Proc. Natd. Acad. Sci. USAVol. 91, pp. 3490-3496, April 1994

Review

Inactivation of gene expression in plants as a consequence of specificsequence duplicationR. B. FlavellJohn Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom

ABSTRACT Numerous examples nowexist in plants where the Insertion of mul-tiple copies of a transgene leads to loss ofexpression of some or all copies of thetransgene. Where the transgene containssequences homologous to an endogenousgene, expression of both transgene andendogenous gene is sometimes found to beimpaired. Several examples of these phe-nomena displaying different features arereviewed. Possible explanatious for theobserved phenomena are outlined, draw-ing on known cellular processes in Dro-sophila, fungi, and mammais as well asplants. It is hypothesized that duplicatedsequences can, under certain circum-stnce, become involved in cycles of hy-brid chromatin formation or other pro-cesses that generate the potential for mod-ification of inherited chromatin structureand cytosine methylation patterns. Theseepigenetic changes could lead to alteredtranscription rates or altered efficienciesofmRNA maturation and export from thenucleus. Where the loss of gene expressionis posttranscriptional, antisense RNAcould be formed on accumulated, ineffi-ciently processed RNAs by an RNA-dependent RNA polymerase or from achromosomal promoter and cause the ob-served loss of homologous mRNAs andpossibly the modification of homologousgenes. It is suggested that the mechanismsevolved to help silence the many copies oftransposable elements in plants. Multi-copy genes that are part ofthe normal genecatalog of a plant species must haveevolved to avoid these silencing mecha-nisms or their consequences.

Frequently, when unexpected phenom-ena are observed they are ignored, thentimidly explored and discussed, and onlylater published and debated with firmconviction in a more coherent frame-work. We have now entered the thirdphase for the unexpected phenomena as-sociated with the silencing of multiplecopies of genes inserted into plants andthe silencing of endogenous plant geneswith sequence homology to the newlyinserted DNA. The discoveries are ex-tremely important because (i) they revealpreviously unrecognized facets of thecontrol of gene expression except as rareisolated instances; (ii) they raise serious

questions for all those wishing to exploittransgenic plants in research laborato-ries, industries, or agriculture; and (iii)they have opened up avenues ofresearchin plant biology, including the means ofmaking mutants.The series of reports that established

the phenomenon were published from1989 to 1991, some 7 years after the firstseries of publications announcing theproduction of transgenic plants (but seerefs. 1 and 2). Prominent early publica-tions were those that showed that inser-tion of an additional copy of a chalconesynthase or dihydroflavonol-4-reductasegene into petunia plants led to the silenc-ing in many but not all the transgenicplants of the inserted gene and its endog-enous homologues (3-5). The coordinatesilencing of the transgene and the homol-ogous endogenous gene gave rise to theterm "cosuppression" (3, 5).

Chalcone synthase facilitates the con-version ofcoumaryol CoA and 3-malonylCoA to chalcone in the pathway of an-thocyanin pigment biosynthesis. Onegene of the small multigene family ofchalcone synthase in petunia is especiallyactive in petunia flowers, where pigmentproduction in the corolla and anthers issubstantial. After insertion of the chal-cone synthase gene under the control ofthe "constitutive" cauliflower mosaic vi-rus (CaMV) 35S promoter, up to 50% ofthe transgenic plants with the new geneshowed sectors of reduced or no antho-cyanin pigment in the flowers, and insome plants the flowers were completelywhite (3). This lack of pigment is corre-lated with very low levels ofmRNA fromthe newly inserted and the endogenouscopies of the chalcone synthase genes. Ina similar but not identical series of plantsMol and coworkers (6, 7) found fromnuclear "run on" experiments that na-scent RNA transcription is unaffected. Ihave confirmed this (unpublished data).

