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Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification.

Lippman Z, May B, Yordan C, Singer T, Martienssen R - PLoS Biol. (2003)

Bottom Line: More recently, transposon silencing has been associated with DNA methylation, histone H3 lysine-9 methylation (H3mK9), and RNA interference (RNAi).According to their pattern of transposon regulation, the mutants can be divided into two groups, which suggests that there are distinct, but interacting, complexes or pathways involved in transposon silencing.Furthermore, different transposons tend to be susceptible to different forms of epigenetic regulation.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

ABSTRACT
Heritable, but reversible, changes in transposable element activity were first observed in maize by Barbara McClintock in the 1950s. More recently, transposon silencing has been associated with DNA methylation, histone H3 lysine-9 methylation (H3mK9), and RNA interference (RNAi). Using a genetic approach, we have investigated the role of these modifications in the epigenetic regulation and inheritance of six Arabidopsis transposons. Silencing of most of the transposons is relieved in DNA methyltransferase (met1), chromatin remodeling ATPase (ddm1), and histone modification (sil1) mutants. In contrast, only a small subset of the transposons require the H3mK9 methyltransferase KRYPTONITE, the RNAi gene ARGONAUTE1, and the CXG methyltransferase CHROMOMETHYLASE3. In crosses to wild-type plants, epigenetic inheritance of active transposons varied from mutant to mutant, indicating these genes differ in their ability to silence transposons. According to their pattern of transposon regulation, the mutants can be divided into two groups, which suggests that there are distinct, but interacting, complexes or pathways involved in transposon silencing. Furthermore, different transposons tend to be susceptible to different forms of epigenetic regulation.

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Related in: MedlinePlus

Southern Blot Analysis(A and B) Genomic DNAs prepared from 4-wk-old plants of the indicated mutant and backcrossed (m/+) genotypes were digested with either HindIII and HpaII (left) or HindIII and MspI (right) and used for Southern blot analysis with a probe specific to the DNA transposons AtMu1 and the retrotransposon ATCOPIA4. The Ler genotype is shown. DNA methylation loss for each element within the mutants and their backcrosses is indicated by loss of band intensity relative to WT as indicated by the arrows or brackets.(C) Genomic DNAs from the same genotypes in (A) and (B) were digested with either HpaII (left) or MspI (right) and used for Southern blot analysis with a probe specific to the ATLINE1-4 element. The probe corresponds to a region flanked on both sides by more than five HpaII/MspI sites within 6 kb. Thus, fragment sizes generated upon digestion of the genotypes tested varied owing to a number of potential methylation changes. The fragments within the brackets depict significant changes in methylation between the genotypes.
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pbio.0000067-g003: Southern Blot Analysis(A and B) Genomic DNAs prepared from 4-wk-old plants of the indicated mutant and backcrossed (m/+) genotypes were digested with either HindIII and HpaII (left) or HindIII and MspI (right) and used for Southern blot analysis with a probe specific to the DNA transposons AtMu1 and the retrotransposon ATCOPIA4. The Ler genotype is shown. DNA methylation loss for each element within the mutants and their backcrosses is indicated by loss of band intensity relative to WT as indicated by the arrows or brackets.(C) Genomic DNAs from the same genotypes in (A) and (B) were digested with either HpaII (left) or MspI (right) and used for Southern blot analysis with a probe specific to the ATLINE1-4 element. The probe corresponds to a region flanked on both sides by more than five HpaII/MspI sites within 6 kb. Thus, fragment sizes generated upon digestion of the genotypes tested varied owing to a number of potential methylation changes. The fragments within the brackets depict significant changes in methylation between the genotypes.

Mentions: In WT, transcripts were low or undetectable by PCR amplifying reverse-transcribed cDNA (RT–PCR), and these loci were associated with elevated levels of H3mK9 and reduced levels of H3mK4 according to chromatin immunoprecipitation (ChIP) analysis. The transposons were also heavily methylated when assayed by modified cytosine restriction McrBC digestion, which cuts DNA at methylated cytosine residues, preventing PCR amplification (Figure 2C), or by DNA gel blot analysis using HpaII and MspI, which are sensitive to both CG and CNG methylation and to CNG methylation alone, respectively (Figure 3). Transcripts, unmethylated DNA, and H3mK4 could be detected in the mutants (see Figure 2) and were indicative of the inheritance of activated transposons in backcrossed plants in all cases except ATGP1, which had substantial levels of H3mK4 in WT plants. Methylated DNA and H3mK9 were also measured, but could not be used to assess inheritance, as these were also inherited from silent elements in the WT parent.


Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification.

