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DNA methylation on N6-adenine in mammalian embryonic stem cells

Nature volume 532, pages 329–333 (2016)Cite this article

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Abstract

It has been widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes. Here we identify N6-methyladenine as another form of DNA modification in mouse embryonic stem cells. Alkbh1 encodes a demethylase for N6-methyladenine. An increase of N6-methyladenine levels in Alkbh1-deficient cells leads to transcriptional silencing. N6-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (<1.5 million years old) but not old (>6 million years old) L1 elements. The deposition of N6-methyladenine correlates with epigenetic silencing of such LINE-1 transposons, together with their neighbouring enhancers and genes, thereby resisting the gene activation signals during embryonic stem cell differentiation. As young full-length LINE-1 transposons are strongly enriched on the X chromosome, genes located on the X chromosome are also silenced. Thus, N6-methyladenine developed a new role in epigenetic silencing in mammalian evolution distinct from its role in gene activation in other organisms. Our results demonstrate that N6-methyladenine constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes.

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Figure 1: A SMRT-ChIP approach identified N6-mA in mammalian genomes.
Figure 2: Alkbh1 is a demethylase for N6-mA in ES cells.
Figure 3: Alkbh1 deficiency silences genes on the X chromosome and young full-length L1 elements.
Figure 4: N6-mA is enriched at young full-length L1 elements, which are located in the vicinity of the downregulated genes in Alkbh1 knockout ES cells.
Figure 5: N6-mA upregulation induced transcriptional silencing on the X chromosome, which is persistent during differentiation.

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Gene Expression Omnibus

Data deposits

All sequencing data were deposited in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE71866.

Change history

  • 20 April 2016

    A sentence about funding was added to the end of the Acknowledgements.

References

  1. Smith, Z. D. & Meissner, A. DNA methylation: roles in mammalian development. Nature Rev. Genet. 14, 204–220 (2013)

    PubMed  CAS  Google Scholar 

  2. Schübeler, D. Function and information content of DNA methylation. Nature 517, 321–326 (2015)

    ADS  PubMed  Google Scholar 

  3. Heyn, H. & Esteller, M. An adenine code for DNA: a second life for N6-methyladenine. Cell 161, 710–713 (2015)

    PubMed  CAS  Google Scholar 

  4. Zhang, G. et al. N6-methyladenine DNA modification in Drosophila . Cell 161, 893–906 (2015)

    PubMed  CAS  Google Scholar 

  5. Greer, E. L. et al. DNA methylation on N6-adenine in C. elegans . Cell 161, 868–878 (2015)

    PubMed  PubMed Central  CAS  Google Scholar 

  6. Fu, Y. et al. N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas . Cell 161, 879–892 (2015)

    PubMed  PubMed Central  CAS  Google Scholar 

  7. Achwal, C. W., Iyer, C. A. & Chandra, H. S. Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA. FEBS Lett. 158, 353–358 (1983)

    PubMed  CAS  Google Scholar 

  8. Ratel, D. et al. Undetectable levels of N6-methyl adenine in mouse DNA: Cloning and analysis of PRED28, a gene coding for a putative mammalian DNA adenine methyltransferase. FEBS Lett. 580, 3179–3184 (2006)

    PubMed  CAS  Google Scholar 

  9. Bourc’his, D. & Bestor, T. H. Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L. Nature 431, 96–99 (2004)

    ADS  PubMed  Google Scholar 

  10. Goodier, J. L. & Kazazian, H. H. Retrotransposons revisited: the restraint and rehabilitation of parasites. Cell 135, 23–35 (2008)

    PubMed  CAS  Google Scholar 

  11. Goodier, J. L., Ostertag, E. M., Du, K. & Kazazian, H. H. A novel active L1 retrotransposon subfamily in the mouse. Genome Res. 11, 1677–1685 (2001)

