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DNA G-quadruplex structures mold the DNA methylome

Nature Structural & Molecular Biology volume 25, pages 951–957 (2018)Cite this article

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Abstract

Control of DNA methylation level is critical for gene regulation, and the factors that govern hypomethylation at CpG islands (CGIs) are still being uncovered. Here, we provide evidence that G-quadruplex (G4) DNA secondary structures are genomic features that influence methylation at CGIs. We show that the presence of G4 structure is tightly associated with CGI hypomethylation in the human genome. Surprisingly, we find that these G4 sites are enriched for DNA methyltransferase 1 (DNMT1) occupancy, which is consistent with our biophysical observations that DNMT1 exhibits higher binding affinity for G4s as compared to duplex, hemi-methylated, or single-stranded DNA. The biochemical assays also show that the G4 structure itself, rather than sequence, inhibits DNMT1 enzymatic activity. Based on these data, we propose that G4 formation sequesters DNMT1 thereby protecting certain CGIs from methylation and inhibiting local methylation.

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Fig. 1: G4 formation is associated with hypomethylation at CGIs.
Fig. 2: DNMT1 is recruited to BG4 peaks associated with low methylation.
Fig. 3: DNMT1 selectively binds and is inhibited by G4 structures.
Fig. 4: Recruitment of DNMT1 by the G4 structures shapes the methylome in G-rich regions.

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Data availability

K562 datasets for DHS (ENCSR000EPC), DNMT1 ChIP-seq (ENCSR987PBI), and whole-genome bisulfite sequencing (ENCSR765JPC) were downloaded from ENCODE. G4-ChIP-seq datasets for K562 and WGBS datasets for entinostat-treated and untreated HaCaT cells are available at the GEO repository under accession GSE107690. G4-ChIP-seq data in entinostat-treated and untreated HaCaT cells were taken from GSE76688. Source data for Figs. 1d,e,h and 3 are available online.

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Acknowledgements

This work is supported by a core CRUK award (C14303/A17197). S.B. is a Senior Investigator of the Wellcome Trust (grant no. 099232/z/12/z). J.S. is a Marie Curie Fellow of the European Union (747297-QAPs-H2020-MSCA-IF-2016).

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  1. Avazeh T. Ghanbarian

    Present address: European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, UK

  2. Dario Beraldi

    Present address: Institute of Cancer Sciences, University of Glasgow, Glasgow, UK

Authors and Affiliations

  1. Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Cambridge, UK

    Shi-Qing Mao, Avazeh T. Ghanbarian, Jochen Spiegel, Sergio Martínez Cuesta, Dario Beraldi, Giovanni Marsico, Robert Hänsel-Hertsch, David Tannahill & Shankar Balasubramanian

  2. Department of Chemistry, University of Cambridge, Cambridge, UK

    Sergio Martínez Cuesta, Marco Di Antonio & Shankar Balasubramanian

  3. School of Clinical Medicine, University of Cambridge, Cambridge, UK

    Shankar Balasubramanian

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Contributions

The project was conceived by S.M. and S.B. S.M. designed and carried out all the experiments with discussions with D.B., J.S., R.H.H., D.T., and S.B. S.M. designed the analysis strategies with input from A.G., D.T., and S.B. J.S. performed the G4-ChIP-seq experiments. A.G. and S.M.C. carried out all the computational analysis with discussions with S.M., D.T., D.B., R.H.H., and G.M. M.D. carried out the circular dichroism spectroscopy and ultraviolet melting experiments. All authors interpreted the results. S.M., D.T., and S.B. wrote the paper with input from all of the authors.

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Correspondence to Shankar Balasubramanian.

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Supplementary Figure 1 BG4 binds unmethylated and methylated G4 structures with the same affinity.

ELISA assays testing binding of BG4 antibody to unmethylated and methylated G4 structures and control non-G4-forming oligonucleotides. CpG sites are highlighted in red. MG denotes methylated CpG. Shown are the mean and s.d. from three measurements.

