The Plant Cell, Vol. 12, 1165–1178, July 2000, www.plantcell.org © 2000 American Society of Plant Physiologists

Endogenous Targets of Transcriptional Gene Silencingin Arabidopsis

Andrea Steimer,

a,1

Paolo Amedeo,

a

Karin Afsar,

a

Paul Fransz,

b

Ortrun Mittelsten Scheid,

a

and Jerzy Paszkowski

a

a

Friedrich Miescher Institute, P.O. Box 2543, CH-4002 Basel, Switzerland

b

Institute of Plant Genetics and Crop Plant Research, Corrensstrasse 3, 06466 Gatersleben, Germany

Transcriptional gene silencing (TGS) frequently inactivates foreign genes integrated into plant genomes but very likelyalso suppresses an unknown subset of chromosomal information. Accordingly, RNA analysis of mutants impaired in si-lencing should uncover endogenous targets of this epigenetic regulation. We compared transcripts from wild-type Ara-bidopsis carrying a silent transgene with RNA from an isogenic transgene-expressing TGS mutant. Two cDNA cloneswere identified representing endogenous RNA expressed only in the mutant. The synthesis of these RNAs was found tobe released in several mutants affected in TGS, implying that TGS in general and not a particular mutation controls thetranscriptional activity of their templates. Detailed analysis revealed that the two clones are part of longer transcriptstermed TSI (for transcriptionally silent information). Two major classes of related TSI transcripts were found in a mutantcDNA library. They are synthesized from repeats present in heterochromatic pericentromeric regions of Arabidopsis

chromosomes. These repeats share sequence homology with the 3

9

terminal part of the putative retrotransposon

Athila

. However, the transcriptional activation does not include the transposon itself and does not promote its move-ment. There is no evidence for a general release of silencing from retroelements. Thus, foreign genes in plants encoun-ter the epigenetic control normally directed, at least in part, toward a subset of pericentromeric repeats.

INTRODUCTION

An appropriate balance between activation and repressionof genetic information is intrinsic to any living cell. Tight con-trol of gene expression is necessary for adaptation to envi-ronmental factors, regulation of physiological parameters,and development of specialized cell types within a multicel-lular organism. Differentiation, in particular, is the result ofmitotically heritable changes in gene expression, where theacquired states of gene activity reach a certain epigeneticstability. This stability arises by strict control of gene activa-tors, by regulation of transcript stability, by altering the tran-scriptional availability of the genetic information itself, or bysome combination of these. Alteration of the transcriptionalavailability of genetic information may be based on stableepigenetic silencing of selected genetic loci. This type ofsilencing is also evident from unexpected repression oftransgene activities in various experimental systems (re-viewed in Depicker and Van Montagu, 1997; Stam et al.,1997; Henikoff, 1998; Vaucheret et al., 1998; Grant, 1999).

The silencing of transgenic loci has received particular at-

tention in plants, because it reduces the reliability of transgenicapproaches in biotechnology. Complex inserts containingrearranged multiple copies of a foreign DNA are particularlyprone to gene silencing. Loss of transgene expression mayoccur by two different mechanisms. The first precludes tran-scription (transcriptional gene silencing, or TGS); the secondtargets selected transcripts for rapid degradation (post-tran-scriptional gene silencing, or PTGS). The triggers of the twoprocesses appear similar, because the onset of both typesof silencing correlates well with redundancy of genetic infor-mation: DNA repeats in the case of TGS (reviewed in Meyer,1996; Henikoff, 1998) and internal RNA repeats or overpro-duction in PTGS (reviewed in Depicker and Van Montagu,1997; Grant, 1999). PTGS events are not meiotically trans-mitted and need to be reestablished in each sexual genera-tion. PTGS is not necessarily coupled with a modification ofthe DNA template, but increased amounts of DNA methyla-tion within the protein-coding region of silenced genes havebeen observed (reviewed in Depicker and Van Montagu,1997). In contrast, TGS is meiotically heritable and corre-lates well with DNA template modification, as manifested byhypermethylation of the promoters of silenced genes (re-viewed in Finnegan et al., 1998), by local changes in chro-matin structure (reviewed in Gregory and Hoerz, 1998), orboth.

1

To whom correspondence should be addressed. E-mail andrea.steimer@fmi.ch; fax 41-61-697-3976.

1166 The Plant Cell

The majority of silencing studies with various plant sys-tems have explored transgenes as indicators of the silencingprocess. Few examples of gene silencing do not involvetransgenic loci. A small gene family in Arabidopsis encodingphosphoribosylanthranilate isomerase (PAI), an enzyme inthe tryptophan biosynthetic pathway, is subject to transcrip-tional regulation of its activity (Bender and Fink, 1995). ThePAI gene family in the ecotype Wassilewskija consists offour gene copies distributed between three chromosomallocations. In a mutant with two linked copies deleted, thelow transcription level of the remaining two ectopic genesleads to the accumulation of a UV-fluorescent precursor oftryptophan. Frequent spontaneous activation of these “inef-ficient” genes, seen as nonfluorescent revertants, occurs at1 to 5% per generation.

A further example of TGS of an endogenous gene wasdiscovered during a search for novel mutant alleles of the

SUPERMAN

gene (Jacobsen and Meyerowitz, 1997). Sev-eral new alleles turned out be the result of epigenetic silenc-ing of the wild-type gene. A similar phenomenon was notedin nature, where an epimutation of the

Lcyc

gene alteredflower morphology (Cubas et al., 1999). These examples ofthe effects of TGS on endogenous loci indicate its impor-tance in the regulation of selected genes; however, the crite-ria for TGS target susceptibility are still poorly understood,and the natural targets of transcriptional silencing in a nor-mal, wild-type plant have not been discovered. Althoughsome have postulated that TGS in plants has a defensefunction against invasive DNA such as transposable ele-ments (Matzke et al., 1996; Martienssen, 1998; Matzke andMatzke, 1998), the experimental data relating to this hypoth-esis are limited (Hirochika et al., 2000).

