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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 the FLC Promoter by Southern Blots.(A) A diagram of the genomic region around FLC promoter is shown above with the positions of restriction sites marked as follows: Fnu4H I (F), Aci I (A), and HpyCH4 IV (H) are sensitive to methylation; Nde I (N) which is not sensitive to methylation is used as a negative control. Red stars highlight the methylated sites. The digested fragments that could be detected by probe covering FLC promoter (gray strip) were diagramed and the size is indicated by numbers (in kilobases) beneath the fragments. A hAT element is represented as gray box (see Figure 4 for more detail). (B) Determination of DNA methylation status at FLC promoter in Ler, hen1-1, Col, and hen1-4. Black arrows indicate the DNA fragments which contain the methylated (and therefore uncut) enzyme recognition sites.
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pgen-1000056-g001: DNA Methylation Analysis of the FLC Promoter by Southern Blots.(A) A diagram of the genomic region around FLC promoter is shown above with the positions of restriction sites marked as follows: Fnu4H I (F), Aci I (A), and HpyCH4 IV (H) are sensitive to methylation; Nde I (N) which is not sensitive to methylation is used as a negative control. Red stars highlight the methylated sites. The digested fragments that could be detected by probe covering FLC promoter (gray strip) were diagramed and the size is indicated by numbers (in kilobases) beneath the fragments. A hAT element is represented as gray box (see Figure 4 for more detail). (B) Determination of DNA methylation status at FLC promoter in Ler, hen1-1, Col, and hen1-4. Black arrows indicate the DNA fragments which contain the methylated (and therefore uncut) enzyme recognition sites.

Mentions: In addition to the previously described Mutator-like transposable element (TE) inserted in the first intron of FLC [19] in Ler, we found that a region located adjacent to the promoter of the FLC was specifically methylated in Ler but not in Col (Figure 1A). We named this region MPF (Methylated region near Promoter of FLC). Restriction enzymes including AciI, HpyCH4 IV and Fnu4HI, which are sensitive to CpG methylation, were able to cut outside of the MPF but not within this region in Ler (Figure 1). Notably different from the TE inserted in FLC-Ler, the MPF of Ler and Col share almost identical sequences (Figure S1). Bisulfite sequencing of MPF (B1 region, Figure 2A) revealed that a small region of less than 100 bp was exhibited a very high level of asymmetric methylation (also called CHH methylation, where H represents A, C or T) (Figure 2C). This region also demonstrated extensive CpG and CNG (where N is any nucleotide) methylation (Figure 2C). In addition, no DNA methylation was found outside the MPF (the B2 and B3 regions, Figure 2A) in Ler (data not shown) or the MPF in Col (Figure 3A) by bisulfite sequencing.


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 the FLC Promoter by Southern Blots.(A) A diagram of the genomic region around FLC promoter is shown above with the positions of restriction sites marked as follows: Fnu4H I (F), Aci I (A), and HpyCH4 IV (H) are sensitive to methylation; Nde I (N) which is not sensitive to methylation is used as a negative control. Red stars highlight the methylated sites. The digested fragments that could be detected by probe covering FLC promoter (gray strip) were diagramed and the size is indicated by numbers (in kilobases) beneath the fragments. A hAT element is represented as gray box (see Figure 4 for more detail). (B) Determination of DNA methylation status at FLC promoter in Ler, hen1-1, Col, and hen1-4. Black arrows indicate the DNA fragments which contain the methylated (and therefore uncut) enzyme recognition sites.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000056-g001: DNA Methylation Analysis of the FLC Promoter by Southern Blots.(A) A diagram of the genomic region around FLC promoter is shown above with the positions of restriction sites marked as follows: Fnu4H I (F), Aci I (A), and HpyCH4 IV (H) are sensitive to methylation; Nde I (N) which is not sensitive to methylation is used as a negative control. Red stars highlight the methylated sites. The digested fragments that could be detected by probe covering FLC promoter (gray strip) were diagramed and the size is indicated by numbers (in kilobases) beneath the fragments. A hAT element is represented as gray box (see Figure 4 for more detail). (B) Determination of DNA methylation status at FLC promoter in Ler, hen1-1, Col, and hen1-4. Black arrows indicate the DNA fragments which contain the methylated (and therefore uncut) enzyme recognition sites.
Mentions: In addition to the previously described Mutator-like transposable element (TE) inserted in the first intron of FLC [19] in Ler, we found that a region located adjacent to the promoter of the FLC was specifically methylated in Ler but not in Col (Figure 1A). We named this region MPF (Methylated region near Promoter of FLC). Restriction enzymes including AciI, HpyCH4 IV and Fnu4HI, which are sensitive to CpG methylation, were able to cut outside of the MPF but not within this region in Ler (Figure 1). Notably different from the TE inserted in FLC-Ler, the MPF of Ler and Col share almost identical sequences (Figure S1). Bisulfite sequencing of MPF (B1 region, Figure 2A) revealed that a small region of less than 100 bp was exhibited a very high level of asymmetric methylation (also called CHH methylation, where H represents A, C or T) (Figure 2C). This region also demonstrated extensive CpG and CNG (where N is any nucleotide) methylation (Figure 2C). In addition, no DNA methylation was found outside the MPF (the B2 and B3 regions, Figure 2A) in Ler (data not shown) or the MPF in Col (Figure 3A) by bisulfite sequencing.

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.

Show MeSH