Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana

Abstract

Background: DNA methylation is an important biochemical mark that silences repetitive sequences, such as transposons, and reinforces epigenetic gene expression states. An important class of repetitive genes under epigenetic control in eukaryotic genomes encodes ribosomal RNA (rRNA) transcripts. The ribosomal genes coding for the 45S rRNA precursor of the three largest eukaryotic ribosomal RNAs (18S, 5.8S, and 25-28S) are found in nucleolus organizer regions (NORs), comprised of hundreds to thousands of repeats, only some of which are expressed in any given cell. An epigenetic switch, mediated by DNA methylation and histone modification, turns rRNA genes on and off. However, little is known about the mechanisms that specify and maintain the patterns of NOR DNA methylation.

Results: Here, we explored the extent of naturally-occurring variation in NOR DNA methylation among accessions of the flowering plant Arabidopsis thaliana. DNA methylation in coding regions of rRNA genes was positively correlated with copy number of 45S rRNA gene and DNA methylation in the intergenic spacer regions. We investigated the inheritance of NOR DNA methylation patterns in natural accessions with hypomethylated NORs in inter-strain crosses and defined three different categories of inheritance in F1 hybrids. In addition, subsequent analysis of F2 segregation for NOR DNA methylation patterns uncovered different patterns of inheritance. We also revealed that NOR DNA methylation in the Arabidopsis accession Bor-4 is influenced by the vim1-1 (variant in methylation 1-1) mutation, but the primary effect is specified by the NORs themselves.

Conclusion: Our results indicate that the NORs themselves are the most significant determinants of natural variation in NOR DNA methylation. However, the inheritance of NOR DNA methylation suggests the operation of a diverse set of mechanisms, including inheritance of parental methylation patterns, reconfiguration of parental NOR DNA methylation, and the involvement of trans-acting modifiers.

Figure 1

DNA methylation in 45S rRNA gene repeats among Arabidopsis natural accessions. (A) A 10-kb rRNA gene repeat unit encoding the 18S, 5.8S, and 25S rRNAs. A bent arrow shows the location of the transcription start site, and the two hybridization probes (IGS and CR) are indicated below the monomer repeat unit as gray bars. (B) A representative genomic DNA blot analysis of Arabidopsis natural accessions using the CR probe. HpaII-digested genomic DNA was size fractionated by gel electrophoresis and transferred to a nylon membrane. The membrane was hybridized with radiolabeled CR probe corresponding to the 5.8S and 25S rRNA coding regions (see the map above). The hybridization signal indicates the extent of DNA methylation in the rRNA gene clusters; unmethylated fragments are cleaved by HpaII to small fragments (open box), whereas methylated genomic fragments remain uncleaved, or partially cleaved, and migrate as larger fragments (solid box). (C) Variation in CR DNA methylation among 88 Arabidopsis natural accessions. CR DNA methylation index (%) was calculated by measuring the percentage of hybridization signal in each lane that corresponded to restriction fragments >1 kb. Means and standard errors were calculated from at least three independent experiments.

Figure 2

Positive correlation between rRNA gene copy number and CR DNA methylation. CR DNA methylation indices were plotted against normalized rRNA gene copy numbers in 41 Arabidopsis natural accessions. Means and standard errors were calculated from at least three independent experiments. The gray line is a linear regression of the data (linear regression coefficient, R = 0.63).

Figure 3

IGS DNA methylation in 45S rRNA gene repeats among Arabidopsis natural accessions. (A) Distributions of relative CR and IGS DNA methylation in 75 natural accessions. The frequency histograms show the range and distribution of CR and IGS DNA methylation in Arabidopsis natural accessions. (B) A correlation between CR DNA methylation and IGS DNA methylation. Means and standard errors were calculated from at least three independent experiments. The gray line is a linear regression of the data (linear regression coefficient, R = 0.68).

Figure 4

CR DNA methylation in F2 populations derived from crosses between Col and accessions with hypomethylated NORs. (A) Plot of CR DNA methylation index versus NOR genotype in a Zdr-1 × Col F2 population (group I). The F2 individuals were classified into nine groups based on their genotypes at NOR2-NOR4 (C = Col/Col, H = Col/Zdr-1, Z = Zdr-1/Zdr-1). (B) Plot of CR DNA methylation index versus NOR genotype in a Ga-0 × Col F2 population (group II) (C = Col/Col, H = Col/Ga-0, G = Ga-0/Ga-0). (C) Plot of CR DNA methylation index versus NOR genotype in a Bay-0 × Col F2 population (group III) (C = Col/Col, H = Col/Bay-0, B = Bay-0/Bay-0). Vertical bars denote standard errors. The values obtained for the parental accessions are indicated by red horizontal lines.

Figure 5

CR DNA methylation in the Bor-4 accession. (A) Increased NOR DNA methylation in Bor-4 plants transformed with a VIM1 transgene under the control of the 35S promoter. Genomic DNA samples of the indicated genotypes were digested with HpaII and used for DNA gel blot analysis with a hybridization probe for the CR region. (B) Plot of CR DNA methylation index versus NOR and VIM1 genotypes in a Bor-4 × Col F2 population. The F2 individuals were classified into nine groups based on their genotypes at NOR2-NOR4 (C = Col/Col, H = Col/Bor-4, R = Bor-4/Bor-4), and then each group was divided into two groups based on the presence of the vim1-1 mutation as a homozygote. Vertical bars denote standard errors. The values obtained for the parental accessions are indicated by red horizontal lines.