To determine the natural targets of transcriptional silenc-ing and thus explore the role of TGS in plants, we examineda set of transcriptional silencing mutants isolated previously.Because these mutations affect the maintenance of TGSand are able to reactivate diverse transcriptionally silenttransgenic loci (Finnegan et al., 1996; Furner et al., 1998;Mittelsten Scheid et al., 1998; Jeddeloh et al., 1999;Amedeo et al., 2000), it has been hypothesized that the mu-tations also reactivate endogenous chromosomal informa-tion that otherwise would be transcriptionally silent. Here,we report the discovery of genetic information under thecontrol of TGS that is expressed in silencing mutants but si-lenced in wild-type plants.

RESULTS

Isolation of Transcriptionally Silent Information

Usually, strains that release TGS accumulate developmentalabnormalities with time, probably because of the ectopicexpression or suppression of various genes that influence

fitness and development (Finnegan et al., 1996; Kakutani etal., 1996). To circumvent a laborious selection against sucha background of activated subsidiary genes, we chose the

mom1

mutant as experimental material. Its normal growthand development over several generations indicate that the

mom1

mutation acts in a relatively specific manner, its reac-tivation being confined to only a subset of transcriptionallysilent chromosomal information, which we aimed to define.Details of

mom1

are described elsewhere (Amedeo et al.,2000).

Populations of mRNA from 2-week-old Arabidopsis seed-lings of the parental line A carrying a silent hygromycin-resistance locus (Mittelsten Scheid et al., 1991) and its mu-tant derivative

mom1

with the transgenic locus reactivatedwere compared by using the method of suppression sub-tractive hybridization (Diatchenko et al., 1996), a procedurethat should enrich for RNA species present only in

mom1

plants or present in greater abundance in

mom1

plants. Thesubtracted cDNA library was screened by inverted RNA gelblot analysis (von Stein et al., 1997). Twelve of 500 cDNA clonesselected initially showed increased abundance when hybrid-ized to labeled

mom1

cDNA. Direct RNA gel blot analysiscomparing total RNA of the parental line A and the mutantline revealed a striking genotype-dependent differential ex-pression for two of the 12 cDNA probes. These cDNA cloneswere named TSI-A and TSI-B (for transcriptionally silent in-formation). Both were abundant in

mom1

RNA but undetect-able in line A (Figure 1A). Results of RNA run-on analysis inisolated nuclei showed that TSI was transcriptionally si-lenced in line A and wild-type plants (data not shown).

Expression of TSI in Other Genotypes AffectingGene Silencing

To exclude the possibility that release of TSI silencing is re-stricted to

mom1

, several Arabidopsis mutants and trans-genic strains affected in this epigenetic regulation wereassayed. Impaired maintenance of transcriptional silencingwas also assigned to eight

som

mutants (

som1

to

som8

)(Mittelsten Scheid et al., 1998), based on the release of si-lencing from an inactive transgenic locus as in

mom1

. In-deed, all

som

mutants showed TSI expression (Figure 1B).The

ddm1

mutation, identified to have decreased DNA meth-ylation (Vongs et al., 1993), also releases TGS from differentloci (Jeddeloh et al., 1998; Mittelsten Scheid et al., 1998).

som4, -5, -7,

and

-8

are alleles of

ddm1

(Jeddeloh et al.,1999), and TSI expression was also found in this mutant, asexpected (Figure 1B). TSI was also derepressed in a trans-genic line having a decreased amount of DNA methylationas a result of overexpression of DNA methyltransferase anti-sense mRNA (Finnegan et al., 1996) and in the

ddm2

mutant(affected in the maintenance DNA methyltransferase gene;E. Richards, personal communication). The silencing mutants

hog1

and

sil1

(Furner et al., 1998) also expressed TSI (Figure1B). Mutant

sil2

showed no sign of TSI expression; however,

Endogenous Targets of Gene Silencing 1167

sil2

was identified on the basis of a rather subtle release-of-silencing phenotype (Furner et al., 1998).

TSI-A hybridization of RNA gel blots with total RNA pre-pared from 2-week-old seedlings of different mutants visual-ized four major transcripts of

z

5000, 4700, 2500, and 1250nucleotides (Figure 1). The TSI-B probe mainly detected twotranscripts of 5000 and 2500 nucleotides (Figure 1A). Inter-

estingly, the polyadenylated RNA fraction contained only the5000- and 2500-nucleotide transcripts that hybridized toboth cDNA probes (TSI-A and TSI-B) (Figure 1A). The TSI ex-pression pattern was heritable and persisted through severalselfed generations of the mutants (data not shown). Com-parison of patterns of TSI-A expression revealed minor geno-type-specific differences in the stoichiometry of the different

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 mg of total RNA and 0.1 mg of poly(A) RNA from2-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 mg 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 Scheidet 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 ofMETas; METas, transgenic for DNA methyltransferase antisense gene (Finnegan et al., 1996); Col, parental wild type of ddm1 (ddm1 and ddm2are mutants with decreased DNA methylation; Vongs et al., 1993).

1168 The Plant Cell

RNA species (Figure 1B). However, it is conceivable thatgeneral features of TSI transcripts are similar in all TGS mu-tants currently available.

