Endogenous Targets of Transcriptional Gene Silencing in Arabidopsis

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In plants, double-stranded RNA that is processed to short RNAs Ϸ21-24 nt in length can trigger two types of epigenetic gene silencing. Posttranscriptional gene silencing, which is related to RNA interference in animals and quelling in fungi, involves targeted elimination of homologous mRNA in the cytoplasm. RNAdirected DNA methylation involves de novo methylation of almost all cytosine residues within a region of RNA-DNA sequence identity. RNA-directed DNA methylation is presumed to be responsible for the methylation observed in protein coding regions of posttranscriptionally silenced genes. Moreover, a type of transcriptional gene silencing and de novo methylation of homologous promoters in trans can occur if a double-stranded RNA contains promoter sequences. Although RNA-directed DNA methylation has been described so far only in plants, there is increasing evidence that RNA can also target genome modifications in other organisms. To understand how RNA directs methylation to identical DNA sequences and how changes in chromatin configuration contribute to initiating or maintaining DNA methylation induced by RNA, a promoter double-stranded RNA-mediated transcriptional gene silencing system has been established in Arabidopsis. A genetic analysis of this system is helping to unravel the relationships among RNA signals, DNA methylation, and chromatin structure.T he term ''RNA silencing'' refers to epigenetic gene silencing effects that are initiated by double-stranded RNA (dsRNA) (1). Discovered independently in plants, fungi, and animals, RNA silencing phenomena are revealing new ways to repress gene expression and to subdue transposable elements and viruses that produce dsRNA during their replication cycle (2-8). A fundamental step in RNA silencing pathways is cleavage of dsRNA into short RNAs (9), which are believed to act as guides for enzyme complexes that either degrade or modify homologous nucleic acids.The most familiar type of RNA silencing occurs primarily in the cytoplasm and is termed posttranscriptional gene silencing (PTGS) in plants, quelling in Neurospora, and RNA interference (RNAi) in animals. PTGS͞RNAi involves a dsRNA that is processed by an RNase III-like enzyme called Dicer into short interfering (si) RNAs 21-22 nt in length. The antisense siRNAs associate with a ribonuclease complex and guide sequencespecific degradation of complementary mRNAs (5-8).A second form of RNA silencing involves sequence-specific changes at the genome level. RNA-directed DNA methylation (RdDM) (10), which has been described so far only in plants, leads to de novo methylation of almost all cytosine residues within the region of sequence identity between the triggering RNA and the target DNA. Similarly to PTGS͞RNAi, RdDM requires a dsRNA that is cleaved to short RNAs Ϸ21-24 nt in length (11). It is not yet certain whether the short RNAs or dsRNA guide methylation of homologous DNA sequences, although the length of short RNAs is consistent with the minimum DNA target size of RdDM (Ϸ30 bp) (12).RdDM is assumed to be the ...

Section: Discussionmentioning

confidence: 91%

RNA-directed DNA methylation inArabidopsis

Aufsatz

Mette

Winden

et al. 2002

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230

“…A chromomethylase (encoded by CMT3), which is required for maintenance of CpXpG methylation, has also been shown to participate in the silencing of retrotransposons (30). Moreover, a truncated Athila (a putative retrotransposon) transcript was induced in several Arabidopsis mutants defective in TGS (27).…”

Section: Discussionmentioning

confidence: 93%

“…In Arabidopsis, Robertson's Mutator transposons and members of the CACTA superfamily are controlled by the SWI2͞ SNF2 chromatin-remodeling gene DDM1 (28,29). DDM1 also has a slight effect on endogenous retrotransposon mobilization (27,54). A chromomethylase (encoded by CMT3), which is required for maintenance of CpXpG methylation, has also been shown to participate in the silencing of retrotransposons (30).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Suppressors of transcriptional transgenic silencing in Chlamydomonas are sensitive to DNA-damaging agents and reactivate transposable elements

