HOMOLOGY-DEPENDENT GENE SILENCING IN...

1040-2519/96/0601-0023$08.00 23

Annu.Rev. Plant Physiol. Plant Mol. Biol. 1996. 47:23–48Copyright© 1996by AnnualReviewsInc. All rightsreserved

HOMOLOGY-DEPENDENT GENESILENCING IN PLANTS

P. Meyer

Max-Delbrück-Laboratorium in der MPG, Carl-von-Linné Weg 10, D-50829 Köln,Germany, and University of Leeds, Centrefor Plant Biochemistry & BiotechnologyandDepartment of Genetics, LeedsLS29JT,UnitedKingdom

H. Saedler

Max-Planck-Institut für Züchtungsforschung, Carl-von-LinnéWeg 10,D-50829 Köln,Germany

KEY WORDS: gene silencing, trans-inactivation, paramutation, cosuppression, DNA methyla-tion

ABSTRACT

Homology-dependentgenesilencing phenomena in plants have receivedcon-siderable attention, especially when it was discovered that the presence ofhomologoussequences not only affected the stability of transgene expression,but that the activity of endogenousgenescould be altered after insertion ofhomologoustransgenesintothegenome.Homology-mediatedinactivationmostlikely comprisesat least twodifferent molecular mechanismsthat induce genesilencing at thetranscriptional or posttranscriptional level, respectively. In thisreview we discuss different mechanistic models for plant-specific inactivationmechanismsand their relationship with repeat-specifi c silencingphenomenainother species.

CONTENTS

INTRODUCTION..................................................................................................................... 24HOMOLOGY-DEPENDENT SILENCING PHENOMENA .................................................. 24

Inactivation of HomologousTransgenes............................................................................. 24Paramutation....................................................................................................................... 26Mutual Inactivation of Transgenesand EndogenousGenes............................................... 27Inactivation MediatedbyRNAViruses............................................................................... 29

MODELSFORHOMOLOGY-BASEDSILENCING............................................................. 29SilencingMediatedVia DNA-DNAPairing........................................................................ 30RNA-MediatedModelsfor Silencing................................................................................... 30

CELLULAR MECHANISMS INVOLVED IN HOMOLOGY-DEPENDENTSILENCING..................................................................................................................... 32

TheRegulatory Functionof Chromatin States.................................................................... 33Posttranscriptional Control of GeneExpression ................................................................ 37Repeat-Specific Control Mechanisms.................................................................................. 39

CONCLUSIONSAND OUTLOOK......................................................................................... 43

INTRODUCTION

With the rapidly increasingapplicationof transgenetechnology in plants,thecontrolof transgeneexpressionhasbecomean importantpoint of concern.Acommonaspectof manycasesof inactivationof transgenesis thepresenceofduplicatedhomologous sequences.Apparentlyhomologyservesas a signalthatcantriggergeneinactivation at eitherthetranscriptionalor posttranscrip-tional level. Homology-dependentgenesilencingis the basicfeaturefor sev-eral phenomenathat apparentlyeachhave distinct regulatorymechanisms.This includes,for example,the inactivationof tandemrepeats,trans-inactiva-tion of allelic or ectopiccopies,andthe coordinatedsilencingof a transgeneandthe endogenous homologous gene.In this reviewwesummarizetherecentdataon homology-dependentgenesilencing,focusingon thedifferentmodelsfor the regulationof transcriptional and posttranscriptional silencing. Mostlikely, transgeneresearchhasuncoveredtheexistenceof anendogenouscon-trol mechanismfor multiple sequences,which doesnot affect transgenesex-clusively. On the basisof the mechanistic modelswe thereforediscussthebiological function of homology-scanning systems and implications forgenomeorganizationand evolution.

HOMOLOGY-DEPENDENT SILENCING PHENOMENA

Inactivationof Homologous Transgenes

The importanceof homologoussequencesfor the induction of genesilencingwasdiscoveredwhentransgenictobaccoplantswereretransformedwith con-structs that were partlyhomologous with the integrated transgene. Inthepresenceof the secondconstruct,the primary transgenebecameinactivatedand hypermethylatedwithin the promoterregion, the site of homology be-

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tween the “suppressor”and the “target” locus (76). Since this remarkablediscovery,numerouscasesof homology-basedsilencingin transgenicplantshavebeenreported.Althoughsingletransgenecopiescanbecomeinactivated(104), the integrationof multiple copiesenhancessilencing efficiencies,par-ticularly if repeatedsequencesare insertedin concatamericarrangementsatonelocus(5, 88), but alsowhenhomologoustransgenesarelocatedat allelesof a locus(83) or are present atunlinkedsites(75). Transgeneinactivationcancomprisebothtranscriptional (83) andposttranscriptional silencing (27, 57)ofmarker genes.

Silencingis influencedby thelengthof thehomologyand especiallyby theposition of the interactingsequences.Linked copiesare more efficiently si-lencedthanunlinkedcopies,andunlinkedloci showcharacteristicdifferencesin silencingcapacity(132)andsusceptibility to beingsilenced(91).Themostefficient exampleof trans-inactivation is a tobaccoline carryinga transgeneinsert with two genesdriven by the 19S and the 35S promoter of CaMV,respectively.Both geneslinked to the two promotersaresuppressed,andthislocustrans-inactivatesnewly introducedconstructsthatprovideat least90 bpof commonhomology (132).

HERITABIL ITY AND REVERSION OF SILENCING Wheninteractingloci aresepa-ratedin geneticcrosses,reversionof thesilencedstateoccursslowlyoverseveralgenerations(91). The silencedstateof concatamerictransgenes,which arealwaysinheritedasa block, is preferentiallytransmittedto theprogeny(5, 63,88). In sometransformants,silencingis progressivelyenhancedin subsequentgenerations(5, 63). Othersilencedtransgenesshowa definedresettingphase.A silencedrolB transgeneis reactivatedandremainsactivein youngseedlings,while silencing occursagain erratically during further developmentof theseedling(27).

In concatemerictransgenes,a reductionof repeatsenhancestheprobabilityof reversionof the silencing event. In Arabidopsis transformantsin whichtransgenecopy numbershad beenreducedbecauseof intrachromosomal re-combination, transgeneinactivation was observedat a lowerfrequency than intheparentalline thatstill containedmultiple repeats(4, 5). A similar observa-tion wasmadeby anotherlaboratoryfor anArabidopsistransformantthathadalso lost severaltransgene repeatsvia intrachromosomal recombination.Whenlines were selectedfrom the parentalplant and the deletion line that hadreactivatedthe transgene,only thedeletionlinesconservedtheactivestateofthe transgeneduringmeiosis(87).

THEROLEOFDNA-METHYLATI ON A correlationbetweengeneinactivation andDNA methylation hasbeenshownfor transgenes(2, 131), transposableele-

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ments(19, 116),andsomeendogenousgenes(123).For othergenesno suchcorrelationwas observed(93). With respectto a correlationbetweenDNAmethylationandhomology-basedsilencing,wecangroupthedifferentsilencingeventsinto threeclasses.SilencingeventsthatshowadirectcorrelationbetweentranscriptionalinactivationandDNA methylation within thepromoter (76,83)or codingregion(57), silencingeventsthatarenot associatedwith detectablechangesin DNA methylation (27,44),andsilencingphenomenawherehyper-methylation patternsbuild up over successivegenerations(86).

