Clustered CTCF binding is an evolutionary mechanism to maintain topologically associating domains
CTCF binding contributes to the establishment of higher order genome structure by demarcating the boundaries of large-scale topologically associating domains (TADs). We have carried out an experimental and computational study that exploits the natural genetic variation across five closely related species to assess how CTCF binding patterns stably fixed by evolution in each species contribute to the establishment and evolutionary dynamics of TAD boundaries. We performed CTCF ChIP-seq in multiple mouse species to create genome-wide binding profiles and associated them with TAD boundaries. Our analyses reveal that CTCF binding is maintained at TAD boundaries by an equilibrium of selective constraints and dynamic evolutionary processes. Regardless of their conservation across species, CTCF binding sites at TAD boundaries are subject to stronger sequence and functional constraints compared to other CTCF sites. TAD boundaries frequently harbor rapidly evolving clusters containing both evolutionary old and young CTCF sites as a result of repeated acquisition of new species-specific sites close to conserved ones. The overwhelming majority of clustered CTCF sites colocalize with cohesin and are significantly closer to gene transcription start sites than nonclustered CTCF sites, suggesting that CTCF clusters particularly contribute to cohesin stabilization and transcriptional regulation. Overall, CTCF site clusters are an apparently important feature of CTCF binding evolution that are critical the functional stability of higher order chromatin structure.