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Measuring quantitative effects of methylation on transcription factor-DNA binding affinity.
Science Advances 2017 November
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.
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