The nuclear protein CCCTC-binding factor (CTCF) contributes as an insulator to chromatin organization in animal genomes. Currently, our knowledge of its binding property is confined mainly to mammals. In this study, we identified CTCF homologs in extant jawless fishes and performed ChIP-seq for the CTCF protein in the Arctic lamprey. Our phylogenetic analysis suggests that the lamprey lineage experienced gene duplication that gave rise to its unique paralog, designated CTCF2, which is independent from the previously recognized duplication between CTCF and CTCFL. The ChIP-seq analysis detected comparable numbers of CTCF binding sites between lamprey, chicken, and human, and revealed that the lamprey CTCF protein binds to the two-part motif, consisting of core and upstream motifs previously reported for mammals. These findings suggest that this mode of CTCF binding was established in the last common ancestor of extant vertebrates (more than 500 million years ago). We analyzed CTCF binding inside Hox clusters, which revealed a reinforcement of CTCF binding in the region spanning Hox1-4 genes that is unique to lamprey. Our study provides not only biological insights into the antiquity of CTCF-based epigenomic regulation known in mammals but also a technical basis for comparative epigenomic studies encompassing the whole taxon Vertebrata.
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