An interestingaspectof DNA methylation in plants is the presenceofmethylatedC residuesoutside of CG or CNG sequences,the symmetricaltargetsequencesfor maintenancemethylation in plants.To date,nonsymmetri-calmethylationpatterns,whichareprobablynotencodedin thesequence—butmore likelyin thesecondary structureof asequence—have only beendetectedin transgenes(57,85) and not in endogenous genes (93).It is thereforeunclearwhetherthey arespecifically imposed on transgenesor genesthat havebeentransferred into new chromosomal environments. In the latter case, theyshouldalsooccur in transposable elements.

Paramutation

An indication thathomology-basedsilencing eventsarenot specificfor trans-geneDNA but reflectan endogenousmechanismcomesfrom theanalysisofparamutation.Paramutationwasdescribedmorethan60 yearsago(139) andhasbeenexaminedin severalspecies(11, 21, 48, 49). The term refersto theinteractionof homologousplant allelesthat leadsto heritableepigeneticef-fects.A detailedreviewof paramutation,in thecontextof geneticimprinting,hasrecentlybeenpublished(74). We thereforeonly summarizea few aspectsrelevantto themechanisticmodelsfor genesilencing.A paramutagenic allelecancauseaparamutablealleleto undergoan epigenetic conversionto becomeaparamutantalleleof lower function.Thenewparamutantstateis metastable,becauseit canbe somatically andgerminally inheritedin the absenceof theparamutagenic allele,but it alsorevertswith different frequencies.Paramuta-tion requires a metastablestateof theparamutableallele, whichis only ampli-fied throughits interactionwith the paramutagenic allele. Paramutationandthefrequenciesof reversionaredependenton environmentalanddevelopmen-tal factors.

Molecular studiesin snapdragon(10, 22, 65), maize(101), and Petunia(83)provided insights into themechanisms involvedin paramutation. Acorre-lation betweenthe expressionof the paramutablegeneand its methylationstatewasobservedfor theR locus in maize(30) andfor anA1maizetransgenein Petunia(83). In contrast,no differencesin cytosinemethylation could bedetectedbetweenparamutagenicand paramutable allelesat the B locus ofmaize,despiteanextensiveanalysisovera distanceof 12 kb (100).Thelatter

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studydoesnotexcludetheinvolvementof DNA modification in paramutation,ascertaintypesof nucleotidemodifications,suchashydroxy-methylcytosine,A-methylation, or methylcytosineslocatedin nonsymmetricalpositionswouldhave gone undetected.Nevertheless,the analysisof B suggestseither thatmethylation is not the causebut a secondaryeffect of paramutation, or thatdifferentclassesandmechanisms of paramutationexist.The latter is a possi-bility becauseB and R paramutation differ in other characteristics (100).Paramutatedallelesof B areextremelystable,whereasR paramutationshowsfrequentreversions.Furthermore,theB locuscontainsa singleallele,whereasin mostR allelesmultiple homologousgenesoccurat the R locus.A similarcomplexityhasbeenfoundfor two semidominantallelesof thenivealocusinAntirrhinummajusthat showstructuralrearrangementssuchasinverteddupli-cationsor concatamerizationof truncatedcopiesof theniveagene (10,22).

Mutual Inactivation of Transgenes and Endogenous Genes

The term cosuppression(59) was coined to describethe inhibition of geneexpressionof an endogenousgeneafter the introduction of a homologoustransgene.This phenomenonwas first describedfor the chalconesynthase(CHS) genein Petunia(90, 129). Up to half of the transformantsthat con-taineda CHS sensecopy producedwhite flowersor floral sectorsbecauseofthe lossof CHS activity. Nuclearrun-onanalysisshowednormalCHS tran-scriptionratesbutareductionin steady-statelevelsof CHSmRNA, apparentlyasa resultof posttranscriptionaleffects(38, 128).Frequently,not all flowersshowed the same cosuppression phenotype.Individual plants developedbrancheswith purple,white, or sectoredflowers.Among the flowersof indi-vidual branches,cosuppressionpatternsusuallyremainedvery similar, whichsuggeststhat cosuppressionwas somatically inherited and initiated duringformationof themeristemof individualbranches (38).

Cosuppressionis not uniqueto CHSbut appearsto bea generalphenome-nonaffectingmanyendogenousgenes.Detectioncanbedifficult if theinhibi-tion of thegenedoesnot producea visible phenotype.Examplesof genesthatshowed an unstable expression after the introduction of homologous se-quencesare dihydroflavanolreductase(129) and the homeotic fbp2 geneinPetunia(3), tomatopolygalacturonidase(120),phytoenesynthase(40), pecti-nesterase(119), an Arabidopsis cab140gene(13), phenylalanine ammonia-lyase(33), β-1,3-glucanase(25), chitinase(50), nitratereductase(24), S-ade-nosyl-L-methionine synthetase(9), and glutaminesynthetasein tobacco(GCoruzzi,unpublisheddata).Theefficiencyof cosuppressionvariesfor individ-ual transformants.The transferof the samesenseconstructoften generatescosuppressed transformants as well as transformantsthat overexpress thesenseconstruct(9, 24). Individual genesshow characteristicdifferencesintheir susceptibility to cosuppression. Two extremeexamplesarethe Petunia


chalconeisomerasegene,for which cosuppression hasnot beenobservedthusfar (28), and the nuclear-encodedcytosolic tobaccoGS2genethat is cosup-pressed,to variable extents,in all transformantstestedso far (G Coruzzi,unpublished data).

It is important to recognizethat therearemultiple stepswithin theexpres-sionpathwaythatcontribute to theproductionandactivity of a geneproduct.This might alsoexplainthepartly contradictoryfeaturesfound for individualcosuppressionevents.Inhibition of geneexpressionat the posttranscriptionallevel hasbeenconfirmedfor manytypesof cosuppression(25, 38, 128),buttranscriptionalsuppression canalsobe found (13). It is possible that cosup-pressionis mediatedby differentmechanismsin differentspeciesor for indi-vidual genes. Al ternatively, for certain genesposttranscriptional silencingcouldbetheprimaryeventthatinduces transcriptional inactivationasasecondstep ofa commoncosuppressionmechanism.

REQUIREMENT FOR TRANSCRIPTION An importantaspectof cosuppressionisthequestion asto whetherthetransgeneandtheendogenousgenesneedto betranscribed.Evidencefor therequirementof mutualtranscription comesfromstudieson cosuppressionof polygalacturonase(PG) in tomato inducedbyconstitutive expressionof a truncatedPG transcript.In ripe fruits wheretheendogenousgeneis active,expressionof bothgenesis reduced,andtranscriptlevelsof theconstitutively expressedtransgenearesignificantly lower in ripefruits comparedwith greenfruits (120).Ontheotherhand,apromoterlessCHSconstructinducedcosuppressioneffectsin 15% of transgenicPetuniaplants(128),which suggeststhatexpressionis not requiredfor cosuppression.How-ever, tracesof CHS antisenseRNA are found in thesetransformants(128),which allow thespeculationthatanendogenouspromoterreadsinto thetrans-gene creating anantisense-mediatedinhibition.

