Conservation and divergence of methylation patterning in plants and animals

Abstract

Cytosine DNA methylation is a heritable epigenetic mark present in many eukaryotic organisms. Although DNA methylation likely has a conserved role in gene silencing, the levels and patterns of DNA methylation appear to vary drastically among different organisms. Here we used shotgun genomic bisulfite sequencing (BS-Seq) to compare DNA methylation in eight diverse plant and animal genomes. We found that patterns of methylation are very similar in flowering plants with methylated cytosines detected in all sequence contexts, whereas CG methylation predominates in animals. Vertebrates have methylation throughout the genome except for CpG islands. Gene body methylation is conserved with clear preference for exons in most organisms. Furthermore, genes appear to be the major target of methylation in Ciona and honey bee. Among the eight organisms, the green alga Chlamydomonas has the most unusual pattern of methylation, having non-CG methylation enriched in exons of genes rather than in repeats and transposons. In addition, the Dnmt1 cofactor Uhrf1 has a conserved function in maintaining CG methylation in both transposons and gene bodies in the mouse, Arabidopsis, and zebrafish genomes.

Fig. 1.

Overall methylation levels in eight eukaryotic organisms. Tree topology is from NCBI Taxonomy (http://www.ncbi.nlm.nih.gov/guide/taxonomy/). Methylation levels shown are from main chromosomes/linkage groups/scaffolds of each organism (Tables S1 and S2). All tissues are wild type. Data from Arabidopsis shoots and mouse embryos are shown here; for other tissues used are described in Materials and Methods. In the case of poplar, more than one third of the genome sequence exists in scaffolds not placed on any of the main linkage groups. These scaffolds have higher methylation levels (Table S2), as well as increased repeat density, increased sequence ambiguity, and low mapability (Fig. S2). This is consistent with the notion that the sequences in these scaffolds are of a highly repetitive nature that prevents them from being assembled properly, and methylation is enriched in repetitive DNA.

Fig. 2.

Distribution of methylation along protein-coding genes. Upstream and downstream regions are the same length as the gene. Only BS-Seq data up to halfway to the next nonoverlapping gene are used in this analysis. Two vertical purple lines mark the gene boundaries. Tissues are the same as in Fig. 1. BS-Seq data annotated to at least one repeat and/or transposon are withheld from this analysis.

Fig. 3.

Distribution of methylation along repetitive DNA. Upstream and downstream regions are the same length as the repeat. Only BS-Seq data up to halfway to the next nonoverlapping repeat are used in this analysis. Two vertical purple lines mark the repeat boundaries. Chlamydomonas repeats shown here are interspersed repeats only. Tissues are the same as in Fig. 1.

Fig. 4.

Comparison of methylation levels across exons and introns. Only internal exons (flanked by introns on both ends) that do not contain any 5′- or 3′-UTR bases are used. Upstream and downstream regions are the same length as the exon. Only BS-Seq data up to halfway to the next exon are used in this analysis. Two vertical purple lines mark the intron–exon and exon–intron boundaries. Tissues are the same as in Fig. 1. BS-Seq data annotated to at least one repeat and/or transposon are withheld from this analysis.

Fig. 5.

Decrease of CG methylation in mutants lacking Uhrf1. (A) Reactivation of a transcriptionally silenced transgene in zebrafish uhrf1 homozygous mutants. WT larvae (3 days postfertilization) carrying the silenced allele of the brain-specific transgene Gt(Gal4-VP16;UAS:EGFP) c269 GFP (off) do not show any GFP labeled cells in the brain (Left) due to methylation of CG sites in the multicopy UAS (33). On the contrary, GFP labeled cells resulting from reactivation of the silenced c269 transgene are readily detected in the brains of uhrf1 homozygous mutant larvae (Right). (B–D) Comparison of CG methylation levels between wild-type and uhrf1 mutant lines from zebrafish (B), mouse (C), and Arabidopsis (D). For Arabidopsis, met1 mutant is used as a control for virtual total loss of CG methylation. Tissues used are mouse embryonic stem cells, Arabidopsis flowers, and zebrafish embryos.