Thank you! 😊

Thanks Simone for all your work and helpful feedback 🫶😊

We also used CRISPR in Euprymna berryi to delete a conserved ~1 kb intronic sequence in a coleoid-specific loop containing neural genes. Many knockout embryos had smaller neural tissues, and genes linked to the loop several Mb away were downregulated, consistent with very long-range regulation.

Our data also support the idea of 'regulatory entanglement': as rearrangements bring regions together, they build up interdependencies and become embedded in nested regulatory architectures, making them increasingly constrained.

Our results point to a ‘hierarchy of regulatory constraint’ across the 3D genome. Some chromatin interactions are stable while others change more readily, so rearrangements selectively retain, rewire or eliminate them depending on their underlying level of constraint.

Surprisingly, octopus has the smallest genome but the largest loops. Many octopus loops connect genes that sit on different chromosomes in squid, so lineage-specific patterns of chromosomal fusion seem to drive loop size more than genome expansion.

Chromatin loops showed different characteristics. Few loops were shared by all three species, and loops varied between tissues & developmental stages. Their anchors are enriched for conserved non-coding elements, TF motifs & open chromatin.

Chromatin domains are often conserved. Pairs of genes that interact in all three species stay close together, are highly and broadly expressed across tissues, and carry out core cellular functions, maintained through hundreds of millions of years of rearrangements.

In the coleoid ancestor, chromosomes broke apart, fused and mixed, bringing previously distant genes and regulatory sequences together. Then the lineages diverged: squid & cuttlefish genomes expanded to >5 Gb, while octopus genomes went through many additional fusions.