    PubMed  PubMed Central  CAS  Google Scholar 

  12. Castro-Diaz, N. et al. Evolutionally dynamic L1 regulation in embryonic stem cells. Genes Dev. 28, 1397–1409 (2014)

    PubMed  PubMed Central  CAS  Google Scholar 

  13. Banaszynski, L. A., Allis, C. D. & Lewis, P. W. Histone variants in metazoan development. Dev. Cell 19, 662–674 (2010)

    PubMed  PubMed Central  CAS  Google Scholar 

  14. Jin, C. & Felsenfeld, G. Nucleosome stability mediated by histone variants H3.3 and H2A.Z. Genes Dev. 21, 1519–1529 (2007)

    PubMed  PubMed Central  CAS  Google Scholar 

  15. Fang, G. et al. Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing. Nature Biotechnol. 30, 1232–1239 (2012)

    CAS  Google Scholar 

  16. Davis, B. M., Chao, M. C. & Waldor, M. K. Entering the era of bacterial epigenomics with single molecule real time DNA sequencing. Curr. Opin. Microbiol. 16, 192–198 (2013)

    PubMed  PubMed Central  CAS  Google Scholar 

  17. Wu, T. et al. Histone variant H2A.X deposition pattern serves as a functional epigenetic mark for distinguishing the developmental potentials of iPSCs. Cell Stem Cell 15, 281–294 (2014)

    PubMed  CAS  Google Scholar 

  18. Lu, K., Collins, L. B., Ru, H., Bermudez, E. & Swenberg, J. A. Distribution of DNA adducts caused by inhaled formaldehyde is consistent with induction of nasal carcinoma but not leukemia. Toxicol. Sci. 116, 441–451 (2010)

    PubMed  PubMed Central  CAS  Google Scholar 

  19. Sedgwick, B. Repairing DNA-methylation damage. Nature Rev. Mol. Cell Biol. 5, 148–157 (2004)

    CAS  Google Scholar 

  20. Flusberg, B. A. et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nature Methods 7, 461–465 (2010)

    PubMed  PubMed Central  CAS  Google Scholar 

  21. Shen, L., Song, C.-X., He, C. & Zhang, Y. Mechanism and function of oxidative reversal of DNA and RNA methylation. Annu. Rev. Biochem. 83, 585–614 (2014)

    PubMed  PubMed Central  CAS  Google Scholar 

  22. Müller, T. A., Yu, K., Hausinger, R. P. & Meek, K. ALKBH1 is dispensable for abasic site cleavage during base excision repair and class switch recombination. PLoS ONE 8, e67403 (2013)

    ADS  PubMed  PubMed Central  Google Scholar 

  23. Nordstrand, L. M. et al. Mice lacking Alkbh1 display sex-ratio distortion and unilateral eye defects. PLoS ONE 5, e13827 (2010)

    ADS  PubMed  PubMed Central  Google Scholar 

  24. Ougland, R. et al. ALKBH1 is a histone H2A dioxygenase involved in neural differentiation. Stem Cells 30, 2672–2682 (2012)

    PubMed  PubMed Central  CAS  Google Scholar 

  25. Abrusán, G., Giordano, J. & Warburton, P. E. Analysis of transposon interruptions suggests selection for L1 elements on the X chromosome. PLoS Genet. 4, e1000172 (2008)

    PubMed  PubMed Central  Google Scholar 

  26. Bailey, J. A., Carrel, L., Chakravarti, A. & Eichler, E. E. Molecular evidence for a relationship between LINE-1 elements and X chromosome inactivation: the Lyon repeat hypothesis. Proc. Natl Acad. Sci. USA 97, 6634–6639 (2000)

    ADS  PubMed  CAS  PubMed Central  Google Scholar 

  27. Chow, J. C. et al. LINE-1 activity in facultative heterochromatin formation during X chromosome inactivation. Cell 141, 956–969 (2010)

    PubMed  CAS  Google Scholar 

  28. Liu, C., Tsai, P., García, A.-M., Logeman, B. & Tanaka, T. S. A possible role of Reproductive Homeobox 6 in primordial germ cell differentiation. Int. J. Dev. Biol. 55, 909–916 (2011)

