of epigenetic marks on DNA.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • CCCTC-Binding Factor / chemistry
  • CCCTC-Binding Factor / metabolism
  • CpG Islands
  • DNA / chemistry
  • DNA / metabolism*
  • DNA Methylation*
  • Fluorescence Polarization
  • Homeodomain Proteins / chemistry
  • Homeodomain Proteins / metabolism
  • Mice
  • Patched-1 Receptor / chemistry
  • Patched-1 Receptor / metabolism
  • Protein Binding
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Repressor Proteins / chemistry
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Transcription Factors / chemistry
  • Transcription Factors / metabolism*


  • CCCTC-Binding Factor
  • Ctcf protein, mouse
  • Homeodomain Proteins
  • Hoxb13 protein, mouse
  • Patched-1 Receptor
  • Ptch1 protein, mouse
  • Recombinant Proteins
  • Repressor Proteins
  • Transcription Factors
  • Zfp-57 protein, mouse
  • DNA