The homie insulator has sub-elements with different insulating and long-range pairing properties.

Chromatin insulators are major determinants of chromosome architecture. Specific architectures induced by insulators profoundly influence nuclear processes, including how enhancers and promoters interact over long distances and between homologous chromosomes. Insulators can pair with copies of themselves in trans to facilitate homolog pairing. They can also pair with other insulators, sometimes with great specificity, inducing long-range chromosomal loops. Contrary to their canonical function of enhancer blocking, these loops can bring distant enhancers and promoters together to activate gene expression, while at the same time blocking other interactions in cis. The details of these effects depend on the choice of pairing partner, and on the orientation specificity of pairing, implicating the 3D architecture as a major functional determinant. Here, we dissect the homie insulator from the Drosophila even skipped (eve) locus, to understand its substructure. We test pairing function based on homie-carrying transgenes interacting with endogenous eve. The assay is sensitive to both pairing strength and orientation. Using this assay, we found that a Su(Hw) binding site in homie is required for efficient long-range interaction, although some activity remains without it. This binding site also contributes to the canonical insulator activities of enhancer blocking and barrier function. Based on this and other results from our functional dissection, each of the canonical insulator activities, chromosomal loop formation, enhancer blocking, and barrier activity, are partially separable. Our results show the complexity inherent in insulator functions, which can be provided by an array of different proteins with both shared and distinct properties.