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

Woo HR, Richards EJ - BMC Plant Biol. (2008)

Bottom Line: 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.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.Our results indicate that the NORs themselves are the most significant determinants of natural variation in NOR DNA methylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Washington University, One Brookings Drive, St. Louis, MO 63130, USA. hrwoo@biology2.wustl.edu

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.

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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.
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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.

Mentions: Our previous study of a limited number of Arabidopsis natural accessions uncovered significant variation in DNA methylation of coding regions of 45S rRNA gene repeats [26]. Here, we examined a larger collection of Arabidopsis accessions to investigate natural variation in NOR DNA methylation in more detail. The extent of rRNA gene methylation was evaluated in 88 accessions of A. thaliana collected by Nordborg et al. [27] using DNA gel blot analysis with the methylation-sensitive restriction endonuclease, HpaII (5'-CCGG-3'), which is inhibited by either 5mCpG or 5mCpHpG (Fig. 1). A hybridization probe corresponding to the 5.8S and 25S rRNA coding sequences (CR) should detect only <1 kb fragments following complete cleavage of the 70 HpaII sites within the entire 10 kb rRNA gene (Fig. 1A). Figure 1B and Additional File 1 show representative genomic DNA blot analyses demonstrating that the extent and pattern of DNA methylation in the CR of the 45S rRNA gene repeats varies greatly among Arabidopsis natural accessions. The examined HpaII sites were largely methylated in a standard laboratory strain Columbia (Col), and these HpaII sites were unmethylated in the Col ddm1-2 (decrease in DNA methylation 1–2) mutant [28]. Based on the distribution of CR hybridization signal in the Col ddm1-2 mutant, we calculated CR DNA methylation indices by measuring the percentage of hybridization signal in each lane that corresponded to restriction fragments larger than 1 kb. Figure 1C and Additional File 2 summarize CR DNA methylation indices in the different Arabidopsis natural accessions examined, which varied widely over a range from 8.1% in Bay-0 to 82.5% in Bil-7. Our broader study of CR DNA methylation among 88 accessions further demonstrates that considerable natural variation exists in rRNA gene methylation in Arabidopsis.


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

Woo HR, Richards EJ - BMC Plant Biol. (2008)

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.
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Related In: Results  -  Collection

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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.
Mentions: Our previous study of a limited number of Arabidopsis natural accessions uncovered significant variation in DNA methylation of coding regions of 45S rRNA gene repeats [26]. Here, we examined a larger collection of Arabidopsis accessions to investigate natural variation in NOR DNA methylation in more detail. The extent of rRNA gene methylation was evaluated in 88 accessions of A. thaliana collected by Nordborg et al. [27] using DNA gel blot analysis with the methylation-sensitive restriction endonuclease, HpaII (5'-CCGG-3'), which is inhibited by either 5mCpG or 5mCpHpG (Fig. 1). A hybridization probe corresponding to the 5.8S and 25S rRNA coding sequences (CR) should detect only <1 kb fragments following complete cleavage of the 70 HpaII sites within the entire 10 kb rRNA gene (Fig. 1A). Figure 1B and Additional File 1 show representative genomic DNA blot analyses demonstrating that the extent and pattern of DNA methylation in the CR of the 45S rRNA gene repeats varies greatly among Arabidopsis natural accessions. The examined HpaII sites were largely methylated in a standard laboratory strain Columbia (Col), and these HpaII sites were unmethylated in the Col ddm1-2 (decrease in DNA methylation 1–2) mutant [28]. Based on the distribution of CR hybridization signal in the Col ddm1-2 mutant, we calculated CR DNA methylation indices by measuring the percentage of hybridization signal in each lane that corresponded to restriction fragments larger than 1 kb. Figure 1C and Additional File 2 summarize CR DNA methylation indices in the different Arabidopsis natural accessions examined, which varied widely over a range from 8.1% in Bay-0 to 82.5% in Bil-7. Our broader study of CR DNA methylation among 88 accessions further demonstrates that considerable natural variation exists in rRNA gene methylation in Arabidopsis.

Bottom Line: 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.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.Our results indicate that the NORs themselves are the most significant determinants of natural variation in NOR DNA methylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Washington University, One Brookings Drive, St. Louis, MO 63130, USA. hrwoo@biology2.wustl.edu

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.

Show MeSH
Related in: MedlinePlus