DNA methylation shapes transcription factor binding beyond canonical CpG contexts

Cytosine methylation is a key epigenetic modification that regulates transcription factor (TF) binding and gene expression. While most current understanding of methylation-sensitive TF binding derives from studies focused exclusively on fully methylated CpG sites, alternative forms-such as non-CpG and hemimethylation-are increasingly recognized as widespread and functionally important, particularly in embryonic stem cells and neurons. However, the direct impact of these alternative methylation contexts on TF-DNA interactions remains poorly defined, largely because current binding assays introduce methylation enzymatically, which precludes strand-specific and position-resolved measurements. Here, we systematically profile the methylation sensitivity of 18 human TFs spanning 11 structural families using chemically synthesized DNA libraries containing position-specific 5-methylcytosines (5mC) in CpG, non-CpG, and hemimethylated contexts, measured via high-throughput protein-binding microarrays. Our results reveal extensive TF sensitivity to methylation state, position, and strand orientation, including strong binding of several TFs to non-CpG and hemimethylated sites. The presence of 5mC can dramatically alter TF-DNA interactions: transforming low-affinity sites into high-affinity ones by enabling new contacts or silencing otherwise favorable motifs through steric hindrance. Genomic analyses further show that the methylation-sensitive sequences identified in vitro are represented within enhancers and regulatory elements, exhibiting distinct methylation patterns across cell types. Together, our findings uncover a previously hidden layer of methylation-dependent TF-DNA recognition, broadening the understanding of epigenetics in transcriptional regulation.