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.