In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases

Abstract

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.

Figure 1. DNA methylation pattern of CpG dyads at repetitive elements in WT ESCs, MEFs, and embryonic liver.

The bars sum up the DNA methylation status of all CpG dyads. The map next to the bar represents the distribution of methylated sites. Each column shows neighboured CpG dyads, and each line represents one sequence read. The reads in the map are sorted first by fully methylated sites and then by hemi-mCpG dyads. Red - fully methylated CpG dyads, light green and dark green - hemi-mCpG dyads on the upper and lower strand, blue - unmethylated CpG dyads, white - mutated or not analysable. *This picture shows the distance of the CpG dyads to each other. In MEFs and embryonic liver, hemimethylated sites are equally distributed all over the repetitive elements, whereas in ESCs elements specific differences in the amount of hemimethylated sites become obvious.

Figure 2. Methylation pattern of 4 single copy genes in WT ESCs, embryonic liver, and MEFs and in Dnmt KO ESCs.

For detailed description see legend Figure 1. In Dnmt1 KO all analyzed regions show a hypomethylated state. In Dnmt3a/3b KO sequence specific differences become obvious.

Figure 3. Methylation pattern of CpG dyads at repetitive elements in ESCs depleted for Dnmts or factors of the methylation machinery.

A: Methylation pattern map. For detailed description see legend Figure 1. In Dnmt1 KO all elements show a hypomethylated state. In Dnmt3a/3b DKO, element specific differences are obvious. Np95 KO (corresponding WT E14) shows a hypomethylated state, comparable to the Dnmt1 KO. B: Relative amount of hemimethylated CpGs. The relative amount of hemimethylated CpGs is the ratio between hemimethylated CpGs and the overall methylation. C: Distribution of methylated/hemimethylated CpG sites along reads. The fraction of reads showing fully methylated sites (+/−unmethylated sites) is colored red. The fraction of reads with hemi- and fully methylated (+/−unmethylated sites) sites is shown in dark green. Reads with hemimethylated site (+/−unmethylated sites) are colored green. Reads in light green have hemimethylated sites on the upper and lower strand at the same time (dispersed hemimethylation).

Figure 4. Non-CpG methylation of major satellites.

A: Major satellite genomic sequence of the hairpin bisulfite PCR product. Cytosines in non-CpG context are marked grey with the corresponding number attached. CpGs are marked red. Purple shows the location of the lower primer. B: Non-CpG methylation of mESCs, differentiated cells and Dnmt and Np95 KOs at major satellites. Only CpA positions show methylation up to ten percent (position 4, 6, 11, 22 and 28). Dnmt3 family KO ESCs show decrease of CpA methylation on different sites. C: The graph represents the relative amount of reads per CpG methylation level: grey - reads showing no CpA methylation, black - reads showing CpA methylation. The reads were grouped into three fraction by CpG methylation level (0–25%, 33–66,7%, 75–100%). Reads showing CpA methylation are depleted in the fraction of reads with low CpG methylation level and enriched in reads showing 50% or more CpG methylation. D: Distribution of un-, hemi- and fully methylated CpG dyads in the reads showing CpA methylation or no CpA methylation. The fraction of reads showing CpA methylation is enriched in fully- and hemimethylated (mainly on the upper strand) CpG dyads. Interestingly, on the upper strand, we also observe the main part of CpA methylation. E: Correlation plot for cytosine methylation at Dnmt1KO ESC in mSat. Methylated CpA positions correlate to neighboured CpG positions on the same DNA strand.

Figure 5. Non-CpG methylation of Afp.

A: Non-CpG methylation of mESCs, Dnmt KOs and differentiated cells. Non-CpG methylation can be found at Afp at 4 CpA positions (16, 23, 34, 45) and on one CpT position (54). Dnmt3a together with Dnmt3L are responsible for this methylation, Dnmt3bKO shows only slight effect. B: Correlation plot for cytosine methylation at Dnmt1KO ESCs at Afp. Methylated CpA positions mostly correlate to methylated neighboured CpG positions.

Figure 6. Estimation of Dnmt efficiencies using a Hidden Markov Model.

A: Dnmt efficiencies. In this diagram the methylation efficiencies for all three Dnmts are given. For all three Dnmts, we discriminate between the activity to methylate unmethylated CpG positions (unmeth.) and to methylate hemimethylated CpGs (hemim.). For Dnmt1, we find element specific methyltransferase activity at unmethylated CpG positions. At L1 and Igf2 sequences, Dnmt1 shows reduced activity at hemimethylated CpG sites. For Dnmt3a/3b, we find for some elements higher activity at hemimethylated positions. For some elements, the efficiencies are not given, since standard deviations were too high (marked with a cross) (for values see Table S3). B: Prediction of WT methylation. Taking the efficiencies, estimated in the HMM with the KO data, we predicted the methylation of the WT ESC line. This prediction (pred.) fits to the real (experimentally observed) methylation data (data).