Lippman Z, May B, Yordan C, Singer T, Martienssen R - PLoS Biol. (2003)

Southern Blot Analysis(A and B) Genomic DNAs prepared from 4-wk-old plants of the indicated mutant and backcrossed (m/+) genotypes were digested with either HindIII and HpaII (left) or HindIII and MspI (right) and used for Southern blot analysis with a probe specific to the DNA transposons AtMu1 and the retrotransposon ATCOPIA4. The Ler genotype is shown. DNA methylation loss for each element within the mutants and their backcrosses is indicated by loss of band intensity relative to WT as indicated by the arrows or brackets.(C) Genomic DNAs from the same genotypes in (A) and (B) were digested with either HpaII (left) or MspI (right) and used for Southern blot analysis with a probe specific to the ATLINE1-4 element. The probe corresponds to a region flanked on both sides by more than five HpaII/MspI sites within 6 kb. Thus, fragment sizes generated upon digestion of the genotypes tested varied owing to a number of potential methylation changes. The fragments within the brackets depict significant changes in methylation between the genotypes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC300680&req=5

pbio.0000067-g003: Southern Blot Analysis(A and B) Genomic DNAs prepared from 4-wk-old plants of the indicated mutant and backcrossed (m/+) genotypes were digested with either HindIII and HpaII (left) or HindIII and MspI (right) and used for Southern blot analysis with a probe specific to the DNA transposons AtMu1 and the retrotransposon ATCOPIA4. The Ler genotype is shown. DNA methylation loss for each element within the mutants and their backcrosses is indicated by loss of band intensity relative to WT as indicated by the arrows or brackets.(C) Genomic DNAs from the same genotypes in (A) and (B) were digested with either HpaII (left) or MspI (right) and used for Southern blot analysis with a probe specific to the ATLINE1-4 element. The probe corresponds to a region flanked on both sides by more than five HpaII/MspI sites within 6 kb. Thus, fragment sizes generated upon digestion of the genotypes tested varied owing to a number of potential methylation changes. The fragments within the brackets depict significant changes in methylation between the genotypes.
Mentions: In WT, transcripts were low or undetectable by PCR amplifying reverse-transcribed cDNA (RT–PCR), and these loci were associated with elevated levels of H3mK9 and reduced levels of H3mK4 according to chromatin immunoprecipitation (ChIP) analysis. The transposons were also heavily methylated when assayed by modified cytosine restriction McrBC digestion, which cuts DNA at methylated cytosine residues, preventing PCR amplification (Figure 2C), or by DNA gel blot analysis using HpaII and MspI, which are sensitive to both CG and CNG methylation and to CNG methylation alone, respectively (Figure 3). Transcripts, unmethylated DNA, and H3mK4 could be detected in the mutants (see Figure 2) and were indicative of the inheritance of activated transposons in backcrossed plants in all cases except ATGP1, which had substantial levels of H3mK4 in WT plants. Methylated DNA and H3mK9 were also measured, but could not be used to assess inheritance, as these were also inherited from silent elements in the WT parent.

Bottom Line: More recently, transposon silencing has been associated with DNA methylation, histone H3 lysine-9 methylation (H3mK9), and RNA interference (RNAi).According to their pattern of transposon regulation, the mutants can be divided into two groups, which suggests that there are distinct, but interacting, complexes or pathways involved in transposon silencing.Furthermore, different transposons tend to be susceptible to different forms of epigenetic regulation.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

ABSTRACT
Heritable, but reversible, changes in transposable element activity were first observed in maize by Barbara McClintock in the 1950s. More recently, transposon silencing has been associated with DNA methylation, histone H3 lysine-9 methylation (H3mK9), and RNA interference (RNAi). Using a genetic approach, we have investigated the role of these modifications in the epigenetic regulation and inheritance of six Arabidopsis transposons. Silencing of most of the transposons is relieved in DNA methyltransferase (met1), chromatin remodeling ATPase (ddm1), and histone modification (sil1) mutants. In contrast, only a small subset of the transposons require the H3mK9 methyltransferase KRYPTONITE, the RNAi gene ARGONAUTE1, and the CXG methyltransferase CHROMOMETHYLASE3. In crosses to wild-type plants, epigenetic inheritance of active transposons varied from mutant to mutant, indicating these genes differ in their ability to silence transposons. According to their pattern of transposon regulation, the mutants can be divided into two groups, which suggests that there are distinct, but interacting, complexes or pathways involved in transposon silencing. Furthermore, different transposons tend to be susceptible to different forms of epigenetic regulation.

Show MeSH
Related in: MedlinePlus