Global identification and functional characterization of Z-DNA in rice

Z-DNA is a left-handed double helix form of DNA that is believed to be involved in various DNA transactions. However, comprehensive investigations aimed at global profiling of Z-DNA landscapes are still missing in both humans and plants. We here report the development of two techniques: anti-Z-DNA antibody-based immunoprecipitation followed by sequencing (ZIP-seq), and cleavage under targets and tagmentation (CUT&TAG) for characterizing Z-DNA in nipponbare rice (Oryza sativa L., Japonica). We found that Z-DNA-IP⁺ (Z-DNA recognized by the antibody) exhibits distinct genomic features as compared to Z-DNA-IP⁻ (Z-DNA not recognized by the antibody). The concomitant presence of G-quadruplexes (G4s) and i-motifs (iMs) may promote Z-DNA formation. DNA modifications such as DNA-6mA/-4acC generally disfavours Z-DNA formation, while modifications like DNA-5mC (CHH) and 8-oxodG promote it, highlighting the distinct roles of DNA base modifications in modulating Z-DNA formation. Importantly, Z-DNA located at transcription start sites (TSSs) enhances gene expression, whereas Z-DNA in genic regions represses it, underscoring its dual roles in regulating the expression of genes involved in fundamental biological functions and responses to salt stress. Furthermore, Z-DNA may play a role in transcriptional initiation and termination rather than in transcriptional elongation. Finally, the presence of Z-DNA in promoters is correlated with the coevolution of overlapping genes, thereby regulating gene domestication. Consequently, our study represents as a pivotal point and a solid foundation for reliably launching genome-wide investigations of Z-DNA, thereby advancing the understanding of Z-DNA biology in both plants and non-plant systems.