homie and nhomie insulators compete with each other for interaction with distant copies, affecting enhancer-promoter interactions and eve function

ABSTRACT Chromatin insulators, a.k.a. boundaries, 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. They also block enhancer-promoter interactions and, somewhat paradoxically, can facilitate other enhancer-promoter interactions, particularly when they stitch together distant regions of the chromosome by pairing with specific insulator partners. Here we explore how long-range interactions facilitated by insulator pairing are affected by the presence of two specifically interacting partners. Our results show that when two partners are present, they compete, reducing each other’s effects, suggesting that interactions tend to be limited to two interacting partners at any given time. In particular, when a distant transgenic copy of an eve insulator ( homie or nhomie ) is present, it can interact with either endogenous insulator. 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. We also 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.