Endogenous targets of transcriptional gene silencing in Arabidopsis.

Transcriptional gene silencing (TGS) frequently inactivates foreign genes integrated into plant genomes but very likely also suppresses an unknown subset of chromosomal information. Accordingly, RNA analysis of mutants impaired in silencing should uncover endogenous targets of this epigenetic regulation. We compared transcripts from wild-type Arabidopsis carrying a silent transgene with RNA from an isogenic transgene-expressing TGS mutant. Two cDNA clones were identified representing endogenous RNA expressed only in the mutant. The synthesis of these RNAs was found to be released in several mutants affected in TGS, implying that TGS in general and not a particular mutation controls the transcriptional activity of their templates. Detailed analysis revealed that the two clones are part of longer transcripts termed TSI (for transcriptionally silent information). Two major classes of related TSI transcripts were found in a mutant cDNA library. They are synthesized from repeats present in heterochromatic pericentromeric regions of Arabidopsis chromosomes. These repeats share sequence homology with the 3' terminal part of the putative retrotransposon Athila. However, the transcriptional activation does not include the transposon itself and does not promote its movement. There is no evidence for a general release of silencing from retroelements. Thus, foreign genes in plants encounter the epigenetic control normally directed, at least in part, toward a subset of pericentromeric repeats.

Disruption of the plant gene MOM releases transcriptional silencing of methylated genes.

Epigenetic modifications change transcription patterns in multicellular organisms to achieve tissue-specific gene expression and inactivate alien DNA such as transposons or transgenes. In plants and animals, DNA methylation is involved in heritability and flexibility of epigenetic states, although its function is far from clear. We have isolated an Arabidopsis gene, MOM, whose product is required for the maintenance of transcriptional gene silencing. Mutation of this gene or depletion of its transcript by expression of antisense RNA reactivates transcription from several previously silent, heavily methylated loci. Despite this, the dense methylation at these reactivated loci is maintained even after nine generations, indicating that transcriptional activity and methylation pattern are inherited independently. The predicted MOM gene product is a nuclear protein of 2,001 amino acids containing a region similar to part of the ATPase region of the SWI2/SNF2 family, members of which are involved in chromatin remodelling. MOM is the first known molecular component that is essential for transcriptional gene silencing and does not affect methylation pattern. Thus, it may act downstream of methylation in epigenetic regulation, or be part of a new pathway that does not require methylation marks.

Involvement of Arabidopsis thaliana ribosomal protein S27 in mRNA degradation triggered by genotoxic stress.

A recessive Arabidopsis mutant with elevated sensitivity to DNA damaging treatments was identified in one out of 800 families generated by T-DNA insertion mutagenesis. The T-DNA generated a chromosomal deletion of 1287 bp in the promoter of one of three S27 ribosomal protein genes (ARS27A) preventing its expression. Seedlings of ars27A developed normally under standard growth conditions, suggesting wild-type proficiency of translation. However, growth was strongly inhibited in media supplemented with methyl methane sulfate (MMS) at a concentration not affecting the wild type. This inhibition was accompanied by the formation of tumor-like structures instead of auxiliary roots. Wild-type seedlings treated with increasing concentrations of MMS up to a lethal dose never displayed such a trait, neither was this phenotype observed in ars27A plants in the absence of MMS or under other stress conditions. Thus, the hypersensitivity and tumorous growth are mutant-specific responses to the genotoxic MMS treatment. Another important feature of the mutant is its inability to perform rapid degradation of transcripts after UV treatment, as seen in wild-type plants. Therefore, we propose that the ARS27A protein is dispensable for protein synthesis under standard conditions but is required for the elimination of possibly damaged mRNA after UV irradiation.

Release of epigenetic gene silencing by trans-acting mutations in Arabidopsis.