    PubMed  CAS  Google Scholar 

  29. Delatte, B. et al. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine. Science 351, 282–285 (2016)

    ADS  PubMed  CAS  Google Scholar 

  30. Lyon, M. F. X-chromosome inactivation: a repeat hypothesis. Cytogenet. Cell Genet. 80, 133–137 (1998)

    PubMed  CAS  Google Scholar 

  31. Fadloun, A. et al. Chromatin signatures and retrotransposon profiling in mouse embryos reveal regulation of LINE-1 by RNA. Nature Struct. Mol. Biol. 20, 332–338 (2013)

    CAS  Google Scholar 

  32. Erickson, I. K., Cantrell, M. A., Scott, L. & Wichman, H. A. Retrofitting the genome: L1 extinction follows endogenous retroviral expansion in a group of muroid rodents. J. Virol. 85, 12315–12323 (2011)

    PubMed  PubMed Central  Google Scholar 

  33. Koziol, M. J. et al. Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications. Nature Struct. Mol. Biol. 23, 24–30 (2016)

    CAS  Google Scholar 

  34. Tomomori-Sato, C., Sato, S., Conaway, R. C. & Conaway, J. W. Immunoaffinity purification of protein complexes from mammalian cells. Methods Mol. Biol. 977, 273–287 (2013)

    PubMed  PubMed Central  CAS  Google Scholar 

  35. Flusberg, B. A. et al. Direct detection of DNA methylation during single-molecule, real-time sequencing. Nature Methods 7, 461–465 (2010)

    PubMed  PubMed Central  CAS  Google Scholar 

  36. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

    PubMed  PubMed Central  Google Scholar 

  37. Zang, C. et al. A clustering approach for identification of enriched domains from histone modification ChIP-seq data. Bioinformatics 25, 1952–1958 (2009)

    PubMed  PubMed Central  CAS  Google Scholar 

  38. Song, Q. & Smith, A. D. Identifying dispersed epigenomic domains from ChIP-seq data. Bioinformatics 27, 870–871 (2011)

    PubMed  PubMed Central  CAS  Google Scholar 

  39. Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009)

    PubMed  PubMed Central  CAS  Google Scholar 

  40. Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature Protocols 7, 562–578 (2012)

    PubMed  PubMed Central  CAS  Google Scholar 

  41. Tackett, A. J. et al. I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions. J. Proteome Res. 4, 1752–1756 (2005)

    PubMed  CAS  Google Scholar 

  42. Byrum, S. D., Taverna, S. D. & Tackett, A. J. Purification of a specific native genomic locus for proteomic analysis. Nucleic Acids Res. 41, e195 (2013)

    PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

We thank Z. Li, K.Hwang and A. Leung for critical reading of the manuscript and the members of the Xiao laboratory for critical discussion. Thanks to L. Geng for helping Hiseq2000 sequencing. This work is funded by R01GM114205-01 (A.X.). T.P.W. is partially supported by CT Stem Cell Foundation (11SCA34). The Fang lab is partially supported by R01 GM114472-01 (G.F.). Mass spectrometry was supported by R01GM106024, S10OD018445 and P20GM103429. The UNC Mass Spectrometry Facility Core was supported by the National Institutes of Environmental Health Sciences (NIEHS) Superfund Basic Research Program (P42 ES005948), and NIEHS Center for Environmental Health and Susceptibility (P30 ES010126).

