Thehomieinsulator has sub-elements with different insulating and long-range pairing properties

Abstract

Chromatin insulators are a major determinant of chromosome architecture. Specific architectures induced by insulators profoundly influence nuclear processes, including how enhancers interact with promoters over long distances and between homologous chromosomes. Insulators can pair with copies of themselves intrans, thereby facilitating homolog pairing. They can also pair with other insulators, sometimes with great specificity, inducing long-range chromosomal loops incis. 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 incis. The details of these effects depend on the choice of pairing partner, and on the orientation specificity of pairing, implicating the 3-dimensional architecture as a major determinant of function. Here we dissect thehomieinsulator from the Drosophilaeven skipped(eve) locus, to understand its complex substructure. We test pairing function incisbased onhomie-carrying transgenes interacting with endogenouseve. The assay is sensitive to both pairing strength and orientation specificity. Using this assay, we found that a consensus Su(Hw) binding site inhomieis 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, enhancer blocking and barrier activities appear to be partially separable. Overall, our results show the complexity inherent in insulator functions, which can be provided by an array of proteins with both shared and distinct properties.