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SUMMARY: Insulators characterized in Drosophila and mammals have been shown to play a key role in the restriction of promiscuous enhancer–promoter interactions, as well as reshaping the topological landscape of chromosomes. Yet the role of insulators in plants remains poorly understood, in large part because of a lack of well‐characterized insulators and binding factor(s). In this study, we isolated a 1.2‐kb RS2‐9 insulator from the Oryza sativa (rice) 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 Nicotiana tabacum (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 O. 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%, whereas the 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 CCCTC‐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. Significance Statement: The role of insualtor in the regulation of enhancer‐promoter interaction and topological architecture of genomes has been well characterized in mammals but rarely in plants largely due to a lack of a true insualtor. We report the isolation and characterization of an insualtor from rice genome and its binding protein prominently associated with the boundaries of chromatin loops. Our findings are of significance in understanding the insualtor‐based gene regulation and genome demarcation in plants [ABSTRACT FROM AUTHOR]

Summary: A single-copy sense Chalcone synthase (Chs) transgene driven by a strong promoter and producing a fully translatable transcript was converted to an allelic antisense Chs transgene by Cre-lox-mediated DNA recombination in petunia. The sense Chs allele suppressed flower pigmentation in a simple pattern determined by cells at the junctions between adjacent petals, as is typical of single-copy sense Chs transgenes of this type, whereas the antisense Chs allele produced a different pattern of Chs suppression with white petal edges and reduced pigmentation throughout the petal limbs, as is typical of antisense Chs transgenes. In plants carrying a lox-flanked Chs transgene, the presence of Cre protein can cause both sense-specific and antisense-specific patterns to be superimposed in the same flower, suggesting that sense and antisense suppression by single-copy transgenes are mediated by different mechanisms or occur in different cellular or developmental compartments. The presence of Cre also causes the production of numerous, non-clonal white spots, suggesting that the turnover state is not cell-autonomous. [ABSTRACT FROM AUTHOR]