Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes

Abstract

Many plant, animal, and fungal genomes contain cytosine DNA methylation in asymmetric sequence contexts (CpHpH, H = A, T, C). Although the enzymes responsible for this methylation are unknown, it has been assumed that asymmetric methylation is maintained by the persistent activity of de novo methyltransferases (enzymes capable of methylating previously unmodified DNA). We recently reported that the DOMAINS REARRANGED METHYLASE (DRM) genes are required for de novo DNA methylation in Arabidopsis thaliana because drm1 drm2 double mutants lack the de novo methylation normally associated with transgene silencing. In this study, we have used bisulfite sequencing and Southern blot analysis to examine the role of the DRM loci in the maintenance of asymmetric methylation. At some loci, drm1 drm2 double mutants eliminated all asymmetric methylation. However, at the SUPERMAN locus, asymmetric methylation was only completely abolished in drm1 drm2 chromomethylase 3 (cmt3) triple mutant plants. drm1 drm2 double mutants also showed a strong reduction of CpNpG (n = A, T, C, or G) methylation at some loci, but not at others. The drm1 drm2 cmt3 triple mutant plants did not affect CpG methylation at any locus tested, suggesting that the primary CpG methylases are encoded by the MET1 class of genes. Although neither the drm1 drm2 double mutants nor the cmt3 single mutants show morphological defects, drm1 drm2 cmt3 triple mutant plants show pleiotropic effects on plant development. Our results suggest that the DRM and CMT3 genes act in a partially redundant and locus-specific manner to control asymmetric and CpNpG methylation.

Figure 1

Diagram represents bisulfite sequencing of a 500-nt region of the top strand of the FWA gene, 219-nt region of the bottom strand of the MEA-ISR region, and 1,028-nt region of the top strand of the SUP gene in different mutant backgrounds. Detailed data supporting the graphical presentation can be found in Table 2 and Fig. 6. For the analysis of FWA and MEA-ISR, the wild-type strain is WS. For the SUP gene, the wild-type strain is clk-st in the Landsberg erecta background. drm1 drm2, cmt3-7, and drm1 drm2 cmt3 mutants are homozygous for the clk-st inverted repeat SUP transgene. Height of the bars represents the percentage of methylation at each site of 15 clones analyzed by bisulfite sequencing.

Figure 2

Southern blot analysis of FWA and MEA-ISR. (A Upper) A diagram of the CfoI and BglII restriction fragments present in the FWA gene. The inner two CfoI sites and the BglII site are within the methylated direct repeats of the FWA promoter, shown as arrows above. The region analyzed by bisulfite sequencing is also noted. (Lower) DNA blots of equivalent amounts of CfoI (Left) and BglII (Right) digested genomic DNA probed with a 1.74-kb fragment corresponding to the diagram on top. The positions of the methylated (M), unmethylated (U), and partially methylated (P) bands are shown, as well as the size of the bands in kilobases. drm1 drm2 cmt3-7 A and B are two independently isolated triple mutant strains. (B Upper) A diagram of the HpaII/MspI restriction fragments present in the MEA-ISR region. The inner HpaII/MspI site lies in the first of seven direct repeats (shown as arrows) and the probe lies in the unique sequence between these repeats and the MEA locus. (Lower) DNA blots of equivalent amounts of HpaII (Left) and MspI (Right) digested genomic DNA probed with a 1.1-kb fragment corresponding to the diagram on top.

Figure 3

Southern blot analysis of the Ta3 retrotransposon and 180-bp centromeric repeat sequences. Each panel shows lanes that contain equivalent amounts of genomic DNA of the indicated genotype digested with HpaII (Left) and MspI (Right). (A Upper) A diagram of the HpaII/MspI restriction fragments present in the Ta3 probe. (Lower) A DNA blot probed with Ta3 probe. (B) Blot probed with a 180-bp centromeric repeat probe.

Figure 4

Genetic redundancy of the DRM and CMT3 genes. (A) Graph showing the level and positions of asymmetric methylation of SUP in the cmt3-7 mutant or the drm1 drm2 double mutant, both in the clk-st background. Height of the bars represents the frequency of methylation at each site of 15 clones analyzed by bisulfite sequencing. The x axis represents the 1,028-nt methylated region on the top strand of SUP. (B) Photographs of a four-week-old wild-type Ler plant (Left) and several drm1 drm2 cmt3-7 triple mutant plants (Right).

Figure 5

Model illustrating the complex relationship between DRM and CMT3 activities. (A) DRM and CMT3 both have the capacity to methylate asymmetric and CpNpG sites, but these activities are locus-specific, and therefore may be regulated by various factors as discussed in the text. (B) Summary of the activities of DRM and CMT3 inferred from the analysis of the types of methylation lost at each locus in the drm1 drm2, cmt3-7, or drm1 drm2 cmt3-7 mutants. Thicker arrows denote more activity, which is inferred from the greater loss of methylation in a particular mutant.