Quantitative Analysis of the DNA Methylation Sensitivity of Transcription Factor Complexes

Abstract

Although DNA modifications play an important role in gene regulation, the underlying mechanisms remain elusive. We developed EpiSELEX-seq to probe the sensitivity of transcription factor binding to DNA modification in vitro using massively parallel sequencing. Feature-based modeling quantifies the effect of cytosine methylation (⁵mC) on binding free energy in a position-specific manner. Application to the human bZIP proteins ATF4 and C/EBPβ and three different Pbx-Hox complexes shows that ⁵mCpG can both increase and decrease affinity, depending on where the modification occurs within the protein-DNA interface. The TF paralogs tested vary in their methylation sensitivity, for which we provide a structural rationale. We show that ⁵mCpG can also enhance in vitro p53 binding and provide evidence for increased in vivo p53 occupancy at methylated binding sites, correlating with primed enhancer histone marks. Our results establish a powerful strategy for dissecting the epigenomic modulation of protein-DNA interactions and their role in gene regulation.

Keywords: 5-methyl-cytosine; ChIP-seq data; SELEX-seq; bZIP; basic leucine zipper proteins; bisulfite sequencing; epigenetic DNA modification; epigenomics; high-throughput in vitro protein-DNA interaction profiling; human Hox protein complexes; integrative analysis; methylome; transcription factors; tumor suppressor protein p53.