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Endogenous targets of transcriptional gene silencing in Arabidopsis - PubMed

Endogenous targets of transcriptional gene silencing in Arabidopsis

A Steimer et al. Plant Cell. 2000 Jul.

Abstract

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.

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Figures

Figure 1.
Figure 1.

Expression of TSI in TGS Mutants. (A) Expression of TSI-A (left) and TSI-B (right) in the mutant mom1. Lanes were loaded with 10 μg of total RNA and 0.1 μg of poly(A) RNA from 2-week-old seedlings of the mutant mom1 and parental line A. (B) Expression of TSI-A in other genotypes affected in gene silencing. Total RNA (5 μg each) from young seedlings was hybridized to TSI-A (top) and RAN (Haizel et al., 1997) as a loading control (bottom). nt, nucleotides; Zh, wild type of ecotype Zürich; A, parental line of som1 to som8 and mom1 (som1 to som8 are silencing mutants; Mittelsten Scheid et al., 1998); C/C, parental line of sil1, sil2, and hog1 (sil1, sil2, and hog1 are silencing mutants; Furner et al., 1998); C24, parental wild type of METas; METas, transgenic for DNA methyltransferase antisense gene (Finnegan et al., 1996); Col, parental wild type of ddm1 (ddm1 and ddm2 are mutants with decreased DNA methylation; Vongs et al., 1993).

Figure 2.
Figure 2.

DNA Gel Blot Analysis in Various Ecotypes and Mutants. (A) Conserved hybridization pattern of TSI-A in various ecotypes. DNA gel blot analysis was performed with ∼5 μg of DNA from various ecotypes (Zh, Zürich; Col, Columbia; Ler, Landsberg erecta; Ws, Wassilewskija; and C24) digested with DraI. The blot was hybridized to a TSI-A probe (left) flanking the DraI site (see map below the gel) and rehybridized to TSI-B (right). Differences in hybridization intensity are the result of loading differences. DNA fragment lengths are indicated in kilobases. (B) TSI methylation states in different mutants. DNA gel blot analysis was performed with ∼5 μg of DNA (Zh, Zürich; line A, parental line of som5 and mom1; som5 and mom1 silencing mutants) digested with the methylation-sensitive restriction enzyme HpaII (left) and the methylation-insensitive restriction enzyme DraI (right) as a control.

Figure 3.
Figure 3.

FISH Images of Pachytene Chromosomes of Landsberg erecta Hybridized to TSI-A and the 180-bp Repeat. (A) 4′,6-Diamidino-2-phenylindole (DAPI)-stained chromosomes. (B) Hybridization signals of TSI-A (green) and the 180-bp repeat (red). (C) Merged DAPI and FISH images showing the TSI-A signals in the pericentromeric region of all chromosomes. Bar in (A) = 5 μm for (A) to (C).

Figure 4.
Figure 4.

RNase Protection Assay with Strand-Specific Probes of TSI-A. Ten micrograms of total RNA of mom1, the parental line A, or yeast was hybridized to antisense (AS) or sense (S) TSI-A probes. Unprotected single-stranded RNA was cleaved by RNase A and RNase T, or RNase T only, as indicated. The integrity of the RNA probes was shown in samples without RNase. nt, nucleotides of the molecular size marker.

Figure 5.
Figure 5.

Organization of TSI Transcripts, BAC F7N22, and Athila. (A) TSI-A and the clones obtained by 5′ extension (clones a and b) and by 3′ extension (clones c and d). The nucleotide sequence of clones a and b are 97% identical to one another, and those of clone c and d are 94% identical. ORFs are indicated by open boxes. Dotted boxes with black triangles symbolize long terminal repeats (LTRs). The TSI-A 5′ extension clones have stop codons (indicated by stars) within their ORFs. (B) Position of TSI sequences on BAC F7N22 (GenBank accession number AF058825) and Athila (Pélissier et al., 1995). The TSI region of BAC F7N22 shares 54% identity with the corresponding region of the retrotransposon Athila. The ORF sequence of BAC F7N22 is interrupted by numerous deletions and insertions marked by triangles (closed for deletions; open for insertions). The transcription start is indicated by a flag with an open triangle. The hatched box between BAC F7N22 and Athila represents the region used for the sequence comparison shown in Figure 6. aa, amino acid; PBS, primer binding site for tRNA priming; PPT, polypurine track. Arrows indicate primers used for 5′/3′ extension reactions (TA-R1 and TA-R2, TA-F1 and TA-F2) and RT-PCR (TS-F2 and TS-R1 for determination of the TSI transcription start; GS-F and GS-R for detection of Athila transcripts). Black boxes indicate fragments used as probes for hybridization.

Figure 6.
Figure 6.

Relationships between TSI Transcripts Isolated from a mom1 cDNA Library and Their Potential Genomic Templates. The comparison is based on a 400-bp sequence within the 3′ noncoding region of TSI (indicated in Figure 5). The numbers indicate the number of cDNA clones with identical sequence. The bar represents 10 substitutions per 100 residues.

Figure 7.
Figure 7.

RNase A/T Protection Assay to Map the 5′ Transcription Start. Ten micrograms of total RNA of ddm1, som7, mom1, and yeast were hybridized to an antisense RNA probe spanning the region of the transcription start as determined by RT-PCR experiments. The protected fragment had a size of ∼480 nucleotides in all mutants assayed. Molecular sizes are indicated (in nucleotides; nt) next to the gel.

Figure 8.
Figure 8.

TSI-A Expression in Suspension Culture. Ten micrograms of total RNA from suspension culture and from wild-type seedlings was loaded and hybridized to the TSI-A probe.

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