Genome-wide mapping of i-motifs reveals their association with transcription regulation in live human cells

Lay Summary Among the secondary structures alternative to the DNA double helix, i-Motifs (iMs) and G-quadruplexes (G4s) are four-stranded non-canonical nucleic acid structures that form in cytosine- and guanine-rich regions, respectively. Because iMs fold in vitro under acidic conditions, they were long thought to form only in vitro. We now show that iMs, like G4s, form in live human cells mainly at gene promoters in open chromatin. iMs that are unstable in vitro still form in cells. iMs and G4s are cell-type specific and associated with increased transcription; however, transcript levels are remarkably different: low for iMs and high for G4s, indicating their distinct activity as regulators of the cell transcriptome. The iM/G4 interplay may represent a novel therapeutic target in disease.i-Motifs (iMs) are four-stranded DNA structures that form at cytosine (C)-rich sequences in acidic conditions in vitro. Their formation in cells is still under debate. We performed CUT & Tag sequencing using the anti-iM antibody iMab and showed that iMs form within the human genome in live cells. We mapped iMs in two human cell lines and recovered C-rich sequences that were confirmed to fold into iMs in vitro. We found that iMs in cells are mainly present at actively transcribing gene promoters, in open chromatin regions, they overlap with R-loops, and their abundance and distribution are specific to each cell type. iMs with both long and short C-tracts were recovered, further extending the relevance of iMs. By simultaneously mapping G-quadruplexes (G4s), which form at guanine-rich regions, and comparing the results with iMs, we proved that the two structures can form in independent regions; however, when both iMs and G4s are present in the same genomic tract, their formation is enhanced. iMs and G4s were mainly found at genes with low and high transcription rates, respectively. Our findings support the in vivo formation of iM structures and provide new insights into their interplay with G4s as new regulatory elements in the human genome.