Organization of Chromosomal TSI Templates

To determine the source of TSI transcripts and the organiza-tion of their template(s), TSI-A and TSI-B were used asprobes for DNA gel blot analysis. The blots revealed thatmultiple copies of TSI-A– and TSI-B–homologous se-quences are present in the genome of Arabidopsis (Figure2). The copy number of TSI-A, as assessed by reconstruc-tion experiments, was

z

200.To examine the degree of evolutionary conservation of the

TSI arrangements, the DNAs of five Arabidopsis ecotypes(Zürich, Columbia, Landsberg

erecta

, Wassilewskija, andC24) were compared by DNA gel blot analysis and by hy-bridization to a TSI-A probe terminated at the DraI site,which has two recognition sequences within TSI-A (Figure2A). An important conservation of the TSI-A pattern amongdifferent ecotypes was observed, with two main DraI re-peats of 4 and 1.3 kb. Some minor differences specific for aparticular ecotype indicated a limited genetic polymorphismwithin TSI-A. Probing the same membrane with TSI-B re-vealed complex banding patterns with differences in eachecotype (Figure 2A), which might indicate a much lower de-gree of conservation for TSI-B. However, the differences inDNA gel blot patterns between TSI-A and TSI-B can also beexplained if TSI-A is an internal part of a longer repeated el-ement and TSI-B is located proximal to a flank between re-peated elements and variable single-copy DNA regions.

TSI repeats were heavily methylated in wild-type Arabidop-sis and line A (Figure 2B); however, they were less methyl-ated in mutants with genome-wide decreased DNA methylation(Figure 2B and data not shown). Importantly, TSI sequencesremained methylated in the

mom1

mutant (Figure 2B), inspite of their expression. This is analogous to the mainte-nance of methylation at the reactivated transgene in the

mom1

background (Amedeo et al., 2000).TSI-A and TSI-B were hybridized to the CIC yeast artificial

chromosome library covering four equivalents of the Arabi-dopsis genome (Creusot et al., 1995). Sixty-two CIC clonesout of 1152 were positive with the TSI-A probe. Twenty-sixof these also contained the pericentromeric 180-bp repeat(Martinez-Zapater et al., 1986; Maluszynka and Heslop-Harrison, 1991), seven contained 5S RNA genes known tobe located in the vicinity of a centromere (Bevan et al.,1999), and 16 clones contained other markers that mappedclose to centromeres. Only four positive clones mappedoutside of centromeric regions. Mapping of TSI-B resulted inhybridization to all TSI-A–positive CIC clones, with an addi-tional seven clones hybridizing to TSI-B only. Thus, both TSIrepeats are concentrated in the pericentromeric regions ofArabidopsis chromosomes. This was confirmed by cytologi-cal analysis of pachytene chromosomes with fluorescent in

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 z5 mg of DNA from vari-ous ecotypes (Zh, Zürich; Col, Columbia; Ler, Landsberg erecta; Ws,Wassilewskija; and C24) digested with DraI. The blot was hybridizedto a TSI-A probe (left) flanking the DraI site (see map below the gel)and rehybridized to TSI-B (right). Differences in hybridization inten-sity are the result of loading differences. DNA fragment lengths areindicated in kilobases.(B) TSI methylation states in different mutants. DNA gel blot analysiswas performed with z5 mg of DNA (Zh, Zürich; line A, parental lineof som5 and mom1; som5 and mom1 silencing mutants) digestedwith the methylation-sensitive restriction enzyme HpaII (left) and themethylation-insensitive restriction enzyme DraI (right) as a control.

Endogenous Targets of Gene Silencing 1169

situ hybridization (FISH). These chromosomes are highly ex-tended and span on average 66

m

m (Fransz et al., 1998).TSI-A hybridization signals were found in the pericentro-meric heterochromatin of all chromosomes (Figures 3A to3C). A similar position was found for TSI-B sequences.Close examination revealed their presence in the hetero-chromatic domains flanking the centromeric 180-bp tandemrepeat region. These regions are known to contain a highdensity of dispersed repeated elements (Copenhaver et al.,1999).

Multicopy TSI Templates AreTranscribed Unidirectionally

To determine the polarity of TSI transcription, RNase T andRNase A/T protection assays were performed with two TSI-Aprobes of opposite polarity. TSI-A was chosen as a probebecause it is present in all transcripts detected on RNA gelblots (Figure 1). There was no evidence for specific protec-tion of any TSI antisense RNA (Figure 4), suggesting a unidi-rectional transcription of the TSI templates. RNase digestionscreated a complex pattern of TSI-A–protected bands (Figure4), which implies that many related but different RNAs hy-bridized to the probe. Furthermore, some of the protectedTSI-A fragments are clearly more abundant than others,pointing toward either structural conservation of particularregions of TSI-A within related RNAs or a greater abundanceof certain transcript subspecies.

Characterization of Transcripts Encoding TSI-Aand TSI-B

The cDNA clones TSI-A and TSI-B had sizes of 903 and 614bp, respectively. From the sizes of TSI transcripts detectedon the RNA gel blots (Figure 1), it was obvious that both TSIclones represent only partial cDNAs. Because only two tran-scripts (5000 and 2500 nucleotides) were detected in thepolyadenylated RNA fraction, this RNA was used for 5

9

and3

9

extension reactions that started from the TSI-A sequence(Figure 5A). Two clones each were analyzed at the level ofnucleotide sequences. The 5

9

extension yielded inserts of2512 and 1997 bp that were 97% identical in nucleotide se-quences. The two clones from the 3

9

extension were 1682and 1652 bp and had 94% identity. Interestingly, both 3

9

ex-tension clones of TSI-A contained a region of 568 bp closelyrelated to TSI-B (77% identity). This explains the detection ofsimilar RNA species on RNA gel blots with TSI-A and TSI-B asprobes and suggests that TSI-A and TSI-B are part of the samepolyadenylated transcript species (Figure 1). To test whether the5

9

extensions of TSI-A are indeed part of the TSI transcript,RNA gel blots were probed with a cDNA fragment close tothe 5

9

end of the extension (Figure 5A, probe TSI-C). Interest-ingly, this probe hybridized only to the largest transcript of

Figure 3. FISH Images of Pachytene Chromosomes of Landsbergerecta Hybridized to TSI-A and the 180-bp Repeat.

(A) 49,6-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 thepericentromeric region of all chromosomes.Bar in (A) 5 5 mm for (A) to (C).