Thereis someevidencethatcritical levelsof transcriptionarerequiredforefficient induction of cosuppression. For severalexamples,silencing wasfound to beenhancedor evendependenton thehomozygousstateof a trans-gene(24, 25, 50). In contrastto meiotically transmittableexamplesof cosup-pression(60), silencing in homozygous lines was not inheritedby outcrossprogenynow containing only onetransgene.Oneinterpretationfor this effectis thattheprimarytransgeneis transcribedat a relativelyhigh rate, andthat byduplication of theserates, transcript levelsin homozygous plants reachacritical threshold.Alternatively, cosuppression might bestimulatedby aDNA-basedinteractionbetweenthe transgeneallelesin homozygous lines.At leastfor cosuppressionof the β-1,3-glucanasein tobacco,the latter assumptioncould beexcluded.In one line, inactivation of the β-1,3-glucanase genesoccurredexclusively in plantshomozygous for a homologous transgene.Inhaploidplantsof this line, suppression was observedregardless of whether the

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transgenederivedfrom homozygousor hemizygoustransformants(25). Thisresultexcludesa function for allelic interactions andsuggestsa dose-depend-ent regulationfor silencingthat is determinedby the ratio betweenthe trans-genetranscripts and the copiesof theendogenous genesor theentiregenomes.

DEVELOPMENTAL AND ENVIRONMENTAL CONTROL OF COSUPPRESSION Sev-eral casesof cosuppressionshowdevelopmental regulationanda dependenceon environmentalfactors.Cosuppression of CHSgenesin Petuniaproducesavarietyof anthocyaninpigmentationpatternsin theflower,amongwhichhighlyorderedpatternscanbefoundthataresomaticallyheritable(90).Theseobser-vationssuggesta linkage betweenregulatorymechanisms of morphologicaldifferentiationandtheinductionof cosuppression(61).Frequently,silencingistriggeredafter a lag period, either stochastically at different stagesduringdevelopment(24,50) or synchronously at aspecificstage ofdevelopment(25;H Vaucheret, unpublished data).

Various cases ofenvironmentalinfluenceson silencinghave beenob-served.Cosuppressionof CHS genesin Petunia (129) and β-1,3-glucanasegenesin tobacco(25) are stimulatedby high light intensities. Silencingofchitinasegenesin Nicotiana sylvestris(50) and nitrate reductasein tobacco(24) are dependent ongerminationand growthconditions.

InactivationMediatedby RNAViruses

An unexpectedlink betweencosuppressionand transgene-mediatedviral re-sistancewasobservedin transgenicplantsresistantto differentmembersof thepotyvirus group (69, 89). Untranslatableconstructsof the viral coat proteingeneor the RNA polymerasegeneof potyvirusesgenerateda strain-specificresistanceagainstthevirus,accompaniedby very low steady-statelevelsof thetransgenicRNA. In virus-resistant lines, homologous transgeneswere alsotrans-inactivated,which suggeststhatviral resistanceis mediatedby a homol-ogy-based inactivation mechanism.

MODELS FOR HOMOLOGY-BASED SILENCING

Thecomplexity of experimentaldetailsconcerninghomology-basedsilencingmakes itdifficult to allocatedefined mechanistic modelsexclusivelyto certainsilencingcategories.We havethereforeavoidedlinking detailedmodelsto thedescriptionof different typesof silencingpresentedabove.In the following,we discussseveralmodelsfor the molecularmechanisms involved in silenc-ing, which are not mutually exclusivebut which may apply individually oreven synergistically for individual silencingtypes.


SilencingMediatedVia DNA-DNA Pairing

Theinteractionof homologousDNA copieshasbeen proposedas a mechanis-tic modelfor certaintypesof cosuppression(59), trans-inactivation (73), andparamutation(83). It wasproposed(61) that silencing reflectschangesin thephysicalstateof a transgeneandthat mutualsilencingof a transgeneandanendogenoushomologuearecausedby regularchangesin theepigeneticstatesof the transgene.A DNA-DNA pairing modelwould providean explanationfor the differencesin efficiency at which silencingoccurswithin individualtransformants,becausethe interactionbetweentwo homologous sequenceswould be determinedby the probability with which the two loci associateininterphasenuclei.This probability shouldbehigherfor tandemly linked cop-ies,comparedwith unlinked,ectopiccopies.Thetandemarrangementof trans-genesmay not only enhancethe efficiency of DNA-DNA pairing, but theformation of stemloopstructureson single strandsof a region carrying in-vertedrepeatscould mediatean efficient spreadof de novo methylation pat-terns.FoldbackDNA is specifically recognizedby the humanmethyltrans-ferase(121).

Individual transgenesdiffer significantly in their capacityto trans-inacti-vatehomologous copies(132),which probablyreflectstheir potentialto scanother chromosomal locationsfor homology. The presenceof very efficienttrans-silencerscloseto thetelomeresuggeststhattelomericregionsarefavor-able sitesfor theinteractionwith homologous sequences(72).

RNA-Mediated Models for Silencing

THE ROLE OF RNA-DNA HYBRIDS The DNA pairing model suggests thatepigenetic patterns,characterizedby a specificstateof DNA methylation orchromatinstructure,areexchangedduringapotentialsomatichybridization(60,75).TheobservationthatRNA moleculescaninducehypermethylationpatternswithin homologousDNA sequencessuggestedthatchangesin epigeneticstatescouldalsobemediatedby DNA-RNA pairing.Evidencefor theparticipationofRNA moleculesin the inductionof DNA methylation camefrom a studyoftobaccotransformantscarryingthecDNA of potatospindletuberviroid. Spe-cific methylation of the viroid DNA was observedwheneverviroid RNAreplicationhadoccurred(135).Thesedatasuggestthat transcriptscaninducemethylationin thehomologousDNA region,whichmightbeespeciallyrelevantfor transformantsthataccumulate largeamountsof nuclear transcripts becauseof high transcription ratesor imperfectRNA processing.Thespecificmethyla-tion of codingregionsin certainposttranscriptionalsilencing events(57)mightreflect such an RNA-mediated inductionof DNA methylation.

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DEGRADATION OF THRESHOLD LEVELS OF RNA The dosage-dependencyofcertainsilencingeffects(25, 27,57) andtheobservationof a linkagebetweensilencingandtheonsetof expressionof theendogenousgene(120)suggestthatsilencingcanbeinducedby theproduction of definedthresholdlevels.Suchamodelmightespeciallyapplyfor particulargenesthatcarrytargetsequencesforRNA degradationto controlhighexpressionlevelsgeneratedby geneinduction(92).

