The roles of DNA methylation on pH dependent i-motif (iM) formation in rice.

I-motifs (iMs) are four-stranded non-B DNA structures containing C-rich DNA sequences. The formation of iMs is sensitive to pH conditions and DNA methylation, although the extent of which is still unknown in both humans and plants. To investigate this, we here conducted iMab antibody-based immunoprecipitation and sequencing (iM-IP-seq) along with bisulfite sequencing using CK (original genomic DNA without methylation-related treatments) and hypermethylated or demethylated DNA at both pH 5.5 and 7.0 in rice, establishing a link between pH, DNA methylation and iM formation on a genome-wide scale. We found that iMs folded at pH 7.0 displayed higher methylation levels than those formed at pH 5.5. DNA demethylation and hypermethylation differently influenced iM formation at pH 7.0 and 5.5. Importantly, CG hypo-DMRs (differentially methylated regions) and CHH (H = A, C and T) hyper-DMRs alone or coordinated with CG/CHG hyper-DMRs may play determinant roles in the regulation of pH dependent iM formation. Thus, our study shows that the nature of DNA sequences alone or combined with their methylation status plays critical roles in determining pH-dependent formation of iMs. It therefore deepens the understanding of the pH and methylation dependent modulation of iM formation, which has important biological implications and practical applications.

The Epigenomic Features and Potential Functions of PEG- and PDS-Favorable DNA G-Quadruplexes in Rice.

A G-quadruplex (G4) is a typical non-B DNA structure and involved in various DNA-templated events in eukaryotic genomes. PEG and PDS chemicals have been widely applied for promoting the folding of in vivo or in vitro G4s. However, how PEG and PDS preferentially affect a subset of G4 formation genome-wide is still largely unknown. We here conducted a BG4-based IP-seq in vitro under K++PEG or K++PDS conditions in the rice genome. We found that PEG-favored IP-G4s+ have distinct sequence features, distinct genomic distributions and distinct associations with TEGs, non-TEGs and subtypes of TEs compared to PDS-favored ones. Strikingly, PEG-specific IP-G4s+ are associated with euchromatin with less enrichment levels of DNA methylation but with more enriched active histone marks, while PDS-specific IP-G4s+ are associated with heterochromatin with higher enrichment levels of DNA methylation and repressive marks. Moreover, we found that genes with PEG-specific IP-G4s+ are more expressed than those with PDS-specific IP-G4s+, suggesting that PEG/PDS-specific IP-G4s+ alone or coordinating with epigenetic marks are involved in the regulation of the differential expression of related genes, therefore functioning in distinct biological processes. Thus, our study provides new insights into differential impacts of PEG and PDS on G4 formation, thereby advancing our understanding of G4 biology.