1170 The Plant Cell

5000 nucleotides, which is also present in the poly(A) frac-tion (data not shown). Because this transcript class also hy-bridized to TSI-A and TSI-B, the 5000-nucleotide transcriptsprobably contain a particular order of all three sequence ele-ments (TSI-C, TSI-A, and TSI-B) (Figure 5).

The results of the RNase protection assay and the markedsequence heterogeneity between the duplicates of the 5

9

and 3

9

extensions of TSI-A indicated that they are tran-scribed from different but related DNA sequences. To deter-mine the composition of TSI transcripts, a cDNA libraryprepared from

mom1

seedlings was screened with two

probes (TSI-A or TSI-B) (Figure 5). Among 5

3

10

5

clonesscreened, 179 were positive with at least one probe. Impor-tantly, 94 clones hybridized to both TSI-A and TSI-B, con-firming the 3

9

TSI-A extension data (Figure 5). Twentyrandom cDNAs averaging 1.7 kb in size were subjected tosequencing. The sequence comparison revealed two majortranscripts, represented by seven and six cDNAs with iden-tical sequences within the overlapping regions (Figure 6).The remaining cDNAs were unique and probably representtranscripts of lesser abundance. Interestingly, the cDNAs fellinto two classes, each showing sequence identity

.

85%among its members but

,

70% identity between the classes(Figure 6).

A search within an Arabidopsis genomic sequence data-base uncovered a chromosomal DNA stretch (bacterial arti-ficial chromosome [BAC] F7N22) identical to one of theabundant cDNAs (Figure 6). This sequence, annotated as

Athila

repeat, is located at the pericentromeric region ofchromosome 5 and was recently used in comparative stud-ies of various

Athila

-like repeats (Wright and Voytas, 1998).

Athila

was isolated in a search for sequences flanking the180-bp repeat; it is classified as a retrotransposon based onthe presence of long terminal repeats, a polypurine track,and a primer binding site for tRNA priming of the reversetranscriptase (Pélissier et al., 1995, 1996). However, the twoopen reading frames (ORF) of

Athila

(Figure 5B, ORF1 andORF2) have no homology with proteins usually encoded byretroelements or any other known protein, and neithermovement nor even transcriptional activity has ever beenobserved.

Both TSIs and all sequenced cDNA clones mapped to the3

9

terminal half of the

Athila

element (Figures 5A and 5B). AllTSI regions homologous to the

Athila

ORF2 appear to bedegenerated coding regions: the ORFs encoded by the twocDNAs derived from the 5

9

extensions of TSI-A are inter-rupted by translational stop codons after 398 (clone a) and83 (clone b) amino acids. The sequence corresponding toORF2 present on the BAC F7N22 is modified by five dele-tions of 2 to 31 bp and five insertions of 3 to 10 bp (Figure5B). This indicates significant deviation of these sequencesfrom that of the putative retrotransposon.

The sequence information of BAC F7N22 was used to de-termine the position of the transcription start for the longestTSI transcript. First, reverse transcription–polymerase chainreaction (RT-PCR) amplifications with primers spanning theregion of the putative start site as expected from the largesttranscript size were performed (Figure 5B). Results indicatedthat transcripts start downstream of position 64,929. Ac-cordingly, an antisense RNA probe for RNase A/T protectionwas produced spanning the 638 nucleotides between posi-tions 64,929 and 65,567. Protected fragments of

z

480

6

10nucleotides (Figure 7) allowed positioning of the TSI tran-scription start on BAC F7N22 to 65,087

6

10 nucleotides.Because RNA preparations from

ddm1

,

som7

, and

mom1

protect fragments of the same size (Figure 7), TSI transcriptsstart at similar sites in different mutant backgrounds.

Figure 4. RNase Protection Assay with Strand-Specific Probes ofTSI-A.

Ten micrograms of total RNA of mom1, the parental line A, or yeastwas hybridized to antisense (AS) or sense (S) TSI-A probes. Unpro-tected single-stranded RNA was cleaved by RNase A and RNase T,or RNase T only, as indicated. The integrity of the RNA probes wasshown in samples without RNase. nt, nucleotides of the molecularsize marker.

Endogenous Targets of Gene Silencing 1171

Selectivity of TSI Activation

The TSI identified in the differential screen and the clonesrecovered from the cDNA library were homologous to onlythe 39 half of the Athila-like element, and the transcriptionstart of the unidirectional transcripts was mapped to themiddle of the putative retroelement. Therefore, we examinedwhether the 59 part of the Athila-like sequence present onBAC F7N22 (including the first ORF; Figure 5) was reacti-vated in TGS mutants. RNA gel blot analysis with a probespanning a 1.6-kb region of ORF1, as well as RT-PCR analy-sis with primers from the same region, gave negative results(data not shown). This suggests that expression of TSI is re-stricted to the 39 half of Athila-like sequences. In addition,we failed to detect extrachromosomal DNA indicative of thereplication of retroelements (Peleman et al., 1991), suggestingthat TSI sequences are not part of a replicative transposon.

The two classes of isolated cDNAs shared only z50%identity with Athila (Figure 6). To address directly the ques-tion of whether Athila is expressed, RT-PCR experiments

were performed with Athila-specific primers in the TSI ho-mologous region (Figure 5). However, the correspondingfragment could not be amplified from RNA of mom1 seed-lings, suggesting that only a subset of Athila-like sequencesbut not the Athila element itself was reactivated in the mu-tant background.

The homology of TSI to the putative retrotransposon alsoraised the question of whether other endogenous retroelementsare transcriptionally reactivated. Reverse transcriptase is themost highly conserved protein encoded by retroviruses andretrotransposons (Doolittle et al., 1989). The same degener-ated primers in a conserved region of the reverse tran-scriptase gene that were applied to clone the Ta superfamilyof Arabidopsis retrotransposons (Konieczny et al., 1991) wereused to investigate whether members of the Ta family are tran-scribed in the mutant background. Although the expected268-bp fragment was amplified from genomic DNA, no amplifi-cation was achieved in RT-PCR with mom1 RNA as template(data not shown). Thus, retrotransposons are not generally re-activated in the mom1 mutant.