In some plants whose flowers are whitedue to the inserted chalcone synthasegene, occasional branches occur withpurple flowers or flowers with purplesectors. All the flowers on such a branchare usually very similar in pattern com-pared with the flowers on other branchesof the plant. Thus, it can be concludedthat a somatically inherited change of

3490

state of the transgene has occurred in thisbranch and, in some cases, during for-mation of the meristem of the branch.The occurrence of such epigeneticchanges during plant or flower develop-ment emphasizes that the silencing phe-nomenon is reversible and can be underdevelopmental control (5, 8). The newphenotype of a flower (and of the branchcontaining the flower) can be inherited (9)through sexual reproduction and seeddevelopment/germination. Many differ-ent "states" of transgene activity in co-suppression are possible based on thevariety of flower pigmentation patternsarising in isogenic progeny. The patternsof floral pigmentation imply that the ca-pacity of the transgene to cause suppres-sion is subject to developmental influ-ences.

This example illustrates many of thefeatures ofcosuppression but many otherexamples are now known in six plantspecies where gene inactivation resultsfrom the introduction of homologous se-quences (reviewed in refs. 10 and 11).Some of these are noted below.Tomato plants transformed with a

truncated gene encoding part of polyga-lacturonase under the control of theCaMV 35S promoter showed only lowlevels of endogenous polygalacturonasemRNA in ripening fruit where it is nor-mally highly induced. Transcripts of thetransgene were similarly reduced butonly in fruit (12). Thus, transcription ofboth genes may be essential for mutualinhibition. Degraded RNA products fromboth genes were found in cosuppressedfruit, suggesting that RNA transcriptionis not inhibited and therefore loss ofmature mRNA is due to posttranscrip-tional turnover.

Multiple copies of transgenes can in-teract to cosuppress each other (13, 14).Some Arabidopsis thaliana plants carry-ing multiple closely linked copies of thehygromycin phosphotransferase genelost resistance to hygromycin during de-velopment (14). This multicopy locusalso suppressed active copies ofthe samegene at another locus introduced bycrossing two strains. The cosuppression

Abbreviations: CaMV, cauliflower mosaic vi-rus; SAR, scaffold attachment region.

Proc. Natl. Acad. Sci. USA 91 (1994) 3491

could be reversed by separating the lociby outcrossing. These results suggestedthat the initial silencing of the multipleinserts at one locus, as well as the silenc-ing/activation of the genes in differentloci, is the consequence of specific se-quence homology-dependent interac-tions.

Similar results were found when a por-tion of the nopaline synthase gene underthe control of the CaMV 35S promoterwas introduced into tobacco plants al-ready containing wild-type nopaline syn-thase genes (15). All progeny containingboth introduced genes expressed no no-paline synthase activity. The lack of no-paline synthase activity was dependenton the presence of the portion of thenopaline synthase gene in the genes in-troduced second. When the wild-typenopaline synthase gene was segregatedaway from the duplicated nopaline syn-thase sequence its activity was fully orpartially restored in most plants. How-ever, suppression of activity was notcorrelated with methylation of cytosinesin the promoter as found in other cases (2,16).Vaucheret (17) has described an exam-

ple of a transgene inserted into tobaccothat contains a chimeric gene of nitritereductase in the antisense direction un-der the control of the CaMV 35S pro-moter linked to a sequence encoding neo-mycin phosphotransferase under the con-trol of the CaMV 19S promoter. Noexpression of either gene could be de-tected and this transgene complex sup-presses any gene under the control of the19S and 35S promoter inserted by sexualcrossing. Thus, the transgene complex isa strong cosuppressor of genes contain-ing related sequences. Cosuppressionwas stable but not observed until tran-sient expression of newly introducedtransferred DNA (T-DNA) had subsid-ed-i.e., cosuppression probably is apostintegration event (but see ref. 16).Ninety base pairs of homology in pro-moter sequences were sufficient to createa cosuppressed condition.These kinds of results help explain and

were reinforced by other examples inwhich transgenes present in one copy ina plant were much more active thantransgenes present in two or more copies(1, 16, 18-21).

