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Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications

Nature Structural & Molecular Biology volume 23, pages 24–30 (2016)Cite this article

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A Correction to this article was published on 23 March 2017

Abstract

Methylation of cytosine deoxynucleotides generates 5-methylcytosine (m5dC), a well-established epigenetic mark. However, in higher eukaryotes much less is known about modifications affecting other deoxynucleotides. Here, we report the detection of N6-methyldeoxyadenosine (m6dA) in vertebrate DNA, specifically in Xenopus laevis but also in other species including mouse and human. Our methylome analysis reveals that m6dA is widely distributed across the eukaryotic genome and is present in different cell types but is commonly depleted from gene exons. Thus, direct DNA modifications might be more widespread than previously thought.

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Figure 1: Identification of m6dA in the genomes of higher eukaryotes.
Figure 2: Genome-wide identification of m6dA marks in X. laevis fat, oviduct and testes, and M. musculus kidney.
Figure 3: Genome-wide distribution of m6dA in the vicinity of genes.
Figure 4: Genome-wide distribution of m6dA peaks in X. laevis testes samples, as determined with three different antibodies (as indicated by zero, one and two asterisks) recognizing m6dA in DIP-seq, and comparison to input-seq or IgG-seq controls.
Figure 5: m6dA-motif identification in bacteria and X. laevis.

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Acknowledgements

M.J.K. was supported by a Long-Term Human Frontiers Fellowship (LT000149/2010-l), a Medical Research Council (MRC) grant (G1001690) and an Isaac Newton Trust Fellowship (RG76588). This work was sponsored by a Biotechnology and Biological Sciences Research Council grant (BB/M022994/1 to J.B.G. and M.J.K.). The laboratory of J.B.G. is funded by Wellcome Trust grant 101050/Z/13/Z (J.B.G.) and is supported by Gurdon Institute core grants, namely a Wellcome Trust Core Grant (092096/Z/10/Z) and a Cancer Research UK grant (C6946/A14492). C.R.B. and G.E.A. are funded by a Wellcome Trust Core Grant (092096/Z/10/Z). A.S.H.C. and C.F. are funded by the MRC Cancer Unit core grant. We are grateful to D. Simpson and R. Jones-Green for preparing X. laevis eggs and oocytes, F. Miller for providing us with M. musculus tissue, T. Dyl for X. laevis eggs and D. rerio samples (all researchers from Gurdon Institute, University of Cambridge), and members of J.B.G.'s laboratory for their critical comments. We thank U. Ruether (Entwicklungs- und Molekularbiologie der Tiere, Heinrich Heine Universitaet Duesseldorf) for providing us with M. musculus kidney DNA. We also thank J. Ahringer, S. Jackson, A. Bannister and T. Kouzarides (Gurdon Institute, University of Cambridge) for critical input and advice, and M. Sciacovelli and E. Gaude (MRC Cancer Unit, University of Cambridge) for suggestions.

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Author notes

  1. Charles R Bradshaw, George E Allen and Ana S H Costa: These authors contributed equally to this work.

Authors and Affiliations

  1. Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK

    Magdalena J Koziol, Charles R Bradshaw, George E Allen & John B Gurdon

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

    Magdalena J Koziol & John B Gurdon

  3. Medical Research Council Cancer Unit, University of Cambridge, Hutchison/MRC Research Centre, Cambridge, UK

    Ana S H Costa & Christian Frezza

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Contributions

A.S.H.C. performed all UHPLC-MS/MS analyses. M.J.K. conceived the study, designed and performed all experiments, analyzed the data, supervised all research and wrote the paper. C.R.B. and G.E.A. performed the bioinformatic analyses, developed ideas and helped to generate figures and to write the paper. C.F. advised on and supervised all UHPLC-MS/MS analyses, and both A.S.H.C. and C.F. helped to design UHPLC-MS/MS studies and to write the paper. J.B.G. assisted with writing the paper and supervised all research.

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Correspondence to Magdalena J Koziol.

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Integrated supplementary information

Supplementary Figure 1 m6dA Ab recognition of DNA oligonucleotides and X. laevis sperm DNA.

a, m6dA Ab dot blot. Dot blot with different amounts of synthetic DNA oligos and m6dA Ab staining. b, Quantification of the m6dA dot blot. Error bars, s.e.m., n=3 technical replicates, *P<0.04, two-sided t-test, AU = arbitrary units. c, m6dA Ab DIP with synthetic DNA oligos. Error bars, s.e.m., n=3 technical replicates, **P<2×10−3, two-sided t-test, AU = arbitrary units. d, Enrichment of X. laevis sperm DNA fragments using m6dA Ab DIP. e, Quantification of the m6dA Ab DIP. Error bars, s.e.m., n=3 technical replicates, **P<5×10−5, two-sided test, AU = arbitrary units. f, X. laevis sperm DNA dot blot in the presence of competitor-antibody solution. As competitors, dA, dC or m5dC and m6dA were used. g, Quantification of the blot exposed to competitor-antibody solution. The level of the m6dA signal shown. Competitors such as m6dA (red), dA (light grey), dC (dark grey) and m5dC (black) are indicated. Error bars, s.e.m., n=9 technical replicates, **P<4×10−17, two-sided t-test. h, DNA recovery after m6dA Ab DIP. Different amounts fragmented sperm DNA were subject to m6dA Ab DIP. Error bars, s.e.m., n=3 technical replicates, **P<0.003, two-sided t-test.

