Chromatin insulators homie and nhomie can interact with distant copies either together or separately, with distinct outcomes for enhancer-promoter interactions.

Chromatin insulators, a.k.a. boundary elements, separate regions of the chromosome with distinct chromatin characteristics, including distinct histone modifications. This activity affects gene expression by allowing chromatin domains to be stably regulated and maintained. Insulators also block enhancer-promoter interactions and, somewhat paradoxically, facilitate other interactions, particularly when they stitch together distant regions of the chromosome by pairing with specific partners. Here we explore how long-range interactions facilitated by insulator pairing are affected by the presence of two potentially competing partners. Our results show that when two partners are present, they can reduce each other's effects on distant gene expression, suggesting that enhancer-promoter interactions are best facilitated by pairwise insulator interactions. When a distant copy of an eve insulator (homie or nhomie) is present, it can interact with either or both endogenous insulators. But when one endogenous insulator is removed, the remaining one interacts more strongly with the transgenic copy, biasing the induced enhancer-promoter interactions toward those nearest the remaining endogenous insulator. On the other hand, physical interaction data suggest that strictly pairwise interactions are not the rule, suggesting a more complex model involving tripartite interactions. We further show that removing one or both endogenous eve insulators significantly reduces endogenous eve function at a critical early stage of development, and that the eve Polycomb domain expands in both directions when its insulator boundaries are removed, showing that insulators in their native context are required for each of the main functions that have been ascribed to them based on transgene assays.