I-motif DNA structures are formed in the nuclei of human cells

Abstract

Human genome function is underpinned by the primary storage of genetic information in canonical B-form DNA, with a second layer of DNA structure providing regulatory control. I-motif structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions; however, in vivo evidence for the existence of such structures has so far remained elusive. Here we report the generation and characterization of an antibody fragment (iMab) that recognizes i-motif structures with high selectivity and affinity, enabling the detection of i-motifs in the nuclei of human cells. We demonstrate that the in vivo formation of such structures is cell-cycle and pH dependent. Furthermore, we provide evidence that i-motif structures are formed in regulatory regions of the human genome, including promoters and telomeric regions. Our results support the notion that i-motif structures provide key regulatory roles in the genome.

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Fig. 1: Characterization of an hTelo i-motif specific antibody fragment (iMab).
Fig. 2: Evaluation of the specificity of iMab for different i-motif and G4 structures.
Fig. 3: Visualization of i-motif structures in the nuclei of human cell lines.
Fig. 4: Visualization and quantification of i-motif structures during cell-cycle progression in HeLa cells.
Fig. 5: Investigation of the effect of pH variation on the formation of i-motif structures in MCF7 cells.
Fig. 6: Evidence of i-motifs formation in regulatory regions.

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Acknowledgements

The authors thank S. Thompson (Leica Microsystems Australia) and P. Young (University of Sydney) for access to the Leica 3XSTED instrument. This work was supported by Program Grants 1113904, Project Grant 1148051, Development Grants 1113790 and 1076356 and Fellowship 105146 from the National Health and Medical Research Council (NHMRC) and Discovery Grants 160104915 and 140103465 from the Australian Research Council (ARC).

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M.Z., M.E.D. and D.C. conceived the project. M.Z. designed and performed the experiments. D.B.L. and R.R. contributed phage display experiments. P.S. contributed in vitro characterization of the antibody. A.L.M. and T.M.B. contributed biophysical studies. W.E.H. contributed microscopy. M.Z., M.E.D. and D.C. analysed the data and wrote the paper. All authors reviewed the manuscript.

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Correspondence to Marcel E. Dinger or Daniel Christ.

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Material and Method section, supplementary tables outlining oligonucleotide sequences and phage display specifics, eight supplementary figures, and a supplementary method describing protocols for the selection of i-motif specific antibody fragments using phage display are described. In addition, we outline methods for counting nuclear foci using the FIJI software package

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Zeraati, M., Langley, D.B., Schofield, P. et al. I-motif DNA structures are formed in the nuclei of human cells. Nature Chem 10, 631–637 (2018). https://doi.org/10.1038/s41557-018-0046-3

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