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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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RNA-directed DNA Methylation and Heterochromatinization at the MPF.(A) Genomic structure of the FLC locus and flanking regions examined by bisulfite sequencing (B1, B2, B3 and B4) or ChIP (C1, C2 and C3). Green box represents the hAT element; pink boxes represent exons; the gray arrow represents the promoter; the orange box represents the TE insertion in Ler. (B) Small RNA tags matched to MPF found from the MPSS (green) or 454 sequencing data (red), and the LNA probe used for small RNA hybridization (blue) are represented with their length indicated by numbers. The color coding of the cytosines in (B) matches the legend in (C). (C) Bisulfite sequencing result of the MPF at the B1 region in Ler. The bars with red stars represent sites that were detected by Southern blot (Figure 1) and n indicates the number of the sequenced clones. (D) Small RNA Northern blots probed with the LNA probe (B) in Ler and Col; tRNA and other RNA bands stained with ethidium bromide (EtBr) were used to indicate the amount of loaded RNA. (E) Chromatin Immunoprecipitation (ChIP) to detect H3K9 mono-, di-, and tri-methylation (represented as H3K9me1, H3K9me2, and H3K9me3, respectively) at MPF (C1) in Ler and Col. Input is saved before immunoprecipitation and “No AB” refers to the sample without antibody. Ta3 served as an internal control for heterochromatic loci.
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pgen-1000056-g002: RNA-directed DNA Methylation and Heterochromatinization at the MPF.(A) Genomic structure of the FLC locus and flanking regions examined by bisulfite sequencing (B1, B2, B3 and B4) or ChIP (C1, C2 and C3). Green box represents the hAT element; pink boxes represent exons; the gray arrow represents the promoter; the orange box represents the TE insertion in Ler. (B) Small RNA tags matched to MPF found from the MPSS (green) or 454 sequencing data (red), and the LNA probe used for small RNA hybridization (blue) are represented with their length indicated by numbers. The color coding of the cytosines in (B) matches the legend in (C). (C) Bisulfite sequencing result of the MPF at the B1 region in Ler. The bars with red stars represent sites that were detected by Southern blot (Figure 1) and n indicates the number of the sequenced clones. (D) Small RNA Northern blots probed with the LNA probe (B) in Ler and Col; tRNA and other RNA bands stained with ethidium bromide (EtBr) were used to indicate the amount of loaded RNA. (E) Chromatin Immunoprecipitation (ChIP) to detect H3K9 mono-, di-, and tri-methylation (represented as H3K9me1, H3K9me2, and H3K9me3, respectively) at MPF (C1) in Ler and Col. Input is saved before immunoprecipitation and “No AB” refers to the sample without antibody. Ta3 served as an internal control for heterochromatic loci.

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)

RNA-directed DNA Methylation and Heterochromatinization at the MPF.(A) Genomic structure of the FLC locus and flanking regions examined by bisulfite sequencing (B1, B2, B3 and B4) or ChIP (C1, C2 and C3). Green box represents the hAT element; pink boxes represent exons; the gray arrow represents the promoter; the orange box represents the TE insertion in Ler. (B) Small RNA tags matched to MPF found from the MPSS (green) or 454 sequencing data (red), and the LNA probe used for small RNA hybridization (blue) are represented with their length indicated by numbers. The color coding of the cytosines in (B) matches the legend in (C). (C) Bisulfite sequencing result of the MPF at the B1 region in Ler. The bars with red stars represent sites that were detected by Southern blot (Figure 1) and n indicates the number of the sequenced clones. (D) Small RNA Northern blots probed with the LNA probe (B) in Ler and Col; tRNA and other RNA bands stained with ethidium bromide (EtBr) were used to indicate the amount of loaded RNA. (E) Chromatin Immunoprecipitation (ChIP) to detect H3K9 mono-, di-, and tri-methylation (represented as H3K9me1, H3K9me2, and H3K9me3, respectively) at MPF (C1) in Ler and Col. Input is saved before immunoprecipitation and “No AB” refers to the sample without antibody. Ta3 served as an internal control for heterochromatic loci.
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Related In: Results  -  Collection

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

pgen-1000056-g002: RNA-directed DNA Methylation and Heterochromatinization at the MPF.(A) Genomic structure of the FLC locus and flanking regions examined by bisulfite sequencing (B1, B2, B3 and B4) or ChIP (C1, C2 and C3). Green box represents the hAT element; pink boxes represent exons; the gray arrow represents the promoter; the orange box represents the TE insertion in Ler. (B) Small RNA tags matched to MPF found from the MPSS (green) or 454 sequencing data (red), and the LNA probe used for small RNA hybridization (blue) are represented with their length indicated by numbers. The color coding of the cytosines in (B) matches the legend in (C). (C) Bisulfite sequencing result of the MPF at the B1 region in Ler. The bars with red stars represent sites that were detected by Southern blot (Figure 1) and n indicates the number of the sequenced clones. (D) Small RNA Northern blots probed with the LNA probe (B) in Ler and Col; tRNA and other RNA bands stained with ethidium bromide (EtBr) were used to indicate the amount of loaded RNA. (E) Chromatin Immunoprecipitation (ChIP) to detect H3K9 mono-, di-, and tri-methylation (represented as H3K9me1, H3K9me2, and H3K9me3, respectively) at MPF (C1) in Ler and Col. Input is saved before immunoprecipitation and “No AB” refers to the sample without antibody. Ta3 served as an internal control for heterochromatic loci.
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
Related in: MedlinePlus