Supplementary Figure 2 Methylation is depleted in BG4 regions.

a, Box-and-whisker plot showing the average methylation for BG4 peaks (n = 8,210), DHSs (n = 142,115) and CGIs (n = 22,673). Similar to Fig. 1e, apart from using the CGI set generated by CpGCluster. b, Box-and-whisker plot showing the methylation levels for BG4 peaks and CGIs at different CpG densities. Similar to Fig. 1h, apart from using the CGI set generated by CpGCluster. c, Box-and-whisker plot showing the average methylation levels for CGIs with or without a BG4 peak at different CpG densities. d, Box-and-whisker plot showing average methylation on CGIs with respect to BG4 peaks in the presence (+) or absence (–) of a DHS or promoter. The number of CGI regions in each category is presented on top of the plot. e, Box-and-whisker plot showing the average methylation for BG4 peaks (n = 17,101), ATAC (ATAC-seq peaks denoting open chromatin, n = 23,217) and CGI regions (n = 26,580) in untreated HaCaT cells. f, Box and scatterplot showing differential percentage methylation in entinostat-treated versus untreated cells for promoter CGIs in open chromatin regions containing sequences with potential to form G4s. i, BG4-negative CGIs without a G4 ChIP-seq peak but having potential to form a G4 structure (n = 1,504); ii, BG4-constant CGIs with a least one high-confidence G4 ChIP-seq peak that does not significantly change before and after treatment (n = 3,261); iii, BG4 increases where a BG4 peak significantly increases in size after treatment (n = 307). Each gray dot represents one CGI region. P values were calculated using a Mann–Whitney U-test.

Supplementary Figure 3 DNMT1 is enriched at BG4 peaks associated with low methylation.

Binding profile of DNMT1 in CGIs with low (less than 20%, n = 14,983), intermediate (between 20% and 80%, n = 4,864) and high (more than 80%, n = 2,826) methylation. Above each plot is a heat map showing the enrichment of BG4 peaks and DHSs across the respective regions. Similar to Fig. 2b, apart from using the CGI set generated by CpGCluster.

Supplementary Figure 4 Structure verification of oligonucleotides used in this study and inhibition of DNMT1 by G4 DNA.

a–c, Circular dichroism spectra of BCL2 and BCL2-mut (a), KIT2 and BKIT2-mut (b), and MYC and MYC-mut (c). Sequences are listed below the graph. d–f, ultraviolet melting profiles of BCL2 (d), KIT2 (e), and MYC (f). Mutated oligonucleotides lose the capacity to form G4s and therefore have no absorbance at 295 nm. g–i, Circular dichroism spectra of BCL2-0CG/2CG/3CG and BCL2-CCC (g), KIT2-0CG/2CG/CGCG and KIT2-CCC (h), and MYC-2CG/CTCA/4CG and MYC-CCC (i). Sequences are listed below the graph. Note that circular dichroism spectra of all G4-forming oligonucleotides show a positive peak at ~263 nm and a negative peak at ~240 nm, which is characteristic of a G4 structures. j, DNMT1 activity in the presence of BCL2 (G4 structure), BCL2-CCC (C-rich, non-G4 forming with 5 CpGs), BCL2-mut (wild-type BCL2 with mutations in G4 tetrad Gs, non-G4 forming), BCL2-2CG (G4 forming with 2 CpGs), BCL2-3CG (G4 forming with 3CpGs) and BCL2-0CG (G4 forming without CpGs). k, KIT2 (G4 structure), KIT2-CCC (C-rich, non-G4 forming with 4 CpGs), KIT2-mut (wild-type KIT2 with mutations in G4 tetrad Gs, non-G4 forming), KIT2-0CG (G4 forming without CpGs), KIT2-2CG (G4 forming with 2 CpGs), and KIT-CGCG (G4 forming with 2 adjacent CpGs). l, MYC (G4 structure), MYC-CCC (C-rich, non-G4 forming with no CpGs), MYC-mut (wild-type MYC with mutations in G4 tetrad Gs, non-G4 forming), MYC-2CG (G4 forming with 2 CpGs), MYC-CTCA (G4 forming without CpGs), and MYC-4CG (G4 forming with 4 CpGs). Sequences of the oligonucleotides used are given below the graphs. Shown are mean±s.d., n = 3 independent experiments. Statistical tests were done using two-way ANOVA.

Supplementary Figure 5 Depletion of DNA methylation is independent of R-loop formation.

Plot showing the average methylation profile centered around BG4 peak regions overlapping with R-loop regions (BG4 + R-loop, red and blue are replicates 1 and 2, respectively, n = 5,464) or BG4 peak regions without overlapping R-loop (BG4 – R-loop, pink and green are replicates 1 and 2, respectively, n = 3,111). The plot extends ±5 kb from the center of BG4 peaks.

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Mao, SQ., Ghanbarian, A.T., Spiegel, J. et al. DNA G-quadruplex structures mold the DNA methylome. Nat Struct Mol Biol 25, 951–957 (2018). https://doi.org/10.1038/s41594-018-0131-8

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