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

Area of Science:

• Epigenetics
• Molecular Biology
• Genomics

Background:

• CpG methylation is a key epigenetic mark influencing gene expression.
• The quantitative impact of DNA methylation on transcription factor (TF) binding is largely unknown.
• Existing knowledge of methylation's effect on TF binding is often qualitative.

Purpose of the Study:

• To introduce Methyl-Spec-seq, a method for quantitatively measuring CpG methylation's effect on TF binding affinity.
• To enable parallel assessment of methylation effects at thousands of DNA sequence variants within binding sites.
• To develop tools for integrating methylation data into TF binding motif analysis.

Main Methods:

• Methyl-Spec-seq: A high-throughput method to measure TF binding affinity changes due to CpG methylation.
• Two-color competitive fluorescence anisotropy: Used for calibrating and validating Methyl-Spec-seq accuracy.
• Bioinformatics software: Extends motif representation, visualization, and searching to include methylation effects.

Main Results:

• Methyl-Spec-seq successfully quantifies methylation-sensitive TF binding affinities across numerous variants.
• Method calibration confirms the accuracy of Methyl-Spec-seq in determining relative binding affinities.
• Developed software provides novel capabilities for analyzing methylation-influenced TF binding motifs.

Conclusions:

• Methyl-Spec-seq offers a quantitative approach to study epigenetic effects on TF binding.
• The developed tools enhance the understanding of how DNA methylation impacts gene regulation.
• This work facilitates comprehensive analysis of epigenetic modifications in gene regulatory processes.