Genome-scale DNA methylation maps of pluripotent and differentiated cells - PubMed (original) (raw)

. 2008 Aug 7;454(7205):766-70.

doi: 10.1038/nature07107. Epub 2008 Jul 6.

Tarjei S Mikkelsen, Hongcang Gu, Marius Wernig, Jacob Hanna, Andrey Sivachenko, Xiaolan Zhang, Bradley E Bernstein, Chad Nusbaum, David B Jaffe, Andreas Gnirke, Rudolf Jaenisch, Eric S Lander

Affiliations

Genome-scale DNA methylation maps of pluripotent and differentiated cells

Alexander Meissner et al. Nature. 2008.

Abstract

DNA methylation is essential for normal development and has been implicated in many pathologies including cancer. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine.

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Figures

Figure 1

Figure 1. CpG methylation levels in ES cells and NPCs for CpGs with ≥10-fold coverage

The top histograms show the distribution of methylation levels (%) across all CpGs, HCPs, LCPs, HCNEs, differentially methylated regions (DMRs), LTRs, SINEs and other genomic features (n, number of CpGs). Methylation levels are bimodal (except at DMRs, which have a unimodal distribution largely consistent with uniform sampling from the maternal and paternal alleles in ES cells and partial hypermethylation in NPCs). The bottom box plots show the distribution of methylation levels conditional on local CpG density (defined as fraction of CpGs in a 300-bp window; shown as percentage). The red lines denote medians, notches the standard errors, boxes the interquartile ranges, and whiskers the 2.5th and 97.5th percentiles.

Figure 2

Figure 2. Correlation between DNA and histone methylation

a, Mean methylation levels across CpGs within each profiled HCP (requiring ≥5-fold coverage of ≥5 CpGs), conditional on their histone methylation state in ES cells and NPC_s_ (n, number of HCPs; those enriched with H3K4me3 are generally also enriched for H3K4me2, but not vice versa). Loss of H3K4 methylation, and to a lesser extent of H3K27me3, is correlated with gain of DNA methylation. b, Methylation levels of individual CpGs outside of HCPs, conditional on enrichment of H3K4me2 (n, number of distinct sites in each category). Changes in histone methylation state are inversely correlated with changes in DNA methylation. c, Methylation levels of CpGs in HCNEs not overlapping CpG islands, conditional on H3K4me2 enrichment. For a–c, the red lines denote medians, notches the standard errors, boxes the interquartile ranges, and whiskers the 2.5th and 97.5th percentiles. All pair-wise comparisons of methylation levels at sites with changing chromatin states are significant (P < 10−20, Mann–Whitney U test).

Figure 3

Figure 3. Developmentally regulated de-methylation of highly conserved non-coding elements

Comparison of histone and DNA methylation levels across the Olig1/Olig2 neural-lineage transcription factor locus. ChIP-Seq tracks for H3K4me1/2/3 and H3K27me3 in ES cells and NPCs are shown. The unmethylated CpG-rich promoters are bivalent and inactive in ES cells and resolve to univalent H3K4me3 on activation in NPCs. H3K4me2 enrichment appears over HCNEs distal to the two genes, and this correlates with CpG de-methylation. Inferred methylation levels for 40 out of 215 sampled CpGs are shown and colour-coded. Red indicates largely methylated (>80%); green indicates largely unmethylated (<20%), and orange indicates intermediate levels (≥20% and ≤80%).

Figure 4

Figure 4. HCP hypermethylation of cultured cells

Inferred mean methylation levels (%) across autosomal HCPs (requiring ≥5-fold coverage of ≥5 CpGs within the CpG island). a, ES-derived astrocytes contains roughly 10 times more hypermethylated HCPs than primary NPC-derived astrocytes after two passages (P) in culture. b, Continued passage of the primary cells lead to gradual hypermethylation of many of the same HCPs. c, Only a handful of mainly germline-specific HCPs display hypermethylation in a whole brain tissue sample. d, Most HCPs are unmethylated in ES cells, but a small subset gain significant methylation on differentiation to NPCs. e, Continued proliferation of NPCs leads to additional HCPs becoming hypermethylated after 18 passages. f, Differentiation of late-stage NPCs into astrocytes by growth factor withdrawal does not lead to additional HCP hypermethylation. g, Expression levels of genes associated with profiled HCPs for ES cells (ES), ES-derived astrocytes (A), primary neocortical astrocytes (AN) and cerebellar astrocytes (AC). Hypermethylation of HCPs is correlated with low expression levels in ES-derived astrocytes. HCPs that are univalent in ES cells and become hypermethylated in ES-derived astrocytes are associated with lower expression levels in both ES cells and primary astrocytes. h, The maximal CpG densities (300-bp window) of hypermethylated HCPs in ES cells or ES-derived astrocytes are significantly lower than for unmethylated HCPs. For g and h, the red lines denote medians, notches the standard errors, boxes the interquartile ranges, and whiskers the 2.5th and 97.5th percentiles.

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