Figure 5. Organization of TSI Transcripts, BAC F7N22, and Athila.

(A) TSI-A and the clones obtained by 59 extension (clones a and b) and by 39 extension (clones c and d). The nucleotide sequence of clones aand 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 withblack triangles symbolize long terminal repeats (LTRs). The TSI-A 59 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 BACF7N22 shares 54% identity with the corresponding region of the retrotransposon Athila. The ORF sequence of BAC F7N22 is interrupted by nu-merous deletions and insertions marked by triangles (closed for deletions; open for insertions). The transcription start is indicated by a flag withan 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 59/39 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 ofAthila transcripts). Black boxes indicate fragments used as probes for hybridization.

1172 The Plant Cell

The repetitive nature of TSI and its pericentromeric loca-tion led to the question of whether other pericentromeric re-peats are expressed in TGS mutants. Athila sequences(Pélissier et al., 1995, 1996) are often flanked by copies ofthe 180-bp repeat (Martinez-Zapater et al., 1986), and tran-scripts could originate from read-through transcription. Notranscripts homologous to these repeats were detectable byRNA gel blot analysis (data not shown). Thus, the release ofsilencing appears to affect selected templates only. Whatdistinguishes these targets from other, apparently relatedrepeats remains to be investigated.

Fast-Growing Suspension Culture Cells Express TSI

TSI expression in the wild-type background was not de-tected either in young seedlings (2 weeks old) or in differenttissues of mature wild-type plants (roots, shoots, leaves,flowers, and siliques). The application of various stresstreatments (increased salinity, UV-C irradiation, or pathogeninfection) also did not activate TSI in wild-type plants (datanot shown). Furthermore, TSI expression was not detectedin freshly initiated callus cultures, and transcriptional sup-pression of TSI was stable even after several in vitro pas-sages of the callus culture. The only exception so far is afast-growing, long-term suspension culture derived fromwild-type Arabidopsis (May and Leaver, 1993). These cellsexpressed TSI to approximately the same extent as the mu-

tants (Figure 8), indicating the release of epigenetic TSI si-lencing under these conditions.

DISCUSSION

The epigenetic control of gene activity in plants can be ex-erted either in a sequence-specific manner, for example, bypolycomb-type proteins (Goodrich et al., 1997), or in abroader way through regional changes in DNA methylationor chromatin properties (reviewed in Richards, 1997). TGSseems to reflect the latter, more global type of epigeneticcontrol (Chen and Pikaard, 1997; Jeddeloh et al., 1998;Mittelsten Scheid et al., 1998). However, even a general sys-tem requires other, probably sequence-independent meansto recognize its targets. A trait suspected to trigger silencing isgenetic redundancy in the form of duplications and repetitivestructures (reviewed in Meyer, 1996; Selker, 1997; Henikoff,1998). Indeed, genetic modifier loci of transcriptional silenc-ing have been identified by their action on a highly repetitivetransgenic locus (Mittelsten Scheid et al., 1998), and someof them were shown to be allelic to the ddm1 mutation(Jeddeloh et al., 1999), which acts primarily and immediatelyon the methylation status of the abundant 180-bp centro-meric repeat (Vongs et al., 1993). Although the activity ofseveral low-copy genes also can be affected by disruptionof genome-wide methylation patterns (Jacobsen and

Figure 6. Relationships between TSI Transcripts Isolated from amom1 cDNA Library and Their Potential Genomic Templates.

The comparison is based on a 400-bp sequence within the 39 non-coding region of TSI (indicated in Figure 5). The numbers indicatethe number of cDNA clones with identical sequence. The bar repre-sents 10 substitutions per 100 residues.

Figure 7. RNase A/T Protection Assay to Map the 59 TranscriptionStart.

Ten micrograms of total RNA of ddm1, som7, mom1, and yeastwere hybridized to an antisense RNA probe spanning the region ofthe transcription start as determined by RT-PCR experiments. Theprotected fragment had a size of z480 nucleotides in all mutantsassayed. Molecular sizes are indicated (in nucleotides; nt) next tothe gel.

Endogenous Targets of Gene Silencing 1173

Meyerowitz, 1997; Jeddeloh et al., 1998), it would be inap-propriate to consider them as primary targets for methyla-tion-mediated epigenetic control, given the stochasticnature and slow progress of silencing. Their altered activityis more likely to be a secondary effect of the accumulationof an epigenetic disorder in the mutant background (Finneganet al., 1996; Kakutani et al., 1996; Jeddeloh et al., 1999). Theprimary endogenous targets of the transcriptional silencingsystem were not known previously, and there was no evi-dence for expression of the demethylated 180-bp repeats inthe mutants (Vongs et al., 1993; Kakutani et al., 1999). Thesearch for plant-specific TGS targets described in this arti-cle has now disclosed a class of repetitive elements as be-ing more promising candidates.

The TSI present in the genome of wild-type plants wasstimulated to produce RNA in a range of mutants that are af-fected in the maintenance of transcriptional silencing. In aninitial search for TSI, we isolated two independent cDNAclones representing RNA specifically expressed in silencingmutants. Because one would anticipate that there are sev-eral DNA templates suppressed by the silencing system inwild-type plants, we find it remarkable that the correspond-

ing cDNAs are closely related to each other or are evenparts of the same transcript. This may indicate that the TSIrecovered here represents a tightly regulated template withno background expression in the wild type, which is thus anoptimal substrate for suppression cloning. Alternatively, theTSI described could be a very efficiently transcribed tem-plate or templates in the absence of the silencing control. Inany case, the expression of TSI seems to be a very specificmarker of disturbed silencing.

