Ectopic enhancer–enhancer interactions as causal forces driving RNA-directed DNA methylation in gene regulatory regions

Authors: YANG, YAZHOU - Northwest A&f University; LIU, JIA - Chongqing University; SINGER, STACY - Agriculture And Agri-Food Canada; YAN, GOUHUA - Beijing Academy Of Agricultural Sciences; Bennett, Dennis; LIU, YUE - Qingdao Agricultural University; WANG, XIPING - Northwest A&f University; HILLY, JEAN-MICHEL - Institute Of Vine & Wine; XU, WEIRONG - Ningxia University; Yang, Yingzhen; Zhong, Gan-Yuan; LIU, ZHONGCHI - University Of Maryland; An, Yong-Qiang - Charles; LIU, HUAWEI - Chinese Academy Of Sciences; Liu, Zongrang

Submitted to: Plant Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/27/2024
Publication Date: 7/17/2024
Citation: Yang, Y., Liu, J., Singer, S., Yan, G., Bennett Jr, D.R., Liu, Y., Wang, X., Hilly, J., Xu, W., Yang, Y., Zhong, G., Liu, Z., An, Y., Liu, H., Liu, Z. 2024. Ectopic enhancer–enhancer interactions as causal forces driving RNA-directed DNA methylation in gene regulatory regions. Plant Biotechnology. https://doi.org/10.1111/pbi.14435.
DOI: https://doi.org/10.1111/pbi.14435

Interpretive Summary: Gene promoter controls gene expression and is an important tool for engineering agronomically important traits. However, epigenetic silencing in promoter often compromises the promoter’s function and the stability and performance of the engineered and improved traits under the field condition. In this work, we examined how silencing state of promoter is influenced/activated by other adjacent genetic elements such as enhancer and promoter and found that adjacent enhancer can significantly enhance the promoter silencing and compromise the promoter’s function. Our finding is of importance for biotech research and is instrumental for future design of next generation of plant transformation vehicle.

Technical Abstract: Cis-regulatory elements (CREs) dictate gene expression and tissue specificity, but in a transgenic state, many of them become epigenetically fragile and prone to RNA-directed DNA methylation (RdDM) in plants. Yet, the precise processes behind this phenomenon remain unknown. Here we report a mechansim underlying a transgene AGAMOUS enhancer (AGe)-specific RdDM (eRdDM) in Arabidopsis. We show that eRdDM is dependent on transcription of AGe-specific RNAs (eRNAs) and ensuing production of 24-nt small interfering RNAs. We further show eRdDM does not requires Pol V, a key component required for all RdDM variants characterized to date, highlighting a major distinction of eRdDM with others. Finally, we show that insertion of a Cauliflower mosaic virus 35S enhancer next to AGe in transgene or distally in AGAMOUS locus, substantially enhances/activates eRdDM and such action is resulted from increase/activation of antisense eRNAs. Our finding reveals a novel epigenetic role of the enhancer-enhancer interaction, which may serve a causal force driving DNA methylation in plant CREs.