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G-quadruplex structures mark human regulatory chromatin

Abstract

G-quadruplex (G4) structural motifs have been linked to transcription1,2, replication3 and genome instability4,5 and are implicated in cancer and other diseases6,7,8. However, it is crucial to demonstrate the bona fide formation of G4 structures within an endogenous chromatin context9,10. Herein we address this through the development of G4 ChIP–seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We find 10,000 G4 structures in human chromatin, predominantly in regulatory, nucleosome-depleted regions. G4 structures are enriched in the promoters and 5′ UTRs of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as MYC. Strikingly, de novo and enhanced G4 formation are associated with increased transcriptional activity, as shown by HDAC inhibitor–induced chromatin relaxation and observed in immortalized as compared to normal cellular states. Our findings show that regulatory, nucleosome-depleted chromatin and elevated transcription shape the endogenous human G4 DNA landscape.

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Figure 1: G4 sites are prevalent in regulatory chromatin regions.
Figure 2: Chromatin relaxation increases G4 prevalence in regulatory chromatin regions.
Figure 3: G4 prevalence is significantly increased in immortalized cells as compared to normal human epidermal keratinocytes.
Figure 4: G4 DNA formation in chromatin.

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Acknowledgements

The authors would like to thank the staff at the Genomic and Light Microscopy and Biorepository core facilities at Cancer Research UK Cambridge Institute. We are grateful to the European Molecular Biology Organization for funding R.H.-H. with the EMBO Long-Term Fellowship. We acknowledge support from University of Cambridge and Cancer Research UK program. The Balasubramanian and Narita laboratories are supported by core funding from Cancer Research UK (C14303/A17197). S.B. is a Senior Investigator of the Wellcome Trust (grant no. 099232/z/12/z).

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Contributions

R.H.-H. developed the G4 ChIP–seq method with help from S.V.L. R.H.-H. carried out all experiments except for the immunofluorescence microscopy and growth-inhibition experimental work and analysis. K.Z., A.P. and M.D.A. carried out immunofluorescence microscopy experiments. M.D.A. performed growth inhibition experiments. R.H.-H., D.B. and G.M. designed, implemented and performed the bioinformatic analysis. R.H.-H., D.B., S.V.L., D.T. and S.B. designed epigenome experiments. R.H.-H., K.Z., A.P., M.D.A. and M.N. designed immunofluorescence experiments. J.P. performed analysis and quantification for colocalization immunofluorescence microscopy experiments. H.K. provided the antibodies to H3K9me3 (clone CMA304), H3K9me3 (clone CMA318) and RNA Pol II C-terminal domain (clone CMA601). All authors analyzed and interpreted the results. R.H.-H. wrote the manuscript with support and contributions from all authors.

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Correspondence to Shankar Balasubramanian.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9. (PDF 4895 kb)

Supplementary Table 1: G4 ChIP–seq association with cancer-related genes.

Table shows the density of HaCaT G4 ChIP–seq peaks in all cancer-related genes; see also Supplementary Figure 6 and Online Methods ‘Cancer-related genes analysis' (XLS 302 kb)

Supplementary Table 2: G4 ChIP–seq association with SCNAs.

Table shows the density of HaCaT G4 ChIP–seq peaks in all SCNAs; see also Supplementary Figure 7 and Online Methods 'SCNAs analysis' (XLS 39 kb)

Supplementary Table 3: ChIP–qPCR primer pair list.

(XLS 27 kb)

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Hänsel-Hertsch, R., Beraldi, D., Lensing, S. et al. G-quadruplex structures mark human regulatory chromatin. Nat Genet 48, 1267–1272 (2016). https://doi.org/10.1038/ng.3662

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