The three main TSI transcripts of 5000, 2500, and 1250nucleotides, originating from unidirectional transcription,contain an element isolated as TSI-A. It is not clear whetherthese represent separate transcriptional units regulated bydifferent promoters or whether they are processing productsof the longest transcript. The 5000- and 2500-nucleotidetranscripts are polyadenylated, but the most abundant tran-script of 1250 nucleotides is absent from the poly(A) fractionand thus possibly is retained in the nucleus. The most abun-dant transcripts have no protein-coding capacity, and onlythe longest mRNA includes sequences related to the ORF2of the putative retrotransposon Athila. However, all TSI se-quences analyzed so far contain only degenerated regionsof ORF2. Therefore, TSI probably belongs to a class of non-coding RNA.

Sequence analysis of 20 randomly chosen TSI cDNAclones derived from mom1 RNA indicated overrepresenta-tion of two transcripts and the presence of several lessabundant but related RNAs. Because no two identical TSIrepeats were found in the Arabidopsis genomic sequenceavailable (covering .80% of the genome), probably variousTSI templates are transcribed with differing efficiencies orperhaps selected TSI transcripts have increased stability.

No putative function for TSI was revealed by sequencecomparison with protein- or RNA-coding sequences. Theonly similarity, limited to z50%, was found with the 39 halfof the putative degenerated retrotransposon Athila (Pélissieret al., 1995, 1996; Wright and Voytas, 1998). The 59 part ofAthila directly adjacent to the TSI template region was notreactivated in the silencing mutant. Furthermore, not allAthila copies are reactivated in the mutant background, andno reactivation of other Arabidopsis retroelements, for ex-ample, the Ta superfamily (Konieczny et al., 1991), was de-tected. This suggests that TGS is not directed towardretrotransposons in general, although its targets may haveoriginated from ancient transposition events. Recently, ahigh selectivity of TGS also in respect to transposon targetshas been revealed (Hirochika et al., 2000).

So far, and based on the complete sequence of two Ara-bidopsis chromosomes (Lin et al., 1999; Mayer et al., 1999),Athila elements were found only in heterochromatic regionsof chromosome 4 (Mayer et al., 1999; Fransz et al., 2000;McCombie et al., 2000). However, none of the TSI cDNAscould be aligned to the sequenced chromosomes withoutnumerous mismatches. Therefore, the presence of TSI tem-plates within chromosome 2 and 4 is still unclear, and thetwo most abundant cDNAs must be transcribed from the

Figure 8. TSI-A Expression in Suspension Culture.

Ten micrograms of total RNA from suspension culture and fromwild-type seedlings was loaded and hybridized to the TSI-A probe.

1174 The Plant Cell

remaining chromosomes. Indeed, the template for one ofthem was found on BAC F7N22 located on chromosome 5.

Repeats derived from degenerated retroelements have of-ten been found in centromeric locations in fungi and plants(Cambareri et al., 1998; Presting et al., 1998; Copenhaver etal., 1999). Whether these repeats are important componentsof centromeric function, either by nature of their sequencesor by their epigenetic modifications, remains a matter for dis-cussion (reviewed in Csink and Henikoff, 1998; Copenhaverand Preuss, 1999). One of the features proposed as a pre-requisite for centromere function is late replication of these het-erochromatic regions (reviewed in Csink and Henikoff, 1998). Ifthis was also true for Arabidopsis centromeres, undue loos-ening of suppressive chromatin in the pericentromeric re-gion leading to TSI expression could cause disturbances inmitosis, which would result in severe phenotypes (Liu andMeinke, 1998). However, sil1 and mom1 mutant plants ex-hibit no abnormalities suggestive of mitotic disorders. It ispossible that TSI is transcribed in spite of the presence ofthe suppressive chromatin environment, as was docu-mented for the Xist locus transcribed from the inactive Xchromosome (Gartler et al., 1999); thus, its activation doesnot interfere with pericentromeric functions. Importantly, notonly strains affected in transcriptional silencing through al-terations in genome-wide DNA methylation but also silenc-ing mutants with unchanged methylation levels (sil1 andmom1) reactivate TSI. This indicates that release of epige-netic suppression from endogenous templates does not re-quire the loss of methylation. Because the latter mutantsexhibit no striking phenotypic alterations, release of TGSand TSI expression per se is not responsible for the multipleand variable morphological and developmental aberrationsseen in methylation-affected mutants. Thus, at this point it isboth evident and surprising that transcriptional reactivationof a subset of usually silent pericentromeric repeats, such asdescribed here, has no immediate consequences. There-fore, their silencing may be important under specific, unde-fined conditions or on a longer time scale. Speculationabout the biological role of TSI and the significance of itsepigenetic suppression system would be premature. How-ever, the epigenetic control of TSI resembles that of othernoncoding RNAs and retroelements with better-definedfunctions, such as Xist involved in X chromosome inactiva-tion (reviewed in Marahrens, 1999) or TART and HeT-A withtelomeric function (reviewed in Pardue et al., 1997). More-over, the TSI expression observed in suspension culturesuggests that long-term, fast growth of dedifferentiated cellscan select for an escape from epigenetic control. Such epi-genetic imbalance could be the main origin of somaclonalvariation (Phillips et al., 1994) and activation of transposableelements (Bretell and Dennis, 1991; Hirochika, 1993; Hirochikaet al., 1996) during prolonged plant tissue culture.

The recognition signals designating a particular set of en-dogenous repeats or transgenes for silencing are still un-known. The affected sequences described here probablyrepresent only the tip of an iceberg, and the discovery of fur-

ther TSI elements should help resolve the role and mecha-nism of transcriptional silencing in plants. At present, theperfect correlation of TSI expression in all mutants known toaffect transcriptional transgene silencing promotes thesemutants to the status of powerful icebreakers. Results of theongoing characterization of the mutated silencer compo-nents will help define their interaction with their targets. Fur-thermore, TSI itself provides a potent tool for uncoveringnovel genes involved in TGS, either in known mutants or intagged mutant populations.

