How is DNA Methylation Maintained in ESCs and Differentiated Cells?
DNA methylation has been shown to be very important for development, differentiation, and aging. But how is DNA methylation maintained? When and how does de novo DNA methylation occur? Are there specific DNA methyltransferases (Dnmts) involved in non-symmetric DNA methylation or DNA methylation outside of the CpG context? There have been many studies involving the DNA methylation machinery in vitro and in situ, but it has never been characterized in vivo until recently.
In order to study these mechanisms, Arand et al. used 454 sequencing (large-scale parallel pyrosequencing) of hairpin-bisulfite amplicons of both single-copy genes and repetitive elements in mouse wild-type Embryonic Stem Cells (mESCs), embryonic liver, mouse embryonic fibroblasts (MEFs), and Dnmt knockout (KO) mESCs. This technique allows for generation of not only single-base resolution DNA methylation data, but also strand-specific data and accurate differentiation between CT conversions and CpG mutations. Using this method in combination with high-throughput 454 sequencing, the group generated a large data set (146,000 CpG sites on both strands). It was confirmed that Dnmt1 is very important for maintenance of DNA methylation because Dnmt1 KO mESCs showed a large increase in hemi-methylated CpG sites. CpA methylation was detected with great confidence in major satellites (75% of mSat reads in J1) in ESCs, while DNA methylation outside of the CpG context was not seen at all in differentiated cells. Non-CpG methylation decreased significantly in Dnmt3a/b DKO mESCs and it was unaffected in Dnmt1 KO mESCs, demonstrating that Dnmt3a/b is necessary for non-CpG methylation. The authors also used a complex biostatistical model (hidden Markov model) to estimate the Dnmt efficiencies for unmethylated and hemi-methylated CpG sites and observed data correlated very well with the model. The model also indicates a “significant methylation probability at unmethylated CpG dyads” for Dnmt1, meaning it is also probably involved in de novo methylation.
The authors determined that all three Dnmts were involved in both de novo and maintenance methylation. Dnmt3a/b were not solely responsible for de novo DNA methylation, nor was Dnmt1 confined to only maintenance methylation. They also discussed the possibility that the hemi-methylated state could be related to the 5hmC mark and a de-methylation pathway, but they will need to create a Tet1+Tet2 double-KO to confirm.
Arand J, Spieler D, Karius T, Branco MR, Meilinger D, Meissner A, Jenuwein T, Xu G, Leonhardt H, Wolf V, Walter J. (2012) In Vivo Control of CpG and Non-CpG DNA Methylation by DNA Methyltransferases. PLoS Genet. June; 8(6): e1002750
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