In another well-investigated example,transgenic tobacco plants were createdthat contained two introduced genes, eachconferring resistance to a different antibi-otic (2, 22-25). The coding sequences ofthe two genes were unrelated in sequencebut they had a common promoter con-tained in two copies of 300 bp taken fromthe nopaline synthase gene ofAgrobacte-rium tumefaciens. One gene was intro-duced at the first transformation step andthe other was introduced at a secondtransformation step. Double transform-

ants were produced readily but, surpris-ingly, in 15% of the double transformantsthe phenotype of the gene conferring ka-namycin resistance (introduced first) waslost after introduction of the second geneconferring hygromycin resistance. In thiscase though, in contrast to the previouslymentioned examples, there was no cosup-pression in that the plants that had lostkanamycin resistance retained hygromy-cin resistance. (This could have been theresult of selection for hygromycin resis-tance in the second transformation step.)Loss ofkanamycin resistance was accom-panied by methylation of specific CpGresidues in its nopaline synthase promotersequence. The dependence of the trans-inactivation on the second gene encodinghygromycin resistance was illustrated byat least partial reactivation ofthe kanamy-cin-resistance gene and loss of cytosinemethylation in its promoter when the hy-gromycin gene was segregated away fromthe kanamycin-resistance gene.When the antibiotic-resistance trans-

genes were combined by sexual crossing,instead of by transformation, similar in-activation of the kanamycin-resistancephenotype in the presence of the hygro-mycin-resistance gene was recorded, thuseliminating the possibility that physiolog-ical states peculiar to the transformationprocedures were the cause of kanamycingene inactivation. Independently inte-grated kanamycin-resistant transgeneswhose activity could not be suppressed byspecific hygromycin-resistance trans-genes introduced by a second transforma-tion event also could not be inactivated bythe bringing together of the two intro-duced genes by sexual crossing. Differenttransgene combinations produced no, par-tial (unstable), or complete (stable) trans-inactivation of kanamycin resistance.These results imply that the ability totrans-inactivate or to be trans-inactivatedis defined by the state of the gene loci,including possibly their position in thechromosome and/or in the nucleus. Ofspecial interest is the observation that insome plants homozygous for kanamycin-resistance genes, complete somaticallystable trans-inactivation of kanamycin re-sistance occurred more readily than inplants heterozygous for the kanamycin-resistance genes (see also ref. 26).Assessment of all these examples indi-

cates that cosuppression and trans-inactivation of genes are dependent onsequence homology, can be epigeneticallyreversible, break down to various extentswhen the homologous loci are segregatedaway from one another, are sometimesdependent on transcription or a promoter,are sometimes under developmental con-trol, do not occur with all copies of trans-genes, and are sometimes associated withchanges in the cytosine methylation pat-tern of genes. In some examples, theinactivation is reciprocal-i.e., all copies

are inactivated while in others only onecopy is inactivated. Homozygous trans-genes are sometimes silenced more effec-tively than hemizygous transgenes. Lossof gene expression appears to be due toinhibition of transcription in some casesand degradation ofmRNA in others. Thisplethora ofunexpected phenomena needsto be explained.The examples quoted above have

emerged from recent research involvingtransgenes. However, a few examples ofallelic interactions leading to silencedstates of endogenous genes, some ofwhich are inherited, have been known inplants for a long time (9-11). These ex-amples include paramutation, in whichone special allele (the paramutable allele)is converted to a state of activity dis-played by the other allele (the paramuta-genic allele). The new state is inheritedbut is often epigenetically unstable andmay revert after the paramutable allelehas been separated from the paramuta-genic allele in progeny segregation (27-29).

Studies of the R locus in maize, whichconfers pigmentation in the aleuronelayer of the seed, have shown that para-mutable alleles are inherently unstableeven in the absence of a paramutagenicallele. This suggests that gene structure isan important element of the potential tointeract with a homologous allele. Thedevelopmental stage when the geneticchange occurs and the subsequent stabil-ity of the paramutated allele also differfrom one paramutable allele to another(29). Meyer et al. (30) discovered para-mutagenic-like versions of the maize Algene inserted into petunia under the con-trol of the CaMV 35S promoter. Epige-netic variants of an unstable transgenewere characterized. One homozygousvariant had lostAl gene expression; bothcopies were hypermethylated in theirpromoter region and showed paramuta-genic behavior that led to a permanent ortemporary inactivation of Al gene ex-pression in heterozgyotes with other Altransgene alleles. This inactivation cor-related with hypermethylation of bothalleles. The extent of paramutation wasvariable during plant development. Thisexample then provides one mechanismfor trans-inactivation: methylation pro-voked by a similarly methylated paramu-tagenic allele.Another discovery that I believe is

