Abstract
Key message
Genetic analysis identifies multiple, potential protein binding sites important for insulator function in Arabidopsis thaliana: Rap1 site in UASrpg, Su(Hw) site in UASrpg, and CTCF site in BEAD1c.
Abstract
Three non-plant insulators UASrpg, BEAD1c, and gypsy isolated from Ashbya gossypii, Homo sapiens and Drosophila melanogaster gypsy retrotransposon, respectively, demonstrate insulator function in transgenic Arabidopsis thaliana. Here, the hypothesis that DNA sequences functional in A. thaliana are the same as those in the original host as previously assumed, was tested. Genetic analyses of the cloned fragments in an enhancer blocking assay system was performed through deletions and mutations to identify more precisely which sequences within the cloned fragments function as insulators. Significant loss of insulator activity was observed when the UASrpg Rap1 binding site R2 was mutated but not R1. Cloned fragments containing BEAD1c are effective insulators in our assay system and the previously investigated gypsy insulator is non-functional. Further analyses identified potential Su(Hw) and CTCF sites within UASrpg, of which only the Su(Hw) site was functional. Thus, the activity of non-plant insulators in A. thaliana is context dependent. These results support the hypothesis that insulator function is conserved across kingdoms.
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Acknowledgements
The authors would like to thank Dr. David Currie for statistical insights and Dr. Allyson MacLean for many helpful suggestions used to improve the manuscript. Several graduate and undergraduate students helped with data collection including Lara Rasooli, Batool Gandorah, Hassan Badreddine, Onkar Bhanushaki, Adina Popescu, Linda Dam, Shukria Ahmadi, Liam McCarthy, Ka Mien Lam, and Krishna Gelda. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (RGPIN/002869-2011) and the University of Ottawa (555256) to DAJ.
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Douglas A Johnson has received research grants from Natural Sciences and Engineering Research Council of Canada (RGPIN/ 002869-2011) and the University of Ottawa (555256).
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AT and DAJ conceived and designed the research, conducted experiments, analyzed data, and wrote the manuscript. All authors read and approved the manuscript.
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Tran, A., Johnson, D.A. Mutational analysis identifies functional Rap1, Su(Hw), and CTCF insulator sites in Arabidopsis thaliana. Plant Cell Rep 39, 1743–1753 (2020). https://doi.org/10.1007/s00299-020-02601-4
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DOI: https://doi.org/10.1007/s00299-020-02601-4