Aberrant DNA Methylation in ES Cells

<div><p>Both mouse and human embryonic stem cells can be differentiated in vitro to produce a variety of somatic cell types. Using a new developmental tracing approach, we show that these cells are subject to massive aberrant CpG island de novo methylation that is exacerbated by differentiation in vitro. Bioinformatics analysis indicates that there are two distinct forms of abnormal de novo methylation, global as opposed to targeted, and in each case the resulting pattern is determined by molecular rules correlated with local pre-existing histone modification profiles. Since much of the abnormal methylation generated in vitro appears to be stably maintained, this modification may inhibit normal differentiation and could predispose to cancer if cells are used for replacement therapy. Excess CpG island methylation is also observed in normal placenta, suggesting that this process may be governed by an inherent program.</p></div

Resetting de novo methylation in vivo.

<p>Blastocysts injected with ES cells carrying a GFP expression vector were transplanted into pseudo-pregnant mice. Whole embryos were isolated at 16 dpc and sorted for GFP⁺ and GFP⁻ cells. DNA from these cells was then treated with bisulfite and deep-sequenced (Ion Torrent) at four different specific CpG island sequences. <b>a</b>. 7,000 individual molecules of island A containing nine individual CpGs with yellow indicating methylation. <b>b</b>. Graph showing percent methylation for islands A, B, C and D.</p

De novo methylation is proportional to H3K27me3 concentration.

<p><b>a</b>. IMS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096090#pone-0096090-g001" target="_blank">Fig. 1</a>) of all 9,500 constitutively unmethylated CpG islands in undifferentiated, endoderm-differentiated ES cells and adult tissue DNA graphed as a function of H3K27me3 density <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096090#pone.0096090-Mikkelsen1" target="_blank">[22]</a>. Each point represents the average IMS within a 5 unit span. <b>b</b>. Average methylation levels of all constitutively unmethylated CpG islands in endoderm (IMS), NPCs (%) and adult tissue (%) were graphed against H3K27me3 density partitioned into ten bins. The X-axis also shows average H3K27me3 levels in each percentile. <b>c</b>. Methylation levels in adult tissue, ES differentiated into endoderm and NPCs of all constitutively unmethylated CpG islands with above background concentrations (>2) of H3K27me3 graphed against their H3K4me3 density in ES.</p

Excess methylation in human ES cells.

<p>DNA from normal human fetal tissues, undifferentiated and in vitro differentiated ES cells were subject to mDIP microarray analysis. Heat map shows 950(IMS>0.75) in at least one of the differentiated ES cell types out of 13,000 CpG islands constitutively unmethylated (IMS<0) in fetal tissue samples. Retinoic acid treated cells and embryoid bodies were derived from CSES2. An estimate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096090#pone.0096090-Straussman1" target="_blank">[10]</a> for the average percent methylation in fetal tissues as compared to in vitro differentiated cells is also shown.</p