METHODS

Plant Material

Arabidopsis thaliana seeds were surface-sterilized twice with cal-cium hypochlorite (5% with 0.1% Tween 80) for 5 min and washedwith sterile double-distilled water. They were then plated in asepticconditions on germination medium (Masson and Paszkowski, 1992)solidified with 0.8% agar. Seedlings were grown in a phytotron withcycles of 16 hr of light at 218C and 8 hr of darkness at 168C.

Differential mRNA Screening and Cloning Procedures

Total RNA was isolated according to Goodall et al. (1990) from 2 g(fresh weight) of 2-week-old seedlings from the mutant mom1(Amedeo et al., 2000) and the parental line A (Mittelsten Scheid et al.,1991). Polyadenylated RNA was prepared by using Dynabeads Oligo(dT)25 (Dynal, Oslo). Suppression subtractive hybridization (Diatchenkoet al., 1996) was performed with 2 mg of poly(A) RNA, using the PCR(polymerase chain reaction)-Select cDNA subtraction kit (Clontech,Palo Alto, CA) according to the supplier’s instructions. The mutantline was used as tester and the parental line A as driver cDNA popu-lation. The subtracted mom1 library was cloned into vector pCR2.1(Invitrogen, Groningen). For the screening procedure, 500 individualbacterial cultures were grown according to the manual of the PCR-Select differential screening kit (Clontech). To reduce the number offalse-positive clones, the library was also screened as described byvon Stein et al. (1997).

59 and 39 extension reactions were performed with the MarathoncDNA amplification kit (Clontech) according to the manufacturer’sprotocol. The sequence-specific primers (see Figure 2) were 59-TGGTTCACCAGATAAGCTCAGTGCCCTC-39 (TA-F1) and 59-CTT-CAGACTGGATAGGACTAGGTGGGCG-39 (nested primer TA-F2)for the 39-extension reaction, and 59-CGCCCACCTAGTCCTATC-CAGTCTGAAG-39 (TA-R1) and 59-CGCATCAAACAACTAACA-ACGAGGGCAC-39 (nested primer TA-R2) for the 59-extension re-action. PCR products were cloned into vector pCR2.1 (Invitrogen).Individual bacterial cultures were grown and subjected to colonyPCR as described in the manual of the PCR-Select differentialscreening kit (Clontech), with the primer combinations used to cre-ate the extension reactions. To screen for positive TSI-A extensionclones, the PCR products were blotted and hybridized to TSI-A. AllPCR reactions used for cloning procedures were performed with apolymerase mix with proof-reading capacity (Advantage cDNA PCRkit; Clontech).

Endogenous Targets of Gene Silencing 1175

RNA and DNA Gel Blot Analysis and Library Screens

Total RNA was isolated either as described by Goodall et al. (1990) orby the RNeasy plant mini kit (Qiagen, Hilden) according to the sup-plier’s instructions. For RNA gel blot analysis, the RNA was electro-phoretically separated after denaturation by glyoxal in a 1.5%agarose gel in phosphate buffer, pH 7.0, and blotted to nylon mem-branes (Hybond N; Amersham) by using standard protocols (Sambrooket al., 1989). The molecular weight marker I (Roche Diagnostics, Rot-kreuz) was used as a size standard. For DNA gel blot analysis, ge-nomic DNA was isolated according to Dellaporta et al. (1983) andseparated electrophoretically after endonucleolytic digestion. DNAfragments were also transferred to nylon membranes (Hybond N) bystandard procedures (Sambrook et al., 1989). Hybridization andwashing of all RNA and DNA gel blots and the filters with the yeastartificial chromosome library was performed according to Churchand Gilbert (1984). Probes were labeled with a-32P-dATP by randomprime DNA polymerization (Feinberg and Vogelstein, 1983). Blotswere exposed to x-ray–sensitive films (Kodak X-OMAT AR).

For the mom1 cDNA library, polyadenylated RNA was preparedfrom 2-week-old mutant seedlings as described above. The cDNA li-brary was prepared by using the Uni-Zap XR library construction kit(Stratagene) according to the manufacturer’s instructions. LongcDNAs were selected by using size fractionation columns fromGibco BRL.

RNase Protection Assays

The RNase protection assays were performed according to Goodallet al. (1990) with minor modifications. To assay the direction of TSItranscription, the pCR2.1 plasmid with the TSI-A insert was cut byEcoRI, creating a fragment of 781 bp, which was ligated into thepGEM-7Zf(1) vector (Promega). To map the 59 transcription start, theprobe was generated by amplifying the bacterial artificial chromo-some (BAC) F7N22 region between positions 64,929 and 65,567 andinserting the product into the pGEM-7Zf(1) vector (Promega). The la-beled probes were synthesized by in vitro transcription of the linear-ized plasmid in the presence of a-32P-UTP by using T7 polymerase(Promega) or Sp6 polymerase (Roche Diagnostics) and purified byelectrophoresis (Goodall et al., 1990). Hybridization was performedat 528C. Single-stranded RNA was cleaved by either 4 mg of RNase Aand 0.6 units of RNase T1 (RNase A/T assay) or by 20 units of RNaseT1 (RNase T assay) for 40 min at 308C. Protected fragments wereseparated on a denaturing 6% polyacrylamide gel. The dried gelswere recorded on a PhosphorImager screen (Amersham PharmaciaBiotech, Uppsala).

Reverse Transcription–PCR and PCR Reactions

Reverse transcription (RT) was performed with 1 mg of total RNAfrom mom1 in the presence of 1 mM dNTPs, 20 units of RNasin(Promega), AM RTase buffer (Roche Diagnostics), and 25 units of AMreverse transcriptase (Roche Diagnostics) at 378C for 1 hr, followedby heat inactivation of the reaction mixture. The template for PCRwas 100 ng of genomic DNA, 100 ng of cDNA, or 50 ng of reverse-transcribed RNA primed by gene-specific antisense primers. PCRwas started with 3-min denaturation at 948C, followed by 30 cycles(denaturation at 948C for 30 sec, annealing at 628C for 30 sec, andelongation at 728C for 30 sec) in the presence of 0.2 mM dNTPs, 0.4

mM forward and reverse primers in Taq DNA polymerase buffer(Roche Diagnostics), and 0.25 units of Taq DNA polymerase (RocheDiagnostics). The gene-specific primers for Athila (X81801) were de-rived from positions 8156 to 8184 (GS-F) and 8430 to 8458 (GS-R).