THE AUTOREGULATION MODEL Inspired by a model for cytokinin habitu-ation (79), an autoregulation model for silencingwasproposed(80). In thismodel, transcriptionof a target gene,susceptibleto silencing, leadsto theproductionof a diffusible activatorthatincreases steady-statemRNA levelsofthetarget gene.Expressionof thetargetgenethereforedependson theconcen-tration of the activatorin a positive feedbackloop. The modelsuggeststhatactivatorsynthesisdependsonthetranscriptionratesof thetargetgeneandthatactivatordegradationis proportional to activatorconcentration.The activatorwill also stabilize mRNA levels of transgenesthat are homologousto theendogenoustarget gene.Transcriptionof homologoustransgenes will enhanceactivatorconcentrations.In this model, the activator thereforemediatesthelinkedstabilizationor repressionof its targetgeneandahomologoustransgene.The systemis stable,whensynthesisratesequalthe degradationratesof theactivator.Variationsin transcriptionrates,however,will induce instabilities inthefeedbackcontrolsystem.Development-dependentchangesin transcriptionrateswould increasetheconcentrationof activatormolecules,which would inturnenhancetheactivatordegradationsystem.Whentranscriptionratesdeclineagain,thehighdegradationrateswould rapidly reducethe numberof activatormolecules,thusdecreasingsteady-statemRNA levelsof thetargetgeneandthehomologous transgene.Steady-statemRNA levels could recoverwhen lowactivator concentrationsraiseagain.The postulation of activatormoleculescould explain the developmental modulation and resettingeffect of certaincosuppressionsystems.

ANTISENSE-MEDIATED RNA DEGRADATION An obviouselementto accountforthesequencespecificityof cosuppressionis theproduction of antisenseRNA.RNA duplexeswouldbetargetsfor RNAseH-likeendogenousenzymes.Antis-ensetranscriptscouldbegeneratedby promoterspresentonthetransgeneDNAor by endogenousplantpromotersat the3′ endof thetransgene(47).Alterna-tively, theycouldbeproducedbyaplantRNA-dependentRNA polymerase(39,69). If antisenseRNA is only producedatparticulardevelopmental stagesor ifpromoterslocated3′ to thesilencedgeneareregulatedbyenvironmentalstimuli,thiswouldexplainthedevelopmental andenvironmentaldependenceof certainsilencingphenomena.


Theproductionof antisensemolecules by an RNA-dependentRNA polym-erasemaydependon theproductionof specificthresholdlevelsof senseRNAor on theaccumulationof RNA intermediatesduring a delayin RNA transportor processing(38, 61). This hypothesisassumesthat RNA-dependentRNApolymerasesrecognize“aberrant” transcripts,which may derive from incor-rect transcription,transport,or translation of thetransgene.TheproductionofaberrantRNA may bemodulatedby changes in epigenetic states ofagenethatinfluence themodeor efficiency of RNAprocessing(38).

Certaintransgenesonly cosuppress sequences that contain ahomologous3′end, whereasgenesonly homologous to the 5′ region are not affected(JEnglish, unpublished data). Thisobservationsuggeststhat antisensetran-scriptsarepreferentiallymadeagainstthe 3′ endregionof the transgene.Ontheotherhand,constructsthat containedthe5′ endof onegeneanda secondgeneat the 3′ end,efficiently silencedboth endogenousgenes,which arguesagainsta generalfunctionof the3′ end(119).Nevertheless, specificmodifica-tionsat the3′ endhavebeendetected,suchastheaccumulation of processingintermediates(J Kooter,unpublisheddata)andincorrectsplicing within the3′end of silencedtranscripts(D Flavelland MMetzlaff, unpublished data).

CELLULAR MECHANISMS INVOLVED INHOMOLOGY-DEPENDENT SILENCING

Many aspectsof the modelslisted aboveare still speculative,and in caseswhereparticularmolecularfeatures,suchas hypermethylation or high tran-scription rates,havebeenassociatedwith silencingevents,their generalim-portanceis still unclear.It would be prematureand possiblydetrimentaltofavor onecommon modelfor themanydifferentsilencingevents,becausewewould narrowthe scopeof our investigations.From the experimentaldetailspublishedfor various genesilencing systemswe can draw two importantconclusions.First, the growing numberof reportson transgenesilencingnolonger correspondwith the early assessmentthat we aredealingwith a fewrareeventsof minor importance.Second,genesilencingwasnot specificallydevelopedfor transgenes,but it reflectsendogenousfunctionsthatmostlikelyparticipatein theregulationof geneexpressionand plantdevelopment.

At present,wecanformulatethreemajorareasfor futureresearchactivities:the function of chromatinin a dynamicregulatorysystemin plant develop-ment, the control of RNA turnover within RNA processingroutesthat areinvolvedin the finetuningof geneexpression,and the importance ofa homol-ogy-detectionmechanismfor geneexpressionandgenomeorganization.Wediscussthe general importance ofthese threeaspects forthe regulation of geneexpression in plants andothereukaryotes.Wedonotknowwhetherandwhichof themechanisms thathavebeenfound in othereukaryotesarealsorelevant

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for plants. Nevertheless, the examples shown below should behelpful todefine primary modelsfor mechanisms of homology-dependentsilencinginplants,and they should explain why we think that an improvementof ourknowledgein the threeareasmentionedabovemight be necessaryto under-standthe controlof gene expressionin plants.

The Regulatory Function of ChromatinStates

It hasbeenproposedthat epigeneticpatternscanbe establishednot only byDNA methylation(54) but alsoby supramolecularchromatinstructures(136).Our presentknowledgeaboutthe formationandcontrol of different statesofchromatinin plantsis still very limited. Assumptionsthat changesin expres-sionpatternsarebasedonmodificationsof chromatinconformationaremainlygroundedin theindirectevidencethatchangesin DNA methylation occurandthatdensemethylationpatternsinducetheformationof highly packedchroma-tin (8).

The mostadvancedstudieson the role of chromatinstructurein heritablegenerepressioncomefrom Drosophila and yeast,two speciesthat lack C-methylation and that haveprovenespeciallysuitablefor chromatinstudies.Their small genomessimplify theanalysisof individual genomicregionsandgeneticanalysishasidentified modifiersof chromatincomplexes.Yeast offerstheadvantagesof rapidgenerationof mutants,easyphysiologicalanalysis,andgenereplacementby homologous recombination.Thepolytenechromosomesin the salivaryglandof Drosophila allow a preciselocalizationof chromatincomplexes.

POSITION-EFFECT VARIEGATION Position-effectvariegation(PEV) is a partialinactivationof geneexpressionin Drosophila causedby a rearrangementthatplacesa normally euchromaticgeneneara heterochromatic region(51, 106).PEV at thewhite locus,involved in eyecolor, canbemonitoredin individualcellsby thereductionof redeyepigment.Inactivationof thegene,whichresultsfrom thespreading oftheheterochromaticstateinto theeuchromaticneighbor-hood,causesamosaicphenotypeof redandwhitecellsthatgavePEVits name(124). PEV demonstratesthe differencesbetweentwo kinds of chromatin:heterochromatin,which is locatedwithin the pericentricregionsand whichremainscondensedthroughout thecell cycle,andeuchromatin, whichis locatedin thechromosome armsanddecondensesduringinterphase.Inactivationof ageneisaccompaniedbycytologicallyvisiblespreadingof heterochromatinover50–100polytenebands,correspondingto hundredsof kilobases.The Droso-philagenomeconsistsof morethan100loci thatsuppressorenhancePEV,someof whichhavebeencharacterizedto encodechromosomal proteins(113).A setof heterochromatin-associatedgenes,theproductsof theSu(var)genes,assem-blecooperativelyto form complexesin heterochromatinregions.Theseprotein


complexescancontinueto expand.Complexformationrequirestheinteractionof largeamountsof different geneproducts,and insufficiencyof oneof theSu(var)genes reduces thespreadingof heterochromatin(70).

