Rice RS2-9, which is bound by transcription factor OSH1, blocks enhancer-promoter interactions in plants.

Insulators characterized in Drosophila and mammals have been shown to play a key role in the restriction of promiscuous enhancer-promoter interactions and also in reshaping the topological landscape of chromosomes. Yet the role of insulators in plants remains poorly understood, in large part due to a lack of well-characterized insulators and binding factors(s). In this study, we isolated a 1.2-kb RS2-9 insulator from the rice (Oryza sativa) genome that can, when interposed between an enhancer and promoter, efficiently block the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and tobacco. In the rice genome, the genes flanking RS2-9 exhibit an absence of mutual transcriptional interactions, as well as a lack of histone modification spread. We further determined that Oryza sativa Homeobox 1 (OSH1) bound two regions of RS2-9, as well as over 50,000 additional sites in the rice genome, the majority of which resided in intergenic regions. Mutation of one of the two OSH1-binding sites in RS2-9 impaired insulation activity by up to 60%, while mutation of both binding sites virtually abolished insulator function. We also demonstrated that OSH1 binding sites were associated with 72% of the boundaries of topologically associated domains (TADs) identified in the rice genome, which is comparable to the 77% of TAD boundaries bound by the insulator-binding factor CTCF in mammals. Taken together, our findings indicate that OSH1-RS2-9 acts as a true insulator in plants, and highlight a potential role for OSH1 in gene insulation and topological organization in plant genomes.