very relevant to the topic under reviewcame from molecular analysis of a seriesof alleles at the niv locus in Antirrhinumthat controls flower pigment production.The series includes closely related semi-dominant and recessive alleles where thesemidominant alleles inactivate wild-type alleles in heterozygotes. Each of thesemidominant alleles investigated has in-versions and multiple copies of niv genesequences. After consideration ofseveral

Rk'eview: Flavell

Proc. Natl. Acad. Sci. USA 91 (1994)

models for how alleles with these se-quence aberrations might confer domi-nance Coen et al. (31, 32) concluded thatthe inactivation of transcription probablywas a consequence of a physical interac-tion between the semidominant and wild-type alleles.The similarities between paramuta-

tion, the behavior of semidominant al-leles, and the trans-inactivation phenom-ena seen with transgenes raise the ques-tion whether all the phenomena areconsequences of the same collection ofmechanisms. This reviewer believes theyare.

Hypotheses to Explain theTrans-Inactivation Phenomena

Four categories of explanation have beenoffered by others to account for the phe-nomena (5-11, 13, 15, 30, 33-37). Theyare not mutually exclusive and no one isapplicable to all the examples surveyedabove. The first hypothesis suggests thatthe genes involved adopt an epigeneticstate that affects gene expression aftertheir physical interaction. In the secondhypothesis gene expression is inhibiteddue to competition between genes fornondiffusible factors, essential for or-dered transcription or translation, such asthe nuclear matrix or nuclear envelope.The third and fourth hypotheses apply tocases in which transcription is not inhib-ited but specific mRNA degradation is thecause of loss of gene expression. Thehypothesis debated most involves the pro-duction ofunintended antisense RNA for-mation and the degradation of mRNAsense-antisense duplexes. The other in-volves the accumulation of higher levelsof a specific RNA due to the addition ofextra copies of its gene and the conse-quential degradation of all of this mRNAspecies by some unknown mechanism.Below I outline nuclear processes that

I believe provide a useful background toconsider these and other explanations forthe whole range of trans-inactivationphenomena reviewed above. This outlineis then followed by a review of some ofthe supporting evidence for its constitu-ent elements.The processes to be considered first in

summary and in more detail later areshown in Fig. 1. Current views of chro-matin behavior and transcription implythat DNA in condensed chromatin is re-cruited into a decondensed form and be-comes attached to the nuclear matrix,and regulated transcription is initiated.Al these processes are programmed andregulated by complex interactions be-tween a large array ofregulatory proteinsand DNA in chromatin.

If it is hypothesized that under certainconditions homologous DNA sequencesinteract in somatic nuclei to form a hybridDNA duplex or triplex, then there could

ywbrd DNA .IIVIIstIto nuclear matrixmodification

I

Hybrid DNA -., '

K duplex/triplex pairing

Cytosine methylationand/or chromatin restructuring

assembly

12Translation

FIG. 1. Schematic illustrating the processes that could suffer aberrations to cause cosup-pression and trans-inactivation. Details are described in the text.

be several unusual consequences. First,a new chromatin/DNA structure wouldresult while the hybrid DNA was sus-tained. Second, there could be an ex-change of chromatin proteins to create anew state of the chromatin/genes in-volved in the duplex. Third, if heterodu-plex DNA were established, then differ-ent patterns of cytosine methylationcould be imprinted into the participatingDNA strands. These events should beviewed as being part of a dynamic reit-erative cycle, as shown in Fig. 1, wherereactions in segments of the cycle canoccur many times per cell cycle or onlyspasmodically in different cells duringdevelopment. On each occasion, thesame or different copies of a sequencecould be involved. The outcome of eachstage could be different on each occa-sion, creating instability of allele chro-matin structure and variation betweenalleles or, alternatively, the outcomecould be constant, leaving allele chroma-tin structure very stable and homoge-neous.Any new chromatin state of a gene