REPRESSION MEDIATED BY MEMBERSOF THE POLYCOMB GROUP Anotherex-amplefor thecontrolof geneactivity by largechromosomalcomplexesis theregulationof homeotic geneexpression.During earlyembryogenesis,thema-ternal and segmentationgeneproductscatalyzethe assemblyof inhibitoryproteinsof thePolycombgroup(Pc-G)andactivatingproteinsof thetrithoraxgroup (trx-G) at homeoticgene loci. Patterns of chromatin conformation,mediatedby Pc-Gandtrx-G proteins,aremitotically transmittedandprovidethebasisfor differentialexpressionof homeoticgenesalongtheanterior-poste-rior axis (98). Pc-Gproteinscanbe localizedat specificregionson polytenechromosomes,which impliesthatthecomplexesrecognizespecificregionsorsecondarystructures.Most likely, thereexistsamolecularrelationship betweenPc-GgenesandSu(var)genes,becausesomemutations in Pc-GgenesaffectPEV, andcertainSu(var)genesinfluencePc-G-mediatedeffects(35). More-over,thePcproteinsharesanamino-acidsequence,calledthechromodomain,with the Su(var)3-9 product,the heterochromatin proteinHP1 (99).

CHROMATIN-M EDIATED REPRESSION IN YEAST In yeast,at leasttwo modesoftranscriptionalrepression are mediatedby chromatin conformation: (a) repres-sion by theglobalregulatorcomplexSsn6/Tup1 (111), and(b) silencing of theyeast matingtype loci(107)and genes attelomeres(96). The gene products oftheTUP1geneandtheSSN6genearephysically associatedin a largeproteincomplex,requiredfor repressionof cell type-specificgenesandgenesrepressedby glucoseor oxygen,respectively.ThecomplexdoesnotbindtoDNA directlybut is targetedto particular promotersvia protein-proteininteractions withspecific promoter binding proteins. In this interaction,Tup1 provides therepressoractivity andSsn6the targetingfunction (127).Repressionby Ssn6/Tup1is mediatedby organizingrepressedchromatindomains,possibly throughinteractionswith histoneH4(111).Silencingattheyeastmatingtypeloci, HMLand HMR, and at telomeresis also regulatedby the creationof a definedchromatinstructure.Formationof a silencingchromatinstructureis mediatedby aninteractionof theSir proteinsandtheir interactionwith histonesH3 andH4 (125). Targetingof the complex to specific regionsis mediatedby theorigin-recognition complexprotein (ORC), the Rap1protein, and the Abf1protein(111).

In summary,we candefinethreeimportantaspectsfor chromatin-mediatedgenerepression:the formationof heterochromatin-relatedproteincomplexes,the targetingof thesecomplexesto particularlocations,and the role of his-tonesas modulators for the formation of certain complexes.Not all three

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aspectsmay be relevantfor potentialchromatin-mediatedsilencingeffectsinplants,but a searchfor modifier functionsof silencing,eitherby mutagenesis(27) or by searchingfor plant proteinsthat sharecommon domains withknown regulatorsof silencingin Drosophila or yeast,might clarify whetherchromatin-mediatedrepressionis a universalfeaturein eukaryotes.Encourag-ing supportfor thisassumptioncomes froma report abouttheidentificationofthe murinebmi-1 gene,a homologueof the Pc-G genePosterior sexcombsgene. Micedeficient for bmi-1 show multiple posterior-directed homeotictransformations(130), causedby ectopicexpressionof genesof the HOX4cluster (95).

A ROLE FOR CHROMATIN-M EDIATED REPRESSION IN HOMOLOGY-DEPENDENT

SILENCING Theformationof chromatinstatescanberelevantfor two aspectsof homology-dependentsilencing mechanisms,the trans-inactivation of ho-mologoussequencesand the developmental regulationof silencing.Even atransientpairing of two homologoussequencescould favor the exchangeofchromatincomponentsthathaveformedarepressedcomplexononecopy.Thus,silencingwouldbetheresultof theestablishmentof arepressedchromatinstatein thetransgeneregion,somepartof which is transferredto otherhomologoustransgenesor endogenesgenes.Supportfor this assumptioncomesfrom theobservationsthatmultiple transgenecopiesarepreferentiallysilencedandthattransgenescanbe specific targetsfor DNA methylation, which is associatedwith chromatin condensation (81). Ithas beenproposed thatDNA methylationactsasa defensemechanismagainstforeignDNA (7, 29). Plantgeneshavearelatively narrow rangeof AT-contentand are embeddedinto 200-kb largechromosomal regionsof amatching AT-content,termedisochores. Monocoty-ledonousand dicotyledonous speciescontaindistinct isochorecompositions(112). Therefore,transgenes with deviant basecompositions may becomespecifictargetsfor denovomethylation. A possiblecase of thisoccurswhen asingle copy of the GC-richA1-gene from maizecan becomespecificallymethylatedin transgenicPetunia(82),whereasits homologue,with aGC-con-tent similar to Petunia, from Gerbera remainsunmethylated(34). Becausechromosomalintegrationsitesdiffer somewhatin basecomposition, transgeneswill becomemethylatedwith differentefficienciesatdifferentsites,andcertaintransgeneswill not beinactivatedat all (26). It is possible thattheinsertionofmultiple copieswill increasethe probability of individual transgenesbeingmethylatedandcondensedheterochromatinbecausetheentireregionisdifferentfrom a typicalisochore.Furthermore,multiple transgenesintegratedin tandemor asinvertedrepeatsmight enhancetheformationof condensedchromosomalcomplexes,if theyprovidetargetchromatin-associatedfactors,similartorepeat-inducedheterochromatinizationprocessesin Drosophila.Transientinteractionsof transgenesequencesinsertedatdifferentchromosomal locationswouldthen


inducea spreadof condensedchromatinstates,which explainsthepreferentialinactivationof multiple transgenecopies, evenat dispersedsites.

CHROMATIN-M EDIATED REGULATION OF CHROMATIN STATES The chromatinconformationmayberegularlymodifiedduringdevelopmentandcouldalsobeinfluencedby environmental conditions.Any changecouldinfluencetranscrip-tional activity of thegene,theefficiencyof RNA transport,or thecompetenceof the locus for somaticpairing with homologoussequences.Any of thesefeaturescould explain the developmental regulationand the environmentaldependence of somehomology-based silencingphenomena.

