I-motif DNA structures form in the nuclei of human cells in a cell-cycle and pH dependent manner.
Major finding: I-motif DNA structures form in the nuclei of human cells in a cell-cycle and pH dependent manner.
Approach: Generation of a selective high-affinity antibody allowed the detection of i-motifs in human cells.
Impact: I-motifs may be potential therapeutic targets in cancer due to telomere and promoter regulatory functions.
In vivo, DNA most commonly adopts the canonical B-form, a right-handed double helix secondary structure, with additional layers of DNA structure providing regulatory control. However, alternative non–B-form conformations have also been described including G-quadruplexes (G4), a regulatory structure formed within guanine (G)-rich regions. A similar structure, the intercalated motif (i-motif), has been identified within C-rich regions in vitro, but its existence has not been confirmed in human cells. I-motif structures form via a stack of intercalating hemiprotonated C-neutral C base pairs, which are stabilized in an acidic pH, and it is not clear if the conditions for i-motif formation occur in cells. Zeraati and colleagues generated and characterized an antibody fragment (iMab) that recognizes i-motif structures with high selectivity and affinity to allow detection of i-motifs in human cells. iMab bound i-motifs with diverse sequences, indicating that it is a structure-specific antibody, and it was able to differentiate i-motifs from G4s. Using iMab, i-motifs were detected in punctate foci in the nuclei of three human cancer cell lines, MCF7, U2OS, and HeLa, indicating that i-motifs can form under cellular conditions. The formation of i-motifs was cell-cycle dependent with increased formation at the G1/S boundary. Further, as had been demonstrated in vitro, the formation of i-motifs was pH-dependent with increased i-motif formation at lower intracellular pH levels. I-motifs were enriched at regulatory regions, colocalizing with TRF2 at telomeres and with E-box transcription factors at gene promoters, suggesting that i-motif structures may have regulatory functions at telomeres and in gene expression. The detection of i-motifs at regulatory regions in the nuclei of human cells suggests a role for i-motifs in genomic regulation and the potential for therapeutic targeting in cancer, though further experiments are needed to validate and characterize the proposed regulatory functions of i-motifs.
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