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Authors and Affiliations

  1. Department of Genetics and Yale Stem Cell Center, Yale School of Medicine, New Haven, 06520, Connecticut, USA

    Tao P. Wu, Tao Wang, Kaixuan Lin, Yifei Liu & Andrew Z. Xiao

  2. Pacific Biosciences, 1380 Willow Road, Menlo Park, 94025, California, USA

    Matthew G. Seetin & Lawrence S. Hon

  3. Environmental Sciences & Engineering, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Yongquan Lai & James A. Swenberg

  4. Department of Genetics and Genomic Sciences and Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, 10029, USA

    Shijia Zhu & Gang Fang

  5. Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72205, USA

    Stephanie D. Byrum, Samuel G. Mackintosh & Alan Tackett

  6. Yale Stem Cell Center and Department of Cell Biology, Yale School of Medicine, New Haven, 06520, Connecticut, USA

    Mei Zhong

  7. Department of Molecular Biophysics & Biochemistry, Yale Center for Genome Analysis, Yale School of Medicine, New Haven, 06520, Connecticut, USA

    Guilin Wang

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Contributions

A.X. conceived the hypothesis, designed the study and wrote the paper, and provided support and guidance for this work; T.P.W. designed and performed the majority of the experiments, analysed the genomic data, generated figures and interpreted the results; T.W characterized the Alkbh1 mutant and performed demethylation assays; K.L. helped with bioinformatics analysis. Y.L. provided technical help. L.H., M.S., S.Z. and G.F. assisted on SMRT sequencing and data analysis. Y.L. and J.A.S. performed the mass spectrometry analysis of N6-mA. S.D.B., S.G.M. and A.J.T. performed MS analysis on histone methylation and recombinant ALKBH1 proteins.

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Correspondence to Andrew Z. Xiao.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Low N6-mA levels in adult tissues and the lack of DNA alkylation adducts in ES cells.

a, A majority of N6-mA peaks identified by SMRT-ChIP is located in H2A.X deposition region in ESCs determined by native ChIP. b, Number of SMRT-ChIP N6-mA sites at different coverage and QV cut-off. c, Top: A DNA motif of H2A.X deposition region determine with standard ChIP-seq. Bottom: sequence motifs for N6-mA peaks at H2A.X deposition regions determined with SMRT-ChIP. d, Distribution of N6-mA peaks at H2A.X deposition regions (P value determined by binomial test).

Extended Data Figure 2 LC-MS/MS data of N6-mA.

a, Experimental workflow for determining N6-mA level with LC-MS/MS. [15N5]N6-mA was used as the internal standard. b, N6-mA levels are ultralow in adult tissues. c, No detection of DNA alkylation adducts, such as N1-mA, N3-mA or N3-mC in mouse ES cells or Alkbh1 knockout cells by MS. d, LC-MS/MS analysis of N1-mA or N6-mA digested from synthetic oligonucleotides (top) and ES cell DNA samples (bottom). e, ESI-QTOF-MS/MS spectra of analytical standard of N6-mA nucleosides (top) and N6-mA containing HPLC fraction from ES cells.

Extended Data Figure 3 Alkbh1 is a specific N6-mA demethylase in vivo and in vitro.

a, Top: schematic of the CRISPR–Cas9 approach. Alkbh1 KO alleles don’t contain the XmaI site at exon 3. Bottom left: PCR-DNA digestion approach indicating the homozygosity of the knockout alleles, which are resistant to Xma1 digestion. Bottom right: western blotting did not detect any ALKBH1 proteins in the KO cells. b, Three additional Alkbh1 knockout ES cell clones show similar levels of N6-mA upregulation. Shown are dot blot results. c, Validating the specificity of anti-N6-mA antibodies with synthetic oligonucleotides. d, Validating the specificity of anti-N6-mA antibodies with DNA samples of different N6-mA/dA ratio. 125 ng of genomic DNA (MEFs) which does not contain any endogenous N6-mA was spiked with N6-mA containing oligonucleotides at the indicated concentration. e, Tandem mass spectrometric analysis shows the lack of H2AK118/119 methylation in wild-type or Alkbh1 knockout ES cells. Spectral counts for H2A peptides containing K118/119 revealed that H2AK118/119 is predominately non-methylated at similar levels between wild-type and Alkbh1 knockout ES cells. Spectral counts are reported as an average with standard deviation from biological triplicate analyses. K118/119: no methylation; K118/119me1: K118/119 monomethylation. f, MS analysis showed that the co-purified factors with recombinant ALKBH1 proteins are mainly heat shock proteins. g, ALKBH1 proteins don’t have noticeable activities towards to dual- or hemi-methylated double-stranded oligonucleotide substrates. h, ALKBH1 activities are dependent on Fe2+ and α-KG. Error bars: standard deviation of triplicates. i, Ectopic expression of wild-type, but not mutant, Alkbh1 (D233A) at the catalytic motif, can rescue the aberrant increase of N6-mA level in Alkbh1 knockout ES cells. The wild-type and mutant Alkbh1 were expressed at similar levels. j, Quantification of three independent rescue experiments in i. P value as labelled, determined by t-test; error bars, s.d. for three biological replicates. k, The demethylation activity of N6-mA by recombinant D233A mutant protein is much reduced in comparison with the wild-type counterpart. l, No significant activities were detected with increasing concentrations of recombinant D233A mutant proteins in demethylation reaction. Error bars, s.d. of triplicates.

Extended Data Figure 4 RNA-seq analysis in Alkbh1 knockout ES cells.

a, RT-qPCR validation of the RNA-seq analysis. Unchanged genes (gene names labelled in black) identified by RNA-seq were unaltered in RT-qPCR analysis. Highly repressed (red), or modestly repressed (green) genes identified by RNA-seq also showed expected levels of repression in RT-qPCR analyses. Of note, the genes (blue) identified as upregulated in RNA-seq; however, they don’t show differential expression (no significance) in RT-qPCR analysis, which further confirmed the suppression function of ALKBH1. Error bars, s.d. of triplicates. b, MA plot of RNA-seq analysed by DESeq2, which shows the similar pattern to that of CuffDiff2 (see Fig. 3a and Methods). c, Gene ontology analysis demonstrated that lineage specifying factors involved in embryonic development are greatly downregulated by Alkbh1 deficiency. d, RNA-seq transcripts of the representative subfamilies in three major retrotransposon superfamilies (LINE, SINE and LTR) in Alkbh1 knockout ES cells (Methods).

Extended Data Figure 5 Validation of N6-mA DIP-seq approach.

a, ‘Spike-in’ experiments for determining the threshold and linear response range of N6-mA DIP. Genomic DNAs were spiked with N6-mA containing oligonucleotides at indicated concentration (x axis). After N6-mA DIP, the relative enrichment of N6-mA over input control was determined by a RT-qPCR approach. Blue line: linear regression based on data points between 20–130 p.p.m. The threshold (the red line) is the background signals detected by RT-qPCR in which unmodified (control) oligonucleotides were spiked in. b, The track of different sequencing method showed N6-mA sites overlapped between SMRT-ChIP and DIP-Seq in Alkbh1 knockout ES cells. c, Number of SMRT-ChIP N6-mA sites in Alkbh1 knockout cells at different coverage and QV cut-off. With rising coverage and QV cut-off, overlap between SMRT-ChIP N6-mA sites and DIP-Seq N6-mA sites also increases. d, The biological replicates of Alkbh1 knockout ES cells N6-mA-DIP peaks show 87.4% overlap. e, A large majority of N6-mA peaks are in the intergenic regions at the whole-genome level or on the X chromosome. f, In Alkbh1 knockout ES cells, N6-mA peaks are mainly targeted to LINE-1 transposons on the X chromosome or genome-wide. g, N6-mA peaks are significantly enriched on full-length, but not on truncated L1 elements (P < 1.0 × 10−5, chi-squared test). h, Enrichment of N6-mA in each full length L1 subfamily. Lx, L1_Mus1-4: >6 million years; L1VL1, L1MdF1-4: 1.5–6 million years; L1MdGf, L1MdA, L1mdT: <1.5 million years.

Extended Data Figure 6 N6-mA enrichment on 5′-end of young full-length L1 elements.

a, Aggregation plot shows that signal intensity of N6-mA at young full-length L1 is enriched at the 5′ UTR and ORF1. b, qPCR analysis of N6-mA DIP samples confirmed the enrichment at the 5′ UTR and ORF1 regions of L1 that are retained in the young full-length L1 elements, but not the 3′ UTR or Nanog promoter.

Extended Data Figure 7 The correlation between N6-mA deposition on young full-length L1 elements and epigenetic silencing.

a, Violin diagram of the density distribution of the distance between L1 and downregulated genes in Alkbh1 knockout cells. b, The distances between ES cells expressing genes in Alkbh1 knockout ES cells and young full-length L1 elements were plotted for indicated chromosomes. c, The distances between downregulated genes in Alkbh1 knockout ES cells and young full-length L1 elements were plotted for indicated chromosomes.

Extended Data Figure 8 N6-mA accumulation correlates with epigenetic silencing.

a, Normalized 5mC levels on gene bodies or promoters in wild-type or Alkbh1 knockout ES cells. b, Histone marks (H2A.X or H3K27Me3) or 5 mC levels on young full-length L1 elements, SINE or LTR transposons. c, Representative sequencing tracks of decommissioned enhancers. H3K27Ac and H3K4me1 levels at this locus are greatly downregulated in Alkbh1 knockout ES cells. See Supplementary Table 2 for all decommissioned enhancers in Alkbh1 knockout ES cells. d, Violin diagram shows the density distribution of the distance between L1 and decommissioned enhancers in Alkbh1 knockout cells. e, ChIP-qPCR approach showed that H3K4me3 levels are decreased at the transcription start sites (TSS) of LINE-1 or Dax1, an X chromosome gene, while unchanged at the control gene TSS. *P < 0.01, t-test; error bars, ± s.e.m. of three technical triplicates.

Extended Data Figure 9 N6-mA accumulation results in imbalanced cell fate decisions during ESC differentiation.

Wild-type or Alkbh1 knockout ES cells were subject to embryoid body differentiation (Methods). mRNA samples were collected at day 1 or day 9. Gene expression levels were quantified by RT–qPCR approaches. *P < 0.01, t-test; error bars, ± s.e.m. of technical triplicates. a, At day 9, Nanog expression is reduced significantly in wild-type ES-cell-derived embryoid bodies as expected, while its level in Alkbh1 knockout ES-cell-derived embryoid bodies is still high. b, Lefty-1 and Lefty-2 are repressed at day 1 or day 9 in Alkbh1 knockout ES-cell-derived embryoid bodies. c, Activation of Cdx2, is insufficient in Alkbh1 knockout ES-cell-derived embryoid bodies. d, However, expressions of other endoderm markers, Foxa2, Gata4, Gata6, are significantly higher in Alkbh1 knockout ES-cell-derived embryoid bodies than wild-type ES-cell-derived embroid bodies. e, Ectoderm markers, Fgf5 and Pax6 are transiently (day 1) overexpressed in Alkbh1 knockout ES-cell-derived embryoid bodies. f, Mesoderm marker, T/Brachyury is similarly expressed in wild-type or Alkbh1 knockout ES-cell-derived embryoid bodies during differentiation.

Supplementary information

Supplementary Table 1

This file contains a list of differential expressed genes in Alkbh1 KO ESCs. (XLSX 2703 kb)

Supplementary Table 2

This file contains the oligos, primers and antibodies used in this study. (XLSX 13 kb)

Supplementary Table 3

This file contains a list of decommissioned enhancers in Alkbh1 KO ESCs. (XLSX 19 kb)

Supplementary Table 4.

This file contains a list of N6-mA peaks in Alkbh1 KO ESCs with DIP-Seq. (XLSX 745 kb)

Supplementary Table 5

This file contains a list of N6-mA sites in WT and Alkbh1 KO ESCs with H2A.X SMRT-ChIP. (XLSX 371 kb)

Supplementary Figure 1

This file contains the gel figures for Extended Data figure 2a and 2h. (PDF 407 kb)

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Wu, T., Wang, T., Seetin, M. et al. DNA methylation on N6-adenine in mammalian embryonic stem cells. Nature 532, 329–333 (2016). https://doi.org/10.1038/nature17640

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