It has beensuggested(61) that cosuppressionof CHS genesin Petuniareflectsdevelopmental andphysiological factorsthat imposeheritablemetas-tablechangesin theplantgenome.This ideawasdeducedfrom modelsdevel-oped from the analysisof transposable elementsin plants (12, 77). Brinkproposedthat the genomepossessesa parageneticfunction that is distinctfrom its geneticfunction.Basedon theconceptof paramutation, hedefinesanorthochromatin that harbors the DNA that is subjectto mutations andaparachromatin that is sensitiveto the cellular environmentand capableofreceiving,recording, andmitotically transmitting information from outside thechromosome.Duringdevelopment, changesin parachromatin would thereforeconditiondifferential activitiesof geneticloci in the orthochromatin (12). Asimilar conceptis found in Mc Clintock’s interpretationof epiallelic statesoftransposableelements.Sheobservedthat individual epiallelesof an elementshowedcharacteristicphasesof activity, which could be influencedby theexpressionof otherelements.This presettingeffectwasmitotically transmit-ted but erasedin the next generation.Sheproposedthat individual genesareembeddedor dissociatedfrom condensedchromatinclustersin a regularman-ner,which regulatestheir differential expressionduring development(77). Ifextracellularsignalsinducechanges inparagenetic states,this could alsomod-ify theposition andassociation of geneswithin suchclustersof a cell anditssomaticderivatives.

It is unknownwhich molecularfactors regulatechanges in chromatinstruc-ture,but it hasbeensuggestedthatparticularchromatinstatesaregeneratedorconservedby changesin DNA methylation (81).EvidencethatDNA methyla-tion is involved in the determination andmodulation of epigenetic statescomesfrom the analysisof the En/Spmtransposableelementin maize(36).This autonomouselementcanexist in threedistinctbut interconvertible formstermedcryptic, programmable,andactive.Theseforms canbe distinguishedby the methylation levels of GC-rich sequencesin the downstreamcontrolregion(DCR), nearthepromotor. Cryptic elementsarealmoststablyinactiveand exhibit somatic reversionfrequenciesto active statesof 10−5: They arehighly methylated.Active elementsareunmethylated.Programmableelements

36 MEYER & SAEDLER

that revertmore frequentlythancryptic elementsand that canbe trans-acti-vatedby anactiveelementhaveanintermediatemethylationstate.Themodu-lation of epigeneticstatesis mediatedby TpnA, an autoregulatoryproteinencodedby the element.TpnA hasthreedistinct functions.It is requiredfortransposition of the element(41), it activatesmethylatedpromotersof pro-grammableelements,and it repressesthe unmethylated promoterof activeelements(115).

Besidesthedetermination of their phasesof activity, transposableelementsareregulatedby a developmental controlmechanismthatdeterminestheheri-tability of the phasesand the activity of the elementsduring development.Therefore,changesin theepigeneticstateinfluencetheactivity of theelementin the next generation(37). A relatedobservationhas beenmade for theactivity of anA1 transgenein Petuniathatwasalsocorrelatedwith changesinDNA methylation. In anF1 progenyfrom onetransgenicline homozygousforA1, plantsderivedfrom pollination of the first flowersshoweda significantlymorestableexpressionof themarkergene,while in progenyfrom pollinationof older flowers the transgenebecameinactivatedand methylatedat highfrequencies(84).Thesedata alsosuggesta developmental regulationof epige-netic patternsthatcan be transferred tothe nextgeneration.

PosttranscriptionalControl of GeneExpression

Posttranscriptional regulatorymechanismshavean importantfunction in thecontrol of geneexpression(43, 52). The efficiency at which a genewill beexpresseddependson mRNA processing,transcriptstability, nucleocytoplas-mic transport,translationefficiency,andproteinmodificationandhalf life. Wedo not discusstheseitems in detail but concentrateon a few examplesthatindicatehow changesin RNA stability mightbe involvedin gene silencing.

RNA TRANSPORT In recentyearsseveralpartly contradictorymodelshaveemergedabout the transportof RNA from the site of transcriptionto thecytoplasm.In analogyto theestablishedmodelof anorganizedmovementofnewly synthesizedpolypeptidesthroughthecytoplasmic secretionmachinery(105), it was proposedthat transcriptsmove to the cytoplasmin an orderedfashion,passingthrough localizedspotsthat harbor individual stepsof theprocessingmachinery(16, 139). This model is supportedby reportsof thelocalizationof splicing componentsin subnucleardomains,calledspecklesandfoci, andby observationsthatintron-containing RNAs are targetedto specklesuponmicroinjection intomammalian nuclei(122).

In contrast with these reports,otherstudies argueagainst acompartmentali-zationof RNA processing,becausesplicingoccurredat thesitesof transcrip-tion. Thesesiteswere not coincidentwith intranuclearspecklesthat harborcomponentsof the splicing machinery(142). Assuming that the position of


certain geneswithin the interphasenucleusdeterminesthe entranceof thetranscript into defined processingroutes,transcriptsof multiple transgeneslocalized at differentpositions may entercommon or separateprocessingroutes.Local concentrationsof homologous transcriptswould be enhancedsignificantly, if transcriptsof the transgeneandthe homologousendogenousgenepassthe sameprocessingtrack.We thereforenot only haveto considerthe generalquantitiesof steady-stateRNA levelswithin the nucleusbut alsohaveto accountfor the local concentrationsof homologousRNA moleculeswithin theprocessingtrack.

RNA STABILITY RNA stability is influencedby anumberof factors.The5′ capstructureand the 3′ poly(A) tail stabilize mRNA againstdegradation.Thepoly(A) tail also regulatesthe efficiency of translation, but only when thetranscriptis capped(42). Other posttranscriptional modifications havebeenproposedto serveassignalsfor degradation.Adenineresiduescanbemethyl-atedor convertedinto inosinesthatmight serveasa tagfor RNA degradation(64). Destabilizing andstabilizing sequenceelementshavebeenidentified inspecific mRNAs. Theseelementseither provide target sequencesfor RNAdegradation(92) or bindingsitesfor stabilizing factors (17).

RNA stability is alsoinfluencedby the efficiencyof translation.An inter-estinglinkagebetweentranslationefficiencyandmRNA metabolism hasbeendetectedfor the humanβ-39 mRNA, a mutation of the β-globin genethatcarriesa stop codon at position 39 (6). The nontranslatability of the β-39mRNA inducedasignificantreduction in mRNA accumulation, althoughtran-scription, splicing, and polyadenylation of the β-39 mRNA are not altered.This observationprompted the ideasthat either a nuclearmechanismexiststhat is capableof sensingnonsensemutations or that there is a feedbackcommunication from thecytoplasmto thenucleus.It hasbeensuggestedthatthis feedbackinteractionoccursat thenuclearmembraneat pointsof contactwith the roughendoplasmatic reticulum,where translation can occur (6).

PRODUCTION OF ANTISENSE RNA As mentionedearlier,it hasbeenproposedthatantisensetranscriptsareinvolvedin posttranscriptionalsilencing.Antisensetranscriptscouldbeproducedby promoterslocatedin the3′ regionof ageneorby an RNA-dependentRNA polymerase.Several examples exist for theuse ofendogenousantisensetranscriptsfor transcriptional(94) or posttranscriptional(53, 55) control of expression.In barley,a lack of alpha-amylaseexpressioncoincideswith theappearanceof atranscriptcomplementary to thealpha-amy-lasemRNA (108).RNA-dependentRNA polymerasesarewidely distributedamongplants,althoughtherehas beena disputeaboutthe possible contamina-tion of thematerialby viral RNA polymerases(39).Theseenzymesareusuallypresentin low amounts, which canbesignificantly increaseduponviral infec-

38 MEYER & SAEDLER

tion.Theenzymesin differentplantsareclearlydistinctivein sizeandtemplatespecificity.Thehost-specificity is conservedafter induction of higherenzymelevelsby infectionswith thesamevirus, andthis supportsthehypothesisthattheenzymesarenot derivedfrom thevirus but areencodedby thehostplant.The biological role of RNA-dependentRNA polymeraseshasnot beenfullyelucidated,but it is obviousthat they cancreateantisensemoleculesagainstexistingcellulartranscripts.