Jeong

Wu-Scharf

Zhang

et al. 2002

In the unicellular green alga Chlamydomonas reinhardtii, the epigenetic silencing of transgenes occurs, as in land plants, at both the transcriptional and posttranscriptional levels. In the case of singlecopy transgenes, transcriptional silencing takes place without detectable cytosine methylation of the introduced DNA. We have isolated two mutant strains, Mut-9 and Mut-11, that reactivate expression of a transcriptionally silenced single-copy transgene. These suppressors are deficient in the repression of a DNA transposon and a retrotransposon-like element. In addition, the mutants show enhanced sensitivity to DNA-damaging agents, particularly radiomimetic chemicals inducing DNA double-strand breaks. All of these phenotypes are much more prominent in a double mutant strain. These observations suggest that multiple partly redundant epigenetic mechanisms are involved in the repression of transgenes and transposons in eukaryotes, presumably as components of a system that evolved to preserve genomic stability. Our results also raise the possibility of mechanistic connections between epigenetic transcriptional silencing and DNA double-strand break repair.

“…This finding suggests a synergistic action of the mutations on release of silencing from the transgenic target locus. The single mutations release silencing also from specific endogenous pericentromeric repeats termed TSI (transcriptionally silent information) at different levels (13). In the double mutant, TSI expression patterns were additive, and individual transcripts appeared even more abundant than additive (Fig.…”

Section: Resultsmentioning

confidence: 99%

Two regulatory levels of transcriptional gene silencing in Arabidopsis

Scheid

Probst

Afsar

et al. 2002

Self Cite

In mammals, some fungi, and plants, DNA methylation plays a central role in the epigenetic control of gene transcription. Recently, however, a subclass of Arabidopsis mutants revealed that the release of transcriptional gene silencing (TGS) does not necessarily require DNA demethylation. Here, we address the fundamental question of whether these mutants delineate a previously uncharacterized, methylation-independent level of epigenetic regulation, or whether they just act downstream of DNA methylation signals. Two mutants described earlier, ddm1 and mom1, reactivate previously silent loci: ddm1 impairs TGS by reducing chromosomal DNA methylation, and mom1 releases TGS without affecting DNA methylation. We examined the epistatic relationship between ddm1 and mom1 by constructing double mutant strains. The synergistic release of TGS revealed by gene expression patterns from silent loci, drastic developmental abnormalities, and characteristic changes in nuclear architecture in these double mutants implies that DDM1 and MOM are likely to operate at independent levels in TGS control. Our results indicate that the methylationindependent silencing mechanism reinforces the methylationbased system and prevents extremely rapid epigenetic deregulation in plants with DNA methylation deficiencies.T he functional relationship, epistatic interaction, and hierarchy of genetic elements can be addressed by combining multiple genetic defects. Combined mutants provide substantial information, even where the number of gene products in a pathway or their modes of action are not well defined (1). We applied this approach to the analysis of transcriptional gene silencing (TGS) in plants. Methylation-dependent and -independent release of TGS is represented by the two recently isolated genes DDM1 (for decreased DNA methylation 1; ref. 2) and MOM (for Morpheus' molecule), respectively (3). Sequence comparison identified DDM1 as a member of the SWI2͞SNF2 family of chromatin remodeling factors (2). MOM defines a new group of proteins with only limited homology to the SWI2͞SNF2 family (3). The properties of these proteins and their modes of action in gene silencing are not yet known. Although mutations in each gene release silencing from a range of common target loci (4), the ddm1 and mom1 mutations have different consequences for plant morphology. Homozygous ddm1 mutants show rapid, progressive loss of vigor on inbreeding (5), whereas mom1 plants are morphologically normal even after many generations of selfing (3). After outcrossing of ddm1 to the wild type, the heterozygous hybrids show no or very slow resilencing and remethylation of the activated targets (6-8), whereas resilencing in mom1 heterozygotes is fast (3). These differences are likely related to the fact that ddm1 affects TGS by reducing DNA methylation levels, whereas mom1 restores transcriptional activity despite maintaining the templates in a hypermethylated state. Thus, mom1 challenges the concept that DNA methylation is an obligate component of the epigenetic switch. Howev...