Supplementary Figure 2 Detection of methylated deoxyadenosine by UHPLC-MS/MS.

a, Fragmentation pathway of m6dA. b, Representative calibration curve (1 out of 4). c, UHPLC-MS/MS EIC chromatogram of m6dA (left) and fragmentation spectrum (right) of representative (1 out of 4) synthetic standard (0.1μM). * Indicates parent ion, n=4 technical replicates, AU = arbitrary units. d, UHPLC-MS/MS EIC chromatogram (left) and fragmentation spectrum (right) of representative (1 out of 4) water control processed without m6dA Ab DIP. n=4 water from different sources, AU = arbitrary units. e, UHPLC-MS/MS EIC chromatogram (left) and fragmentation spectrum (right) of representative (1 out of 4) water control processed following m6dA Ab DIP. Only a spurious m6dA signal is detected. * Indicates parent ion, n=4 water from different sources, AU = arbitrary units. f-g, UHPLC-MS/MS EIC chromatogram (left) and fragmentation spectrum (right) of representative (1 out of 4) genomic DNA from Dam+ and Dam– bacteria isolated following m6dA Ab DIP. * Indicates parent ion, n=4 bacteria from different cultures, AU = arbitrary units.

Supplementary Figure 3 Distribution of m6dA in the genome in X. laevis and M. musculus.

a, Distribution of m6dA peaks around TSS, from 20kb 5’ to 20kb 3’, identified in X. laevis fat, oviduct, testes and in M. musculus kidney. One biological replicate from one animal is shown in each graph. b-e, Density of m6dA versus unmethylated dA in distinct areas of the genome of X. laevis and M. musculus. Only the regions shown in dark grey are statistically significant. One biological replicate from one animal is shown in each graph, **P<0.005, *P<0.05, binomial test on m6dA peaks.

Supplementary Figure 4 Genome-wide distribution of m6dA in the vicinity of genes, on the basis of comparisons of m6dA Ab* and m6dA Ab** DIP-seq versus input-seq or IgG-seq.

a, Distribution of m6dA peaks around TSS, from 20kb 5’ to 20kb 3’, identified in X. laevis testes. The m6dA peaks were obtained from m6dA Ab* and m6dA Ab** DIP-seq versus input-seq or IgG-seq controls. One biological replicate from one animal is shown in each graph. b, Density of m6dA versus unmethylated dA in distinct areas of the genome of X. laevis testes. The m6dA peaks were obtained from m6dA Ab* and m6dA Ab** DIP-seq versus input-seq or IgG-seq controls. Only the regions shown in dark grey are statistically significant. One biological replicate from one animal is shown in each graph, **P<0.005, *P<0.05, binomial test on m6dA peaks.

Supplementary Figure 5 m6dA motifs identified in X. laevis from shifted m6dA peaks.

MEME motifs on shifted X. laevis m6dA peaks. Overlaps between biological replicates from different animals were used for analysis. Same tissue n=2 biological replicates from different animals, different tissue overlaps n=6 biological replicates for different animals, E-value < 1.2×10−8, statistics by MEME, AU = arbitrary units.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1, 3 and 4 (PDF 1211 kb)

Supplementary Data Set 1

Uncropped images for dot blots shown in Figure 1b,g (PDF 93 kb)

Supplementary Table 2

Dataset of all m6dA peaks identified by comparing m6dA Ab DIP-seq with input-seq samples from X. laevis and M. musculus biological replicates (XLSX 16237 kb)

Supplementary Table 5

Data set of all m6dA peaks identified by comparing m6dA Ab DIP-seq, m6dA Ab* DIP-seq, m6dA Ab** DIP-seq with input-seq and IgG-seq samples from X. laevis testis biological replicates (XLSX 15638 kb)

Supplementary Table 6

Data set of all m6dA peaks identified in Dam+ and Dam– E. coli (XLSX 260 kb)

Supplementary Table 7

Data set of enrichment values found in X. laevis m6dA peaks for all 256 4bp motifs (XLSX 110 kb)

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Koziol, M., Bradshaw, C., Allen, G. et al. Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications. Nat Struct Mol Biol 23, 24–30 (2016). https://doi.org/10.1038/nsmb.3145

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