The TA-F1 and TA-R1 primers were used as a positive control forall RT-PCR reactions. The reverse transcriptase region of the polgene of Arabidopsis Ty1/copia–like retrotransposon family was am-plified as described by Konieczny et al. (1991) with minor modifica-tions.

Stress Application (Salinity, UV-C, or Pathogen)

Induction of TSI by UV-C irradiation was tested on RNA gel blots withRNA samples from 1-week-old seedlings subjected to UV-C treat-ment of 1 or 5 kJ/m2 and collected at several time points within 1 hr(Revenkova et al., 1999). The effect of osmotic stress was tested onRNA gel blots with RNA from 1-week-old seedlings that were trans-ferred for 24 hr to medium with NaCl concentrations of 0, 0.04, 0.08,or 0.12 M (Albinsky et al., 1999). To test TSI expression in conditionsof pathogen stress, RNA samples from 3-week-old seedlings eithermock-treated or infected with Peronospora (provided by R. Reist,Novartis Crop Protection, Basel) were analyzed by RNA gel blot anal-ysis. To verify the appropriate pathogen response, induction of PR1expression was monitored by reprobing the membrane with a PR1probe (provided by R. Reist).

DNA Sequencing

Sequencing reactions were performed with Dye Terminators (con-ventional rhodamine terminators or dRhodamine terminators) fromPE Applied Biosystems (Foster City, CA) using a Perkin-Elmer Gene-Amp PCR system model 2400, 9600, or 9700 thermocycler and ana-lyzed with an ABI PRISM 377 DNA sequencer.

Cytogenetic Analysis of TSI-A on Pachytene Chromosomes

Flower buds from wild-type Landsberg erecta were fixed in ethanol/acetic acid (3:1 [v/v]), washed in water, and incubated for 2 hr at 378Cwith a combination of 0.3% (w/v) pectolyase (Sigma), 0.3% (w/v) cy-tohelicase (BioSepra, Idstein), and 0.3% (w/v) cellulase (Sigma) in 10mM sodium citrate, pH 4.5, to remove the cell wall (Ross et al., 1996).The pachytene chromosomes were spread as described by Fransz etal. (1998). Pachytene chromosome preparations were baked at 608Cfor 30 min. After incubating with RNase (10 mg/mL in 2 3 SSC [1 3SSC is 0.15 M NaCl and 0.015 M sodium citrate]), the slides wererinsed twice in 2 3 SSC buffer for 5 min and twice in PBS (10 mM so-dium phosphate, pH 7.0, and 143 mM NaCl) for 5 min. The prepara-tions were then fixed in 1% (v/v) paraformaldehyde in PBS for 10min, rinsed twice in PBS for 5 min, dehydrated through an ethanolseries (70, 90, and 100%; each 2 min), and air-dried.

Plasmid DNA with TSI-A or TSI-B was labeled with biotin-dUTP ordigoxygenin-dUTP using the High Primed labeling kit of BoehringerMannheim. The hybridization mix contained z100 ng of labeled DNAin 50% formamide, 2 3 SSC, 50 mM sodium phosphate, pH 7.0, and10% dextran sulfate. Twenty microliters of hybridization mix wasadded to each slide. After denaturation at 808C for 2 min, the slideswere incubated at 378C for 18 hr. Posthybridization washes wereperformed in 50% formamide in 2 3 SSC, pH 7.0, three times for 5min at 428C followed by 2 3 SSC at room temperature three times for

1176 The Plant Cell

5 min. Immunodetection of the hybridization signals was performedaccording to Fransz et al. (1998). The fluorescence patterns were ex-amined with a Zeiss (Jena) Axioplan, using filter blocks for 49,6-dia-midino-2-phenylindole and fluorescein isothiocyanate. Selectedimages were recorded by conventional and charge-coupled devicecameras. The digital images were processed to enhance brightnessand contrast by using Adobe Photoshop (Adobe Systems, USA)computer software.

Database Searches

Sequence analysis was performed with the GCG software (Wiscon-sin Package Version 10.0; Genetics Computer Group, Madison, WI).For homology searches, GenEMBL, the Arabidopsis database (http://genome-www.stanford.edu/), the Kazusa Arabidopsis opening site(http://zearth.kazusa.or.jp/arabi/), and SwissProt (http://www.expasy.ch/sprot/) were used. Peptide sequences were analyzed by Gene-Quiz (http://columba.ebi.ac.uk:8765/gqsrv/submit) or Expasy (http://www.expasy.ch/).

ACKNOWLEDGMENTS

We owe special thanks to Yoshiki Habu for his contribution to themanuscript. We thank Barbara Hohn, Thomas Hohn, Witold Filipowicz,Patrick King, Danielle Conroy, and Diana Pauli for helpful commentson the manuscript. We also thank Eric Richards, Ian Furner, JeanFinnegan, Roland Reist, Doris Albinsky, and Ekaterina Revenkova forproviding biological material. Further, we acknowledge Herbert Angliker,Maciej Pietrzak, and Peter Müller for sequencing and primer service.This work was supported by the Swiss Federal Office for Educationand Science (BBW Grant No. 96.0250-1) and the European Union(EU Grant No. BIO4CT960253).

Received January 24, 2000; accepted May 18, 2000.

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DOI 10.1105/tpc.12.7.1165 2000;12;1165-1178Plant Cell

PaszkowskiAndrea Steimer, Paolo Amedeo, Karin Afsar, Paul Fransz, Ortrun Mittelsten Scheid and Jerzy

Endogenous Targets of Transcriptional Gene Silencing in Arabidopsis

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