To evaluatethe importanceof posttranscriptional control mechanismsforsilencing we still needto answerseveralkey questions. We do not knowwhethertranscription, translation,or polysome-associationis aprerequisiteforsilencing.We needto definewhatdeterminesthe“aberrant”stateof RNA andwhetherthis inducesRNA degradationor the productionof antisensetran-scripts.We also needto understandhow transcriptsare transportedinto thecytoplasmandwhether and whichfactors exist that induce feedbackresponsesat theDNA level whenRNA processingor translationis disturbed.

Repeat-Specific Control Mechanisms

Theparticipationof repeatedsequencesin geneinactivation phenomenais notlimitedto plantsbutcanbefoundin severalothereukaryotes.A comparisonofhomology-dependentsilencingin plantswith othereukaryoticsilencingsys-temsilluminatessomeinterestingsimilarities thatsuggestcommon biologicalfunctions.

THE RIP- AND MIP-MECHANISMS OF FILAMENTOUS FUNGI In the filamentousfungiNeurosporacrassaandAscobulusimmersus,thepresenceof DNA repeatstriggersmethylation and inactivationof the repeatedregions.In Neurosporatransformantsthat containlinked or unlinkedduplicatedsequences,a mecha-nism namedRepeatInducedPoint Mutation (RIP) inducesmethylation ofC-residuesfollowed by mutationof C to T, preferentiallyat CA dinucleotides(14, 117). In Ascobolus, geneduplication leadsto de novo methylation andpremeioticinactivation becauseof a mechanismtermedMethylation InducedPremeiotically(MIP) (45).Theefficiencyof MIP dependsonagene’s locationandon thelengthof homologousrepeats.Clusteredrepeatsof a critical lengthalwaysbecomemethylated,whereasefficienciesfor ectopichomologuesvaryconsiderably(110).Methylation not only encompassesC residueswithin CpGdinucleotides, it also extendsto C residueslocated in nonsymmetrical se-quences,which implies anoveltypeof methyltransferaseactivity (46).Similarnonsymmetrical 5mC patternshave beenobservedfor sequencesthat hadundergoneRIP in Neurospora (118)andfor planttransgenesthathadbecometranscriptionally (85) or posttranscriptionallysilenced (57),respectively.

An inversecorrelationbetweencopy numberandthe expressionof trans-geneswasalsoshownfor theasexualcycleof Neurospora (97).After transfor-


mationwith a resistancemarker,vegetativeandreversibleinactivationof themarkeroccurredin multicopy transformants. Inactivation wasaccompaniedbyhypermethylation of markergenes.Treatmentwith 5-azacytidineinducedastablereactivationof the marker in some,but not all, transformants,whichsuggestsa functionalimpactof DNA methylationon geneexpression.Methy-lation might also be involved in a unidirectional si lencing event inNeurospora,termedquelling (109).Theexpressionof endogenousgeneswasimpairedwhenseveralhomologouscopieswereintegratedat an ectopicsite.Reversionof thequelling effectwascorrelatedwith a reductionin thenumberof ectopical ly integrated gene fragments. These data demonstrate thatNeurosporaandAscoboluscontainhomology-searchingmechanismsrespon-sible for the specific methylation of repeatedsequences.Theseshow somesimilarities to silencingphenomenain plants.Becausefilamentousfungi areexcellentsubjectsfor mutationanalysis,it is very likely thatendogenousgenesinvolvedin theregulationof silencingwill befirst identifiedin NeurosporaorAscobolusbefore theyarefoundin plants.

HOMOLOGY-MEDIATED REPRESSION IN DROSOPHILA In Drosophila, repeatedsequences areinvolvedin the initiation ofposition-effectvariegation, intrans-effects of PEV, and in transvection.PEV-like effects were inducedwhentandemlylinked copiesof a P-transposoncarrying a white transgenewereintegratedinto the Drosophila genome(31), which suggeststhat pairing ofrepeatscontributesto heterochromatinformation.Homologous chromosomesof nondividing nuclei are physically paired in somaticcells of Drosophila,providing thebasisfor the transmissionof heterochromatinstatesto homolo-gousalleles.Oneexamplefor pairing-dependenttrans-inactivationis dominantPEV at the brown locus.Heterochromatinthat wasimposedon a rearrangedbrownallelewastransmittedto theunrearrangedhomologouscopy,whichalsobecame inactivated(32).

Other pairing-dependentmodulations of geneexpressionhave beende-scribedunderthe term transvection(68). Like paramutation, transvectionde-pendson the interactionof susceptible alleles,but the effect is not preservedaftersegregationof theinteractingallelesduringmeiosis.A well-characterizedexample forallelic interaction is the regulationof the white geneby theproductof the zestegene(103).The white genecarriesa setof zestebindingsitesin its promotor,in theeyeenhancer region.A lackof zestefunctionleadsto amoderatedecreasein whiteactivity.Thez1 mutation of zesteis responsiblefor the zesteproduct forming hyperaggregates(20), and this causessevererepressionin lines that containtwo pairedcopiesof white.Repressionis notobservedfor single white copies,but if a homologouswhite gene,togetherwith thezestebindingsites,is insertedat ectopicpositions, aboutonethird oftheselinesshowrepressionin a z1 background.This observationis interpretedasindicatingthatspecificloci caninteractwith thewhite locusto formz1-spe-

40 MEYER & SAEDLER

cific aggregates.Zeste-whiteinteractionsarefurther modified by severalge-netic loci,mostof whicharemembersof thepolycombgroup.

REPEAT-INDUCED DNA METHYLATION IN MAMM ALS In contrastto the largenumberof repeat-inducedinactivationeventsfoundin transgenicplants,it hasbeenunclearwhethera similar mechanismexistsin mammals.Senseinactiva-tion of anendogenousgene(15)andaninversecorrelationbetweenthenumberof transgenecopiesandtheirmethylationstatehavebeenreported(78).Becausesucheventsarerare,it wasdoubtfulwhethertheseeventsreflectedthepresenceof a homology-searchmechanismas has beenpostulatedfor plantsandfungi.

