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Small RNA-directed epigenetic natural variation in Arabidopsis thaliana.

Zhai J, Liu J, Liu B, Li P, Meyers BC, Chen X, Cao X - PLoS Genet. (2008)

Bottom Line: Here, we report that, in the model plant Arabidopsis thaliana, a cluster of approximately 24 nt siRNAs found at high levels in the ecotype Landsberg erecta (Ler) could direct DNA methylation and heterochromatinization at a hAT element adjacent to the promoter of FLOWERING LOCUS C (FLC), a major repressor of flowering, whereas the same hAT element in ecotype Columbia (Col) with almost identical DNA sequence, generates a set of low abundance siRNAs that do not direct these activities.We have called this hAT element MPF for Methylated region near Promoter of FLC, although de novo methylation triggered by an inverted repeat transgene at this region in Col does not alter its FLC expression.A genome-wide comparison of Ler and Col small RNAs identified at least 68 loci matched by a significant level of approximately 24 nt siRNAs present specifically in Ler but not Col, where nearly half of the loci are related to repeat or TE sequences.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Progress in epigenetics has revealed mechanisms that can heritably regulate gene function independent of genetic alterations. Nevertheless, little is known about the role of epigenetics in evolution. This is due in part to scant data on epigenetic variation among natural populations. In plants, small interfering RNA (siRNA) is involved in both the initiation and maintenance of gene silencing by directing DNA methylation and/or histone methylation. Here, we report that, in the model plant Arabidopsis thaliana, a cluster of approximately 24 nt siRNAs found at high levels in the ecotype Landsberg erecta (Ler) could direct DNA methylation and heterochromatinization at a hAT element adjacent to the promoter of FLOWERING LOCUS C (FLC), a major repressor of flowering, whereas the same hAT element in ecotype Columbia (Col) with almost identical DNA sequence, generates a set of low abundance siRNAs that do not direct these activities. We have called this hAT element MPF for Methylated region near Promoter of FLC, although de novo methylation triggered by an inverted repeat transgene at this region in Col does not alter its FLC expression. DNA methylation of the Ler allele MPF is dependent on genes in known silencing pathways, and such methylation is transmissible to Col by genetic crosses, although with varying degrees of penetrance. A genome-wide comparison of Ler and Col small RNAs identified at least 68 loci matched by a significant level of approximately 24 nt siRNAs present specifically in Ler but not Col, where nearly half of the loci are related to repeat or TE sequences. Methylation analysis revealed that 88% of the examined loci (37 out of 42) were specifically methylated in Ler but not Col, suggesting that small RNA can direct epigenetic differences between two closely related Arabidopsis ecotypes.

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DNA Methylation Analysis of 44 loci Varied in Small RNA Abundance between Col and Ler using Real-time McrBC-PCR.(A) Real-time PCR results using McrBC non-digested (white bar) and digested (black bar) DNA from both Col (under the axis) and Ler (above the axis) as the PCR templates. For the comparison, the Non-digested result of each locus was normalized to 1. N/A means PCR amplification failed. If the value of McrBC digested sample at certain locus is significantly lower than McrBC non-digested one, then this locus is methylated, otherwise it is unmethylated. Locus #60 which is methylated in both Col and Ler is used as the positive control and unmethylated Actin is used as the negative control. (B) Summary of the McrBC results. “Methylated” is defined as the value of McrBC non-digested sample at a certain locus is lower than 0.5, and “unmethylated” is defined as the value of McrBC non-digested sample at a certain locus is higher than 0.5.
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pgen-1000056-g007: DNA Methylation Analysis of 44 loci Varied in Small RNA Abundance between Col and Ler using Real-time McrBC-PCR.(A) Real-time PCR results using McrBC non-digested (white bar) and digested (black bar) DNA from both Col (under the axis) and Ler (above the axis) as the PCR templates. For the comparison, the Non-digested result of each locus was normalized to 1. N/A means PCR amplification failed. If the value of McrBC digested sample at certain locus is significantly lower than McrBC non-digested one, then this locus is methylated, otherwise it is unmethylated. Locus #60 which is methylated in both Col and Ler is used as the positive control and unmethylated Actin is used as the negative control. (B) Summary of the McrBC results. “Methylated” is defined as the value of McrBC non-digested sample at a certain locus is lower than 0.5, and “unmethylated” is defined as the value of McrBC non-digested sample at a certain locus is higher than 0.5.

Mentions: We investigated the methylation pattern of locus #10 as an example using bisulfite sequencing. Extensive methylation was found in Ler (Figure S8), whereas the same region in Col remained unmethylated (data not shown). Other eight randomly selected loci were tested using methylation sensitive McrBC-PCR, and all of them, even those with the minimal number of three unique siRNAs, were methylated in Ler but not Col (Figure S9). Furthermore, we tested the methylation status of 44 loci (in which 42 have successful amplification results), including all the loci on Chromosome I and II,, by real-time McrBC-PCR (Figure 7A). From these analyses, 88% of the loci (37 out of 42) were found to be specifically methylated in Ler but not Col, and no locus was found only methylated in Col, strongly supporting the role of ∼24 nt siRNA in triggering epigenetic natural variation (Figure 7B).


