Investigation of Enhancer-Blocking DNA Insulators in Arabidopsis thaliana

Abstract

Currently research has focused on insulators from non-plant species such as the fruit fly, Drosophila melanogaster. The accumulated data suggests that many different insulator sequences exist in D. melanogaster, each one containing its own different primary binding protein, while sharing similar secondary binding proteins. Together, they produce chromatin loops separating enhancers and promoters into distinct domains preventing cross-talk between them. Is this the case in plants? To approach this question, we have investigated enhancer-blocking insulators in the model plant Arabidopsis thaliana using two unrelated approaches. Firstly, we have developed an assay for the direct selection of insulators in Arabidopsis thaliana using a random oligonucleotide library. This assay helped us to define four novel insulator sequences named InI-3, InII-12, InIII-50, and InIII-78. Secondly, we have used genetic analyses to characterize potential insulator sequences originally from three non-plant species: UASrpg from the fungus Ashbya gossypii, BEAD1c from human T-cell receptors, and gypsy from D. melanogaster, that have been reported to function in A. thaliana. Our findings suggest that non-plant insulators and their protein binding sites function in plants and support the model of multiple, functional, different insulator sequences as was found in D. melanogaster. They also argue for the conservation of insulator mechanisms across species.