In Vivo Control of CpG and Non-CpG DNA Methylation by DNA Methyltransferases

The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position–, cell type–, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs. Author Summary: DNA methylation is a stable covalent epigenetic modification of cytosines mostly confined to CpG-dinucleotides in mammals. In general, it is associated with silencing of genomic DNA regions. Three catalytically active DNA methyltransferases (Dnmts) set and maintain CpG methylation in cooperation with other (co-)factors. The in vivo contribution of the Dnmts to maintain CpG and non-CpG methylation following rounds of DNA replication are not well understood, particularly since in vivo DNA methylation patterns can be highly dynamic. In our work, we use ultradeep sequencing to determine the methylation status of both DNA strands in ESCs depleted for Dnmts 1, 3a, 3b, and 3L, respectively. Using hidden Markov models, we calculate the relative contribution of each of the enzymes for the maintenance of DNA methylation patterns using parameter estimated fitting. While in general the modelling supports a classification of Dnmts into maintenance and de novo functions, it argues against a strict enzyme specific functional categorisation. We observe evidence for a context-dependent contribution of Dnmts to set and maintain CpG and non-CpG methylation at distinct classes of repetitive elements and selected single copy genes. We furthermore unambiguously identify Dnmt3a/3b and 3L dependent non-CpG methylation at specific sequence positions and confined to ESCs.