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. 2003 Dec;1(3):E67.
doi: 10.1371/journal.pbio.0000067. Epub 2003 Dec 22.

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

Zachary Lippman  1 , Bruce May, Cristy Yordan, Tatjana Singer, Rob Martienssen
Affiliations

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

Zachary Lippman et al. PLoS Biol. 2003 Dec.

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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Conflict of interest statement

The authors have declared that no conflicts of interest exist.

Figures

Figure 1
Figure 1. Inheritance of Transposon Activity
Pollen from homozygous mutant plants (m/m) was crossed onto WT (+/+) to generate backcrossed BC heterozygous seed (m/+). The parents were also self-pollinated as a control. Each class of progeny was then tested for expression of transposon mRNA, loss of DNA methylation, and changes in histone H3 methylation. Accumulation of transposon mRNA (+) or lack thereof (−) in each progeny genotype was used to determine whether the elements were silent (“cryptic”), reversibly activated, or heritably activated (“preset”).
Figure 2
Figure 2. Inheritance of Transposon Modification
Reverse-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).
Figure 3
Figure 3. 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.
Figure 4
Figure 4. siRNA Differentially Accumulates in Chromatin Mutants
siRNA Northern blots were hybridized with sense RNA probes for each of the transposons indicated in (A) and (B) in order to detect 25 nt antisense siRNA from each of the sequences tested. AtMu1 is single copy so that autoradiographic exposure was increased substantially. A 22 oligonucleotide size marker is indicated, and the 21 nt miRNA miR-171 was used as a loading control. It is unaffected in the mutants tested.
Figure 5
Figure 5. Model of Transposon Silencing Complexes
(A) DDM1, MET1, and SIL1 are all required for transposon silencing and may interact. MET1 and DDM1 are also required for siRNA accumulation (shown in red). AGO and KYP have similar effects on transposon activation and may also interact. They impact DNA methylation via CMT3 (Cao and Jacobsen 2002; Jackson et al. 2002). (B) siRNA, histone H3 methylation, and DNA methylation interact to silence transposons. Silencing is maintained by the MET1/DDM1/SIL1 complex. A possible network is shown.
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