CW198 acts as a genetic insulator to block enhancer-promoter interaction in plants

Authors: JIANG, LI - Nanjing Agricultural University; LIU, YUE - Qingdao Agricultural University; WEN, ZHIFENG - Fujian Agricultural & Forestry University; YANG, YINGJUN - Henan University Of Science And Technology; SINGER, STACY - Agriculture And Agri-Food Canada; Bennett, Dennis; XU, WENYING - China Agricultural University; SU, ZHEN - China Agricultural University; YU, ZHIFANG - Nanjing Agricultural University; COHN, JOSH - Syngenta Crop Protection; CHAE, HYUNSOOK - Syngenta Crop Protection; QUE, QIUDENG - Syngenta Crop Protection; Liu, Zongrang

Submitted to: Transgenic Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/15/2022
Publication Date: 9/2/2022
Citation: Jiang, L., Liu, Y., Wen, Z., Yang, Y., Singer, S., Bennett Jr, D.R., Xu, W., Su, Z., Yu, Z., Cohn, J., Chae, H., Que, Q., Liu, Z. 2022. CW198 acts as a genetic insulator to block enhancer-promoter interaction in plants. Transgenic Research. https://doi.org/10.1007/s11248-022-00326-6.
DOI: https://doi.org/10.1007/s11248-022-00326-6

Interpretive Summary: Promoter-promoter crosstalk in transgene often leads to mis-regulation that compromises the stability and persistent performance of the biotech engineered traits, thereby imminently requiring an insulator to remedy the problem. However, such insulator is not available yet. In this work, we set out to search for such insulator. Through development of a sophisticated assay system, we successfully isolated one from plant and demonstrated it can efficiently block the enhancer-promoter interference in transgene. We also analyzed the binding of protein factors to CW198 and other genetic factors. Our work provide a new insulator that can be immediately used for remedying the problem associated with promiscuous interactions and interference among transgene elements in biotech engineered agronomical traits to ensure their genetic stability and persistent performance under the field conditions.

Technical Abstract: Insulators in vertebrates play a role in genome architecture and orchestrate temporo-spatial enhancer-promoter interactions. In plants, insulators and their associated binding factors have not been documented as of yet, largely as a result of the lack of characterized insulators. In this study, we took advantage of a comprehensive strategy to identify and validate the enhancer-blocking insulator CW198. We show that a 1.08-kb CW198 fragment from Arabidopsis can, when interposed between enhancer and promoter, efficiently abrogate the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and tobacco plants. In plants, both transcriptional crosstalk and spreading of histone modifications were rarely detectable across CW198, which resembles the insulation pattern observed across the CTCF insulator in mammalian genome. Further analyses reveal that LFY, a master regulator of floral development, strongly binds CW198, as well as an additional 31,650 sites in the Arabidopsis genome, with a preference for intergenic regions, consistent with the general binding pattern of the well-known vertebrate insulator-binding factor CTCF. Interestingly, LFY binds to the regions between adjacent genes arranged only in tandem and head-to-head, but rarely in a tail-to-tail gene pairs. Furthermore, the LFY binding is abundantly enriched in the genome region covering AGL8 promoter that lacks interactive activity in its native chromosome position but gains it when the LFY binding cluster is truncated in transgene plants. Taken together, our findings support an insulation function for CW198 bound by LFY in plants. The significance of the present findings and their relevance to the topological architecture of genomes in plants, as well as gene regulation, will be discussed.