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

Abstract

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 <i>Drosophila even skipped</i> (<i>eve</i>) gene is formed by the head-to-tail pairing of the <i>nhomie</i> and <i>homie</i> boundaries. To explore the relationship between loop topology and the physical and regulatory landscape, we flanked the <i>nhomie</i> boundary region with two attP sites. The attP sites were then used to generate four boundary replacements: <i>λ DNA</i>, <i>nhomie forward</i> (WT orientation), <i>nhomie reverse</i> (opposite of WT orientation), and <i>homie forward</i> (same orientation as WT <i>homie</i>). The <i>nhomie forward</i> replacement restores the WT physical and regulatory landscape: in MicroC experiments, the <i>eve</i> TAD is a 'volcano' triangle topped by a plume, and the <i>eve</i> gene and its regulatory elements are sequestered from interactions with neighbors. The <i>λ DNA</i> replacement lacks boundary function: the endpoint of the 'new' <i>eve</i> TAD on the <i>nhomie</i> side is ill-defined, and <i>eve</i> stripe enhancers activate a nearby gene, <i>eIF3j</i>. While <i>nhomie reverse</i> and <i>homie forward</i> restore the <i>eve</i> TAD, the topology is a circle-loop, and this changes the local physical and regulatory landscape. In MicroC experiments, the <i>eve</i> TAD interacts with its neighbors, and the plume at the top of the <i>eve</i> 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 <i>eve</i> enhancers weakly activate genes in the neighboring TADs. Conversely, <i>eve</i> function is partially disrupted.