Gene silencing in plants inactivates trans-genes introduced into plants and/or endogenous homologous genes. This stable but potentially reversible loss of gene activity resembles epigenetic changes that occur in normal development. The stability of silencing implies the involvement of trans-acting components, although none of them have been identified so far. Here we report the finding of second-site mutations interfering with maintenance of the silent state. We mutagenized Arabidopsis thaliana plants carrying a silent transgene encoding hygromycin phosphotransferase (hpt) and therefore show a heritable hygromycin-sensitive phenotype. The M2 generation was screened for hygromycin resistance. Eight putative mutants (som1 to 8) were found that expressed the transgene and transmitted the expressed state to their progeny. All mutations were shown to reactivate a silent transgenic test locus in trans. The level of DNA methylation at the hpt locus and at centromeric repeats was found to be reduced in the som mutants. Complementation crosses indicated complex epigenetic interactions among the som mutant alleles and with the previously described ddm1 allele, which elicits DNA hypomethylation [Vongs, A., Kakutani, T, Martiensen, R.A. & Richards, E.J. (1993) Science 260, 1926-1928]. Som mutants can be classified into three groups: (i) allelic or interacting with ddm1 and with each other (som 1,4, and 5), (ii) nonallelic with ddm1 and som mutants of group A (som2), and (iii) mutants with slow resilencing after out-crosses, which hinders their classification (som 3, 6, 7 and 8).

Methylation of cytosines in nonconventional methylation acceptor sites can contribute to reduced gene expression.

Epigenetic silencing of gene expression is often correlated with extensive DNA methylation at cytosine residues in the promoter and the coding region of silenced genes. Increasing evidence indicates that, in such cases, DNA methylation can also occur in sequence contexts other than CG and CNG, resulting in genomic regions with almost complete modification of cytosines. Whether this nonconventional methylation at CNN sites also contributes to gene repression is not known. We constructed genes with a promoter and a coding region devoid of the conventional methylation acceptor sites CG and CNG in addition to constructs with the corresponding wild-type sequences containing these sites. We generated unmethylated and completely methylated DNA by the polymerase chain reaction and performed expression assays in plant protoplasts. Quantification of transcript levels by RNase protection assay demonstrated that DNA methylation at positions other than CG or CNG sites contributes to the reduction in gene expression.

A change of ploidy can modify epigenetic silencing.

A silent transgene in Arabidopsis thaliana was reactivated in an outcross but not upon selfing of hemizygous plants. This result could only be explained by assuming a genetic difference between the transgene-free gametes of the wild-type and hemizygous transgenic plants, respectively, and led to the discovery of ploidy differences between the parental plants. To investigate whether a change of ploidy by itself can indeed influence gene expression, we performed crosses of diploid or tetraploid plants with a strain containing a single copy of a transgenic resistance gene in an active state. We observed reduced gene expression of the transgene in triploid compared with diploid hybrids. This led to loss of the resistant phenotype at various stages of seedling development in part of the population. The gene inactivation was reversible. Thus, an increased number of chromosomes can result in a new type of epigenetic gene inactivation, creating differences in gene expression patterns. We discuss the possible impact of this finding for genetic diploidization in the light of widespread, naturally occurring polyploidy and polysomaty in plants.

Gene inactivation in Arabidopsis thaliana is not accompanied by an accumulation of repeat-induced point mutations.

Chromosomal integration of multicopy transgene inserts in higher plants is often followed by loss of expression. We have analysed whether this inactivation can trigger repeat-induced point mutations (RIP) as has been observed in Neurospora crassa. We have previously characterized transgenic lines of Arabidopsis thaliana containing the hygromycin phosphotransferase (hpt) gene either as a unique sequence in plants expressing the gene, or as multimeric, closely linked repeats in clones that were resistant to hygromycin directly after transformation but exhibited gene inactivation in the subsequent generation. At the sequence level, we have determined the mutation frequencies in the promoter and coding regions of active and inactive copies of transgene inserts after passage through three sexual generations. No RIP-like mutations were found in inactivated genes. Comparison of our data with those from Neurospora suggest that sequence divergence within plant repetitive DNA is either much slower than in Neurospora or is generated by a different mechanism.