More compelling evidencethat repeat-inducedinactivationis alsopresentin mammalscamefrom theanalysis ofhuman geneticdiseasesassociatedwiththe amplificationof triplet repeats(67). Most of thesegenes,which encodetranscriptionfactors,carrymultimersof CAG- or CCG-tripletsthatarerespon-sible for poly-glutamine or poly-proline stretcheswithin thecodingsequence.Thetriplet repeatsarevariablein lengthandcan expand or contractin somaticcells or when passedto the next generation.As the repeatexpansionis notuniform, individuals carry a mosaicpattern;individual cells harborvariousrepeat lengths in the affected gene (102). Excessive repeat amplificationcausesdisease.The onsetof diseaseis developmentally regulated;for mostdiseasesthereseemsto be an inverserelationbetweenthe numberof tripletrepeatsandtheageat which the first symptomsof thediseasebecomemani-fest (56, 126). Becausethe probability and the length of repeatextensionsignificantly increasein cells that showdeficienciesin DNA repair (1), it isunclearwhether diseasesarecaused byadefective mismatchrepairor whetherthe repeatelementsarecausallyinvolved.For triplets locatedwithin protein-coding regions,repeatamplification results in a significant increasein thelength ofthesingle aminoacidstretches.It hasbeenshownthatlong poly-glu-taminestretchesreducethe activity of transcriptionfactors(18), andthe dis-easephenotypecould reflect a lack of function of the protein. Amplifiedtripletsarenot restrictedto protein-coding regions,however,but arefound inthe5′ noncodingregion(141)or in 3′ untranslatedDNA of somegenes(138).For thesediseases,therepeat-amplificationdoesnotaffectproteinfunctionbuttranscription.Theexpansionof triplet repeats in theFMR1gene(140) actuallyleadsto a reductionof geneexpression.The increasein CGG repeatsmakestheCpGisland-typepromoterof theFMR1 gene,which is normallyunmethy-lated, susceptible to methylation. The level of methylation is not uniform.Individualshavedifferent levelsandthe mentalretardationphenotype corre-lateswith theextentof methylation (71). In analogyto repeat-inducedhetero-chromatinization inDrosophila (31), it is temptingto speculatethatamplifica-tion of triplet repeatsin humansinducesheterochromatinformation,which isinducedor followed by cytosinemethylation, convertingthe geneinto con-densed andtranscriptionally impairedchromatin.


Indirect evidencefor the existenceof a mechanismfor homology-basedDNA methylation camefrom a studyof thedistribution of CpGdinucleotidesin themammalian genome(66). Basedon thecriterion thatCpGdepletionandTpG overrepresentationfor a particulargenomicregionindicatesa prior his-tory of high methylation, repetitive sequenceswere identified as preferredtargetsfor methylation. Thesedatasuggestthat sequencerepeatsarespecifi-cally recognizedto becomemethylated.This would not only affect highlyrepetitiveDNA but alsohomologous membersof genefamilies andpseudo-genes.It wasproposed(66) thattheparentalsequencesareprotectedfrom themethylation mechanism,becausethe insertionof introns into their sequencemasksthe sequence homology withtheirpseudogenes.

A ROLE FOR REPEAT-INDUCED METHYLATION IN GENOME EVOLUTION If weconsiderthat manyplant genomescarry a largeproportionof duplicatedloci(137),homology-dependentsilencingmechanismsshouldinfluencetheexpres-sionof endogenousduplicatedgenes.It isconceivablethatduplicatedsequencesescapesilencingif theyareembeddedin noninteractingchromosomalenviron-mentsor if they containa significantdegreeof sequencedivergence.In thisrespect,the presenceandefficiencyof repeat-dependentsilencing mechanismsshould influence the potential of the plant genometo developnew allelicvariationsduringevolution. In this context,repeat-inducedmethylationcoun-terbalances DNA amplification processes generating heterogeneous epigeneticpatternsin repeated sequencesthat can befurther modulatedby environmentalstress.In mammalian tissuecultures,certainresistancegenescan beamplifiedunderselection pressure(114).In plants,themostdramaticexamplesfor DNAamplification aretheenvironmentally inducedmorphologicalchangesin flax.Flax plantstreatedwith high levelsof fertilizers amplified specificgenomicsubsets(23).It hasbeenproposed(58)thatchangesin heterochromatincontentandDNA methylation arealsoassociatedwith this phenomenon.Epigeneticstatesarealsoresponsivetochangingenvironmentalconditionsasdemonstratedby the activationof transposable elements(133, 134) or the changeof DNAmethylation patternsin tissueculture (62) or in field-grown plants (84). Acombination of environmentally regulatedmechanisms of geneamplificationand epimutation would provide the cell with an efficient systemto adapttochanging external conditions.Becauseof thestochastic characterof amplifica-tion andepigeneticmodificationmechanisms, theplantwould bea chimeraofdistinctivesomaticsectorssubjecttoselection.Novelphenotypesof thegenomecouldbemanifested but also correctedin subsequent generations.In mammalsthe separationof germlineand somaticcells, as well as the high degreeofcomplexityof differentiatedcells,putssevereconstraints on theefficiencyofsuchamechanism. Forplantscells,however,totipolenceandtheproductionofgermcellslatein somaticdevelopment, abalancedactivityof amplificationandepimutation shouldhavea considerable evolutionary advantage.

42 MEYER & SAEDLER

CONCLUSIONSAND OUTLOOK

The term homology-dependentsilencingrefers to inactivation eventsat thetranscriptionalor posttranscriptional level that differ in efficiency, resetting,andheritability. Besidesthe requirementfor homology, a commonfeatureofall homology-dependentinactivationeventsis thedisturbanceof thesequencecontextbecauseof DNA rearrangements.This appliesfor the inactivation oftransgenesthatintegraterandomlyinto thegenomevia illegitimaterecombina-tion andfor nontransgenicsilencingeventssuchastransposoninactivation andparamutation.On the other hand,not every illegitimately recombinedtrans-geneis subjectto silencing,which suggeststhat the chromosomal locationplays an important role. The efficient silencingactivity of certain loci mayresultfrom a particularsequencecontext,a specificDNA rearrangement,or asecondarystructure.Alternatively, the position of a locuswithin the nucleusmay determinetheefficiency of homology pairingor RNA processing.

To clarify theunderlyingmechanisms of genesilencingandto understandtheir importancein the regulationof plant development,we mustexpandourvery limited knowledgeabout the location of geneswithin the nucleus,thefactorsinvolved in chromatinconformation, and the regulationof transcripttransportandprocessing.Finally, we haveto understandhow developmentalprogramsandenvironmentalconditionsinfluencethe formationof epigeneticpatterns atthe DNAlevelandwhethertherearefeedbackcontrol signalsin theRNA processing pathway. Most certainly, we can expect morethan onemechanismof genesilencing to exist, and it is unlikely that the silencingphenomenareportedso far alreadyrepresenta complete selection.For thescientificcommunity, it will berewardingto noteandpursuesilencing eventswith greater emphasisthanin previousyears,whensilencedgeneswere outofthe scopeof many scientists.Understandingthe molecularbasisof genesi-lencing will not only improve the control of the application of transgenetechnology,it will most likely unveil new endogenouscontrol mechanismsinvolvedin the regulationof plantdevelopmentand genomeevolution.

ACKNOWLEDGMENTS

We thankB. Daviesfor critical readingof the manuscriptandthe followingcolleaguesfor communicating unpublished resultsor supplying preprints:D.Baulcombe,G. Coruzzi, P. Elomaa,A. Depicker,J. English, R. Flavell, R.Jorgensen,M. Metzlaff, andH. Vaucheret.We alsothankthemembersof theHumanCapitol andMobility network (grantnumberERBCHRXCT940530)on gene silencing for continuousdiscussion.

Any Annual Reviewchapter, aswell asany arti clecited in an Annual Reviewchapter,may bepurchased fromthe Annual ReviewsPreprints and Reprints service.

1-800-347-8007; 415-259-5017; email: [email protected]


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