Quantitative visualization of DNA G-quadruplex structures in human cells - PubMed (original) (raw)

Quantitative visualization of DNA G-quadruplex structures in human cells

Giulia Biffi et al. Nat Chem. 2013 Mar.

Abstract

Four-stranded G-quadruplex nucleic acid structures are of great interest as their high thermodynamic stability under near-physiological conditions suggests that they could form in cells. Here we report the generation and application of an engineered, structure-specific antibody employed to quantitatively visualize DNA G-quadruplex structures in human cells. We show explicitly that G-quadruplex formation in DNA is modulated during cell-cycle progression and that endogenous G-quadruplex DNA structures can be stabilized by a small-molecule ligand. Together these findings provide substantive evidence for the formation of G-quadruplex structures in the genome of mammalian cells and corroborate the application of stabilizing ligands in a cellular context to target G-quadruplexes and intervene with their function.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Structure specificity of the BG4 antibody for G-quadruplex structures

Binding curves as determined by ELISA showing that the BG4 antibody has high affinity for intramolecular and intermolecular DNA G-quadruplex structures with negligible binding to a RNA hairpin, double-stranded and single-stranded DNA. BG4 does not show a preference for any particular structural conformation, binding with similar affinity to parallel propeller (KIT1, KIT2 and MYC), anti-parallel (SPB1 and TBA), mixed parallel/anti-parallel propeller (hTELO) and intermolecular (intermolec hTELO) G-quadruplex structures. Dissociation constants (Kd) are indicated. Error bars represent the standard error of the mean calculated from 3 replicates.

Figure 2

Figure 2. Visualization of DNA G-quadruplex structures in nuclei of human cancer cells

a, Immunofluorescence showing BG4 foci (red) in U2OS osteosarcoma cell nuclei. b, Loss of BG4 foci in U2OS cells after pre-incubation of the antibody with pre-folded G-quadruplex oligonucleotides. c, Loss of BG4 foci in U2OS cells after DNase I treatment. The dotted lines are the boundary of the nuclei. d, Increase in BG4 foci number after transfection with pre-folded G-quadruplex oligonucleotides. Nuclei are counterstained with DAPI (blue). Scale bars correspond to 20 μm. e, The graph shows the quantification of BG4 foci number per nucleus for a-d. 100-200 nuclei were counted per condition and the standard error of the mean calculated from a set of 3 replicates. These observations support the targeting and visualization of DNA G-quadruplex structures in human cells by the BG4 antibody.

Figure 3

Figure 3. Localization of G-quadruplex structures in chromosomes

a, Immunofluorescence for BG4 on metaphase chromosomes of HeLa cells. Discrete BG4 foci (red) were observed both within interstitial regions (i, ii, iii) and at telomeres (iv, v), a well-characterised site of G-quadruplex formation. Note the symmetrical appearance of foci in some sister chromatids (v), which supports G-quadruplex formation within the same genomic locations in newly replicated DNA. Chromosomes are counterstained with DAPI (blue), scale bars correspond to 2.5 μm. b, Absence of large co-localization of telomeric TRF2 protein (green) and G-quadruplexes (red) in U2OS cells. This suggests that endogenous G-quadruplex structures are largely present outside telomeres. Nuclei are counterstained with DAPI (blue). Scale bar corresponds to 20 μm.

Figure 4

Figure 4. Modulation of G-quadruplex structures during cell cycle progression

a, BG4 staining in synchronized MCF-7 mammary adenocarcinoma cell populations at the G0/G1 and G1/S boundaries and during S phase. Nuclei are counterstained with DAPI (blue). Scale bars correspond to 20 μm. b, Quantification of BG4 foci number per nucleus for a. 100 nuclei were counted per stage and the standard error of the mean calculated from a set of 3 replicates. c, Over 2-fold reduction in BG4 foci number after inhibition of DNA synthesis by aphidicolin treatment (5 μm2h). d, Quantification of BG4 foci number with or without aphidicolin treatment. 100 nuclei were counted per phase and the standard error of the mean calculated from a set of 3 replicates. These experiments demonstrate that G-quadruplex structures are modulated during the cell cycle and, in particular, support the replication-dependent formation of endogenous DNA G-quadruplexes.

Figure 5

Figure 5. Stabilization of endogenous G-quadruplex structures by a small molecule ligand

a, Increase in BG4 foci number in U2OS cells after treatment with the G-quadruplex binding ligand pyridostatin (PDS). Nuclei are counterstained with DAPI (blue). Scale bar corresponds to 20 μm. b, Quantification of BG4 foci number per nucleus with or without PDS treatment. 200 nuclei were counted per condition and the standard error of the mean calculated from 3 replicates. After PDS treatment, a marked increase (~ 2.9-fold) in nuclear staining was observed. These results indicate that, at pertinent sites, PDS traps endogenous G-quadruplex structures to increase the number of BG4 targets available.

Comment in

References

    1. Sen D, Gilbert W. Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis. Nature. 1988;334:364–6. - PubMed
    1. Lipps HJ, Rhodes D. G-quadruplex structures: in vivo evidence and function. Trends Cell Biol. 2009;19:414–22. - PubMed
    1. Cahoon LA, Seifert HS. An alternative DNA structure is necessary for pilin antigenic variation in Neisseria gonorrhoeae. Science. 2009;325:764–7. - PMC - PubMed
    1. Cheung I, Schertzer M, Rose A, Lansdorp PM. Disruption of dog-1 in Caenorhabditis elegans triggers deletions upstream of guanine-rich DNA. Nat. Genet. 2002;31:405–9. - PubMed
    1. Rodriguez R, et al. Small-molecule-induced DNA damage identifies alternative DNA structures in human genes. Nat. Chem. Biol. 2012;8:301–10. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources