DNA cytosine methylation is a central epigenetic modification that has essential roles in cellular processes including genome regulation, development and disease. Here we present the first genome-wide, single-base-resolution maps of methylated cytosines in a mammalian genome, from both human embryonic stem cells and fetal fibroblasts, along with comparative analysis of messenger RNA and small RNA components of the transcriptome, several histone modifications, and sites of DNA–protein interaction for several key regulatory factors. Widespread differences were identified in the composition and patterning of cytosine methylation between the two genomes. Nearly one-quarter of all methylation identified in embryonic stem cells was in a non-CG context, suggesting that embryonic stem cells may use different methylation mechanisms to affect gene regulation. Methylation in non-CG contexts showed enrichment in gene bodies and depletion in protein binding sites and enhancers. Non-CG methylation disappeared upon induced differentiation of the embryonic stem cells, and was restored in induced pluripotent stem cells. We identified hundreds of differentially methylated regions proximal to genes involved in pluripotency and differentiation, and widespread reduced methylation levels in fibroblasts associated with lower transcriptional activity. These reference epigenomes provide a foundation for future studies exploring this key epigenetic modification in human disease and development.

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Gene Expression Omnibus

Data deposits

Sequence data is available under the GEO accessions GSM429321-23, GSM432685-92, GSM438361-64, GSE17917, GSE18292 and GSE16256, and the SRA accessions SRX006782-89, SRX006239-41, SRX007165.1-68.1 and SRP000941. Analysed data sets can be obtained from


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We thank A. Elwell and A. Hernandez for assistance with sequence library preparation and Illumina sequencing. R.L. is supported by a Human Frontier Science Program Long-term Fellowship. R.D.H. is supported by an American Cancer Society Postdoctoral Fellowship. This work was supported by grants from the following: Mary K. Chapman Foundation, The National Institutes of Health (U01 ES017166 and U01 1U01ES017166-01), the California Institute for Regenerative Medicine (RS1-00292-1), the Australian Research Council Centre of Excellence Program (CE0561495, DP0771156) and Morgridge Institute for Research, Madison, Wisconsin. We thank the NIH Roadmap Reference Epigenome Consortium ( and C. Gunter (Hudson-Alpha Institute) for assistance. This study was carried out as part of the NIH Roadmap Epigenomics Program.

Author Contributions Experiments were designed by J.R.E., B.R., R.L., J.A.T. and R.D.H. Cells were grown by J.A.-B. and Q.-M.N. MethylC-Seq, RNA-Seq and smRNA-Seq experiments were conducted by R.L. and J.R.N. ChIP-Seq experiments were conducted by R.D.H., L.L. and Z.Y. ChIP-Seq data analysis was performed by G.H., R.D.H. and L.E. BS-PCR validation was performed by R.H.D. Sequencing data processing was performed by R.L., J.T.-F., L.E., V.R. and G.H. Bioinformatic and statistical analyses were conducted by M.P., R.L., G.H., J.T.-F., R.H.D., R.S. and A.H.M. AnnoJ development was performed by J.T.F and A.H.M. The manuscript was prepared by R.L., M.P., R.H.D., A.H.M. and J.R.E.

Author information

Author notes

    • Ryan Lister
    •  & Mattia Pelizzola

    These authors contributed equally to this work.


  1. Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA

    • Ryan Lister
    • , Mattia Pelizzola
    • , Robert H. Dowen
    • , Joseph R. Nery
    •  & Joseph R. Ecker
  2. Ludwig Institute for Cancer Research,

    • R. David Hawkins
    • , Gary Hon
    • , Leonard Lee
    • , Zhen Ye
    • , Que-Minh Ngo
    • , Lee Edsall
    •  & Bing Ren
  3. Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA

    • Bing Ren
  4. ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Western Australia 6009, Australia

    • Julian Tonti-Filippini
    •  & A. Harvey Millar
  5. Morgridge Institute for Research, Madison, Wisconsin 53707, USA

    • Jessica Antosiewicz-Bourget
    • , Ron Stewart
    • , Victor Ruotti
    •  & James A. Thomson
  6. Genome Center of Wisconsin, Madison, Wisconsin 53706, USA

    • Jessica Antosiewicz-Bourget
    • , Ron Stewart
    • , Victor Ruotti
    •  & James A. Thomson
  7. Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, Wisconsin 53715, USA

    • James A. Thomson
  8. Department of Anatomy, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA

    • James A. Thomson


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Corresponding author

Correspondence to Joseph R. Ecker.

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