Abstract

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. Thirty-four years have passed since it was proposed that cytosine DNA methylation in eukaryotes could act as a stably inherited modification affecting gene regulation and cellular differentiation Single-base-resolution maps of DNA methylation for two human cell lines Single-base DNA methylomes of the flowering plant Arabidopsis thaliana were previously achieved using MethylC-Seq 15 or BS-Seq 16 . In this method, genomic DNA is treated with sodium bisulphite (BS) to convert cytosine, but not methylcytosine, to uracil, and subsequent high-throughput sequencing. We performed MethylC-Seq for two human cell lines, H1 human embryonic stem cells 17 and IMR90 fetal lung fibroblasts 18 , generating 1.16 and 1.18 billion reads, respectively, that aligned uniquely to the human reference sequence (NCBI build 36/HG18). The total sequence yield was 87.5 and 91.0 gigabases (Gb), with an average read depth of 14.23 and 14.83 per strand for H1 and IMR90, respectively ( We detected approximately 62 million and 45 million methylcytosines in H1 and IMR90 cells, respectively (1% false discovery rate (FDR), see Supplementary Information and