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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

Inheritance of Transposon ModificationReverse-transcribed cDNA (A), ChIP (B), and McrBC-digested genomic DNA (C) were amplified by PCR using primers from five retroelements and one DNA transposon in mutant (m/m) and backcrossed plants (m/+). Primers corresponded to transcribed ORFs for each element except for AtMu1 ChIP, which was done on the terminal inverted repeat (TIR). For ATLANTYS2, the larger product is ATLANTYS2-1 and smaller product is ATLANTYS2-2. Input RNA was normalized for each genotype using actin primers.(A) Mock RT–PCR was performed without reverse transcriptase (−RT) using primers specific for the Cen180 repeat, which can detect trace amounts of contaminating DNA due to its high-copy number.(B) ChIP was performed with antibodies recognizing dimethyl lysine-9 (K9) and dimethyl lysine-4 (K4) of histone H3 along with no antibody (na) and total (T) DNA controls. ChIP analysis for AtMu1 and ATCOPIA4 was performed using reduced cycles of PCR and Southern blotting (see Materials and Methods).(C) McrPCR was carried out on untreated (−) and McrBC-treated (+) DNA (see Materials and Methods).
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pbio.0000067-g002: Inheritance of Transposon ModificationReverse-transcribed cDNA (A), ChIP (B), and McrBC-digested genomic DNA (C) were amplified by PCR using primers from five retroelements and one DNA transposon in mutant (m/m) and backcrossed plants (m/+). Primers corresponded to transcribed ORFs for each element except for AtMu1 ChIP, which was done on the terminal inverted repeat (TIR). For ATLANTYS2, the larger product is ATLANTYS2-1 and smaller product is ATLANTYS2-2. Input RNA was normalized for each genotype using actin primers.(A) Mock RT–PCR was performed without reverse transcriptase (−RT) using primers specific for the Cen180 repeat, which can detect trace amounts of contaminating DNA due to its high-copy number.(B) ChIP was performed with antibodies recognizing dimethyl lysine-9 (K9) and dimethyl lysine-4 (K4) of histone H3 along with no antibody (na) and total (T) DNA controls. ChIP analysis for AtMu1 and ATCOPIA4 was performed using reduced cycles of PCR and Southern blotting (see Materials and Methods).(C) McrPCR was carried out on untreated (−) and McrBC-treated (+) DNA (see Materials and Methods).

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)

Inheritance of Transposon ModificationReverse-transcribed cDNA (A), ChIP (B), and McrBC-digested genomic DNA (C) were amplified by PCR using primers from five retroelements and one DNA transposon in mutant (m/m) and backcrossed plants (m/+). Primers corresponded to transcribed ORFs for each element except for AtMu1 ChIP, which was done on the terminal inverted repeat (TIR). For ATLANTYS2, the larger product is ATLANTYS2-1 and smaller product is ATLANTYS2-2. Input RNA was normalized for each genotype using actin primers.(A) Mock RT–PCR was performed without reverse transcriptase (−RT) using primers specific for the Cen180 repeat, which can detect trace amounts of contaminating DNA due to its high-copy number.(B) ChIP was performed with antibodies recognizing dimethyl lysine-9 (K9) and dimethyl lysine-4 (K4) of histone H3 along with no antibody (na) and total (T) DNA controls. ChIP analysis for AtMu1 and ATCOPIA4 was performed using reduced cycles of PCR and Southern blotting (see Materials and Methods).(C) McrPCR was carried out on untreated (−) and McrBC-treated (+) DNA (see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC300680&req=5

pbio.0000067-g002: Inheritance of Transposon ModificationReverse-transcribed cDNA (A), ChIP (B), and McrBC-digested genomic DNA (C) were amplified by PCR using primers from five retroelements and one DNA transposon in mutant (m/m) and backcrossed plants (m/+). Primers corresponded to transcribed ORFs for each element except for AtMu1 ChIP, which was done on the terminal inverted repeat (TIR). For ATLANTYS2, the larger product is ATLANTYS2-1 and smaller product is ATLANTYS2-2. Input RNA was normalized for each genotype using actin primers.(A) Mock RT–PCR was performed without reverse transcriptase (−RT) using primers specific for the Cen180 repeat, which can detect trace amounts of contaminating DNA due to its high-copy number.(B) ChIP was performed with antibodies recognizing dimethyl lysine-9 (K9) and dimethyl lysine-4 (K4) of histone H3 along with no antibody (na) and total (T) DNA controls. ChIP analysis for AtMu1 and ATCOPIA4 was performed using reduced cycles of PCR and Southern blotting (see Materials and Methods).(C) McrPCR was carried out on untreated (−) and McrBC-treated (+) DNA (see Materials and Methods).
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