DNA methylation patterns of transcription factor binding regions characterize their functional and evolutionary contexts

Background DNA methylation is an important epigenetic modification which has numerous roles in modulating genome function. Its levels are spatially correlated across the genome, typically high in repressed regions but low in transcription factor (TF) binding sites and active regulatory regions. However, the mechanisms establishing genome-wide and TF binding site methylation patterns are still unclear. Results We used a comparative approach to investigate the association of DNA methylation to TF binding evolution in mammals. Specifically, we experimentally profiled DNA methylation and combined this with published occupancy profiles of five distinct TFs (CTCF, CEBPA, HNF4A, ONECUT1, FOXA1) in the liver of five mammalian species (human, macaque, mouse, rat, dog). TF binding sites were lowly methylated, but they often also had intermediate methylation levels. Employing a classification and clustering approach, we extracted distinct and species conserved patterns of DNA methylation levels at TF bound regions. CEBPA, HNF4A, ONECUT1 and FOXA1 shared the same methylation patterns, while CTCF’s differed. These patterns characterize alternative functions and chromatin landscapes of TF bound regions. Leveraging our phylogenetic framework, we found DNA methylation gain upon evolutionary loss of TF occupancy, indicating coordinated evolution. Furthermore, each methylation pattern has its own evolutionary trajectory reflecting its genomic contexts. Conclusions Our epigenomic analyses found that specific DNA methylation profiles characterize TF binding, and are associated to their regulatory activity, chromatin contexts, and evolutionary trajectories. ### Competing Interest Statement P.F. is a member of the Scientific Advisory Boards of Fabric Genomics, Inc. and Eagle Genomics, Ltd. All other authors declare no competing interests.