Stem-loop and circle-loop TADs generated by directional pairing of boundary elements have distinct physical and regulatory properties.

The chromosomes in multicellular eukaryotes are organized into a series of topologically independent loops called TADs. In flies, TADs are formed by physical interactions between neighboring boundaries. Fly boundaries exhibit distinct partner preferences, and pairing interactions between boundaries are typically orientation-dependent. Pairing can be head-to-tail or head-to-head. The former generates a stem-loop TAD, while the latter gives a circle-loop TAD. The TAD that encompasses the () gene is formed by the head-to-tail pairing of the and boundaries. To explore the relationship between loop topology and the physical and regulatory landscape, we flanked the boundary region with two attP sites. The attP sites were then used to generate four boundary replacements: , (WT orientation), (opposite of WT orientation), and (same orientation as WT ). The replacement restores the WT physical and regulatory landscape: in MicroC experiments, the TAD is a 'volcano' triangle topped by a plume, and the gene and its regulatory elements are sequestered from interactions with neighbors. The replacement lacks boundary function: the endpoint of the 'new' TAD on the side is ill-defined, and stripe enhancers activate a nearby gene, . While and restore the TAD, the topology is a circle-loop, and this changes the local physical and regulatory landscape. In MicroC experiments, the TAD interacts with its neighbors, and the plume at the top of the triangle peak is converted to a pair of 'clouds' of contacts with the next-door TADs. Consistent with the loss of isolation afforded by the stem-loop topology, the enhancers weakly activate genes in the neighboring TADs. Conversely, function is partially disrupted.