Abstract
BackgroundGenomic methylation patterns are established during gametogenesis, and perpetuated in somatic cells by faithful maintenance methylation. There have been previous indications that genomic methylation patterns may be less stable in embryonic stem (ES) cells than in differentiated somatic cells, but it is not known whether different mechanisms of de novo and maintenance methylation operate in pluripotent stem cells compared with differentiating somatic cells.ResultsIn this paper, we show that ablation of the DNA methyltransferase regulator DNMT3L (DNA methyltransferase 3-like) in mouse ES cells renders them essentially incapable of de novo methylation of newly integrated retroviral DNA. We also show that ES cells lacking DNMT3L lose DNA methylation over time in culture, suggesting that DNA methylation in ES cells is the result of dynamic loss and gain of DNA methylation. We found that wild-type female ES cells lose DNA methylation at a much faster rate than do male ES cells; this defect could not be attributed to sex-specific differences in expression of DNMT3L or of any DNA methyltransferase. We also found that human ES and induced pluripotent stem cell lines showed marked but variable loss of methylation that could not be attributed to sex chromosome constitution or time in culture.ConclusionsThese data indicate that DNA methylation in pluripotent stem cells is much more dynamic and error-prone than is maintenance methylation in differentiated cells. DNA methylation requires DNMT3L in stem cells, but DNMT3L is not expressed in differentiating somatic cells. Error-prone maintenance methylation will introduce unpredictable phenotypic variation into clonal populations of pluripotent stem cells, and this variation is likely to be much more pronounced in cultured female cells. This epigenetic variability has obvious negative implications for the clinical applications of stem cells.
Highlights
Genomic methylation patterns are established during gametogenesis, and perpetuated in somatic cells by faithful maintenance methylation
We found that human embryonic stem (ES) and induced pluripotent stem cells tend to lose DNA methylation spontaneously in a process that is independent of sex and passage number
Ablation of DNMT3L largely prevented the passage-dependent silencing of retroviral transcription that occurs in the presence of DNMT3L, and this lack of silencing activity was associated with a failure to methylate the long terminal repeat (LTR) of the Mo-MLV42bp/GFP retrovirus (Figure 1c)
Summary
Genomic methylation patterns are established during gametogenesis, and perpetuated in somatic cells by faithful maintenance methylation. Maintenance methylation is very stable in differentiated/somatic cells, and DNA that is methylated in predetermined patterns maintains this methylation pattern for >80 cell divisions in transfected cells [10]. This stability is a consequence of recognition of hemimethylated DNA after DNA replication by DNMT1 and the regulatory factor UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) [11]. Both DNMT1 and UHRF1 bind to hemimethylated CpG dinucleotides, and deficiency in either factor results in genomewide demethylation and embryonic lethality [12,13,14]. The observation that UHRF1 is able to bind to histone H3 that is di- or trimethylated at lysine 9 [15] implies the involvement of other chromatin factors
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