A single three-dimensional chromatin compartment in amphioxus indicates a stepwise evolution of vertebrate Hox bimodal regulation

José Luis Gómez-Skarmeta, Hector Escrivá, Ignacio Maeso, Damien Devos and colleagues perform 4C-seq profiling of the Hox cluster in amphioxus embryos and find that, unlike in vertebrate embryos, the cluster is organized into a single chromatin interaction domain. They suggest that the vertebrate Hox bipartite regulatory system is an evolutionary novelty. The HoxA and HoxD gene clusters of jawed vertebrates are organized into bipartite three-dimensional chromatin structures that separate long-range regulatory inputs coming from the anterior and posterior Hox-neighboring regions 1 . This architecture is instrumental in allowing vertebrate Hox genes to pattern disparate parts of the body, including limbs 2 . Almost nothing is known about how these three-dimensional topologies originated. Here we perform extensive 4C-seq profiling of the Hox cluster in embryos of amphioxus, an invertebrate chordate. We find that, in contrast to the architecture in vertebrates, the amphioxus Hox cluster is organized into a single chromatin interaction domain that includes long-range contacts mostly from the anterior side, bringing distant cis -regulatory elements into contact with Hox genes. We infer that the vertebrate Hox bipartite regulatory system is an evolutionary novelty generated by combining ancient long-range regulatory contacts from DNA in the anterior Hox neighborhood with new regulatory inputs from the posterior side.