Small RNA-directed epigenetic natural variation in Arabidopsis thaliana.

Zhai J, Liu J, Liu B, Li P, Meyers BC, Chen X, Cao X - PLoS Genet. (2008)

DNA Methylation Analysis of 44 loci Varied in Small RNA Abundance between Col and Ler using Real-time McrBC-PCR.(A) Real-time PCR results using McrBC non-digested (white bar) and digested (black bar) DNA from both Col (under the axis) and Ler (above the axis) as the PCR templates. For the comparison, the Non-digested result of each locus was normalized to 1. N/A means PCR amplification failed. If the value of McrBC digested sample at certain locus is significantly lower than McrBC non-digested one, then this locus is methylated, otherwise it is unmethylated. Locus #60 which is methylated in both Col and Ler is used as the positive control and unmethylated Actin is used as the negative control. (B) Summary of the McrBC results. “Methylated” is defined as the value of McrBC non-digested sample at a certain locus is lower than 0.5, and “unmethylated” is defined as the value of McrBC non-digested sample at a certain locus is higher than 0.5.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2289841&req=5

pgen-1000056-g007: DNA Methylation Analysis of 44 loci Varied in Small RNA Abundance between Col and Ler using Real-time McrBC-PCR.(A) Real-time PCR results using McrBC non-digested (white bar) and digested (black bar) DNA from both Col (under the axis) and Ler (above the axis) as the PCR templates. For the comparison, the Non-digested result of each locus was normalized to 1. N/A means PCR amplification failed. If the value of McrBC digested sample at certain locus is significantly lower than McrBC non-digested one, then this locus is methylated, otherwise it is unmethylated. Locus #60 which is methylated in both Col and Ler is used as the positive control and unmethylated Actin is used as the negative control. (B) Summary of the McrBC results. “Methylated” is defined as the value of McrBC non-digested sample at a certain locus is lower than 0.5, and “unmethylated” is defined as the value of McrBC non-digested sample at a certain locus is higher than 0.5.
Mentions: We investigated the methylation pattern of locus #10 as an example using bisulfite sequencing. Extensive methylation was found in Ler (Figure S8), whereas the same region in Col remained unmethylated (data not shown). Other eight randomly selected loci were tested using methylation sensitive McrBC-PCR, and all of them, even those with the minimal number of three unique siRNAs, were methylated in Ler but not Col (Figure S9). Furthermore, we tested the methylation status of 44 loci (in which 42 have successful amplification results), including all the loci on Chromosome I and II,, by real-time McrBC-PCR (Figure 7A). From these analyses, 88% of the loci (37 out of 42) were found to be specifically methylated in Ler but not Col, and no locus was found only methylated in Col, strongly supporting the role of ∼24 nt siRNA in triggering epigenetic natural variation (Figure 7B).

Bottom Line: Here, we report that, in the model plant Arabidopsis thaliana, a cluster of approximately 24 nt siRNAs found at high levels in the ecotype Landsberg erecta (Ler) could direct DNA methylation and heterochromatinization at a hAT element adjacent to the promoter of FLOWERING LOCUS C (FLC), a major repressor of flowering, whereas the same hAT element in ecotype Columbia (Col) with almost identical DNA sequence, generates a set of low abundance siRNAs that do not direct these activities.We have called this hAT element MPF for Methylated region near Promoter of FLC, although de novo methylation triggered by an inverted repeat transgene at this region in Col does not alter its FLC expression.A genome-wide comparison of Ler and Col small RNAs identified at least 68 loci matched by a significant level of approximately 24 nt siRNAs present specifically in Ler but not Col, where nearly half of the loci are related to repeat or TE sequences.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Progress in epigenetics has revealed mechanisms that can heritably regulate gene function independent of genetic alterations. Nevertheless, little is known about the role of epigenetics in evolution. This is due in part to scant data on epigenetic variation among natural populations. In plants, small interfering RNA (siRNA) is involved in both the initiation and maintenance of gene silencing by directing DNA methylation and/or histone methylation. Here, we report that, in the model plant Arabidopsis thaliana, a cluster of approximately 24 nt siRNAs found at high levels in the ecotype Landsberg erecta (Ler) could direct DNA methylation and heterochromatinization at a hAT element adjacent to the promoter of FLOWERING LOCUS C (FLC), a major repressor of flowering, whereas the same hAT element in ecotype Columbia (Col) with almost identical DNA sequence, generates a set of low abundance siRNAs that do not direct these activities. We have called this hAT element MPF for Methylated region near Promoter of FLC, although de novo methylation triggered by an inverted repeat transgene at this region in Col does not alter its FLC expression. DNA methylation of the Ler allele MPF is dependent on genes in known silencing pathways, and such methylation is transmissible to Col by genetic crosses, although with varying degrees of penetrance. A genome-wide comparison of Ler and Col small RNAs identified at least 68 loci matched by a significant level of approximately 24 nt siRNAs present specifically in Ler but not Col, where nearly half of the loci are related to repeat or TE sequences. Methylation analysis revealed that 88% of the examined loci (37 out of 42) were specifically methylated in Ler but not Col, suggesting that small RNA can direct epigenetic differences between two closely related Arabidopsis ecotypes.

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