Dnmt2-dependent methylomes lack defined DNA methylation patterns

Abstract

Several organisms have retained methyltransferase 2 (Dnmt2) as their only candidate DNA methyltransferase gene. However, information about Dnmt2-dependent methylation patterns has been limited to a few isolated loci and the results have been discussed controversially. In addition, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which raised the possibility that Dnmt2-only genomes might be unmethylated. We have now used whole-genome bisulfite sequencing to analyze the methylomes of Dnmt2-only organisms at single-base resolution. Our results show that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in Dnmt2-deficient flies. Furthermore, genetically modified Dnmt2-only mouse embryonic stem cells lost the DNA methylation patterns found in wild-type cells. Our results thus uncover fundamental differences among animal methylomes and suggest that DNA methylation is dispensable for a considerable number of eukaryotic organisms.

Keywords: RNA methylation; epigenetics.

Fig. 1.

Characterization of the Schistosoma mansoni methylome. (A) Average methylation levels were determined for all cytosine residues with a methylation ratio >0.1 and then distributed into bins with increasing methylation ratios (red bars). For comparison, the corresponding data are also shown for honey bee worker brains (gray bars), an established Dnmt1/3-dependent methylome with a very low DNA methylation level (30). (B) Dinucleotide sequence contexts of unconverted cytosines in Schistosoma (red) and in honey bees (gray). (C) Position-specific nonconversion ratios (red) and coverages (gray) of the Schistosoma forkhead gene. The specific region previously reported to be methylated (23) is indicated as a green bar. Sequence position numbers refer to GenBank accession JF781495.

Fig. 2.

Characterization of the Drosophila melanogaster methylome. (A) Average methylation levels were determined for all cytosine residues and then distributed into bins with increasing methylation ratios (blue bars). For comparison, the corresponding data are also shown for human sperm DNA that was spiked into the Drosophila sample before bisulfite conversion (black bars). The actual numerical values of the first bins are 99.7% (Drosophila) and 92.9% (human sperm). (B) Dinucleotide sequence context of unconverted cytosines in Drosophila (blue) and in human sperm (black). (C) Position-specific nonconversion ratios (red) and coverage (gray) of the Drosophila Invader4 element. Results are shown for the sequence with the lowest conversion rate among genomic Invader4 elements. The specific region previously reported to be methylated (24) is indicated as a green bar. Sequence position numbers refer to GenBank accession AE014135.3.

Fig. 3.

Characterization of DNA methylation in the TKO mouse ES cell model. (A) Schematic illustration of Dnmt genotypes in wild-type and TKO mouse ES cells. (B) Average methylation levels were determined for all cytosine residues and then distributed into bins with increasing methylation ratios (orange bars). For comparison, the corresponding data are also shown for wild-type mouse ES cells (gray bars). The actual numerical values of the first bins are 86.9% (wild type) and 96.5% (TKO). (C) Fractions of nonconverted (ratio >0.1) CpN dinucleotides in wild-type cells (gray bars) and TKO cells (orange bars).

Fig. 4.

Characterization of the Dnmt2 mutant Drosophila methylome. (A) Average methylation levels were determined for all cytosine residues that were covered by more than three sequence reads and then distributed into bins with increasing methylation ratios (green bars). The actual numerical value of the first bin is 99.0%. (B) Histograms showing the number of nonconverted (ratio >0.5) cytosine residues in 1-kb windows. For comparison, the corresponding data are also shown for the wild-type Drosophila methylome (blue). (C) Venn diagram showing overlapping windows with >2 (Left) or >20 (Right) nonconverted cytosine residues in wild-type (blue) and Dnmt2 mutant (green) embryos.