created via hybrid association or by gainof a protein or methylated cytosines viaother routes could influence the subse-

quent chromatin condensation pattern asdepicted in Fig. 1, with some states caus-ing the gene to remain in condensed chro-matin and silent. Hybrid DNA structuresor those of the new chromatin statesmight interfere with binding of the genesto the nuclear matrix or with subsequenttranscription to result in aberrant expres-sion of some or all copies of the genes.The trans-inactivation would thereforeresult from interference in steps labeled 1in Fig. 1.When trans-inactivation is posttran-

scriptional, two routes can be hypothe-sized, affecting steps labeled 2 in Fig. 1.It can be envisaged that altered allelicchromatin states are transcribed, but inthe wrong segment of the nucleus, andthe mRNA-protein complexes are im-properly or inefficiently transportedthrough the nucleus, processed, and ex-ported through the nuclear pore. Theyare consequently degraded. In an elabo-ration of this model, one could envisagethat if mRNA transport processing, ex-port, and possibly translation were inef-ficient, or excess mRNA accumulateddue to aberrantly high levels oftranscrip-tion, then an RNA-dependentRNA poly-merase could synthesize antisense RNA

3492 Review: FlaveH

Proc. Natl. Acad. Sci. USA 91 (1994) 3493

molecules on the mRNA templates. Theantisense RNAs would form complexeswith all the homologous sense mRNAs inthe cell, and the complexes would bedegraded before or after export from thenucleus. Alternatively, the RNA-RNAduplexes could be formed in the cyto-plasm and prevent translation. The in-volvement of an RNA-dependent RNApolymerase has been recently postulatedby Lindbo et al. (38) to explain the loss oftransgene mRNA and viral RNA in virus-resistant tobacco plants containing a nu-clear transgene encoding tobacco etchvirus coat protein and infected with to-bacco etch virus.

In the second route, it is envisaged thattransgenes become incorporated intochromosomes under the control of pro-moters that generate antisense RNA tothe transgene. The antisense RNA wouldinhibit RNA survival and translation asdescribed above.These scenarios could explain changes

in the state of certain homologous genes,their imprinting and the stability/insta-bility of new states, failure to be tran-scribed, or turnover of their mRNA.The hypotheses have been divided into

those that affect transcription and thosethat are posttranscriptional. These can belinked if a feedback system exists suchthat an accumulation of primary tran-scripts or antisense RNAs in the nucleusinfluences the chromatin or methylationstate of a gene to affect its ability toefficiently participate in transcription. Ifthis is the case, then inherited trans-inactivation of genes with sequence ho-mology could occur due to excess onRNA buildup without hybrid DNA for-mation. The cycles of modification ofchromatin structure shown in Fig. 1 with-out hybrid DNA formation are depictedto include this sort of possibility.Evidence for the principal steps envis-

aged in these hypotheses is reviewednext.

(i) Interactions Between Loci with Ho-mologous DNA Sequences. For interac-tions between loci with homologous se-quences to occur, the DNA must be ac-cessible. This suggests that the chromatinmust be decondensed and DNA strands ofthe interacting loci be opened up-presumably during transcription, regula-tory protein binding, replication, or re-combination. This might explain the needfor a promoter for trans-inactivation/cosuppression in some cases. CertainDNA structures such as the presence ofinverted repeats might provoke alterna-tive forms of genes that facilitate accessi-bility ofDNA (32). Sequence-specific in-teraction implies hybrid chromatin orDNA duplex or triplex (39) formation, atleast transiently, but this would need to bevery efficient to account for the frequencyof trans-inactivation observed, unless thechromatin/DNA that interacted was her-

itably imprinted. There is little evidence,direct or indirect, for such regular inter-actions involving all or most accessiblechromatin sectors in somatic plant cells.

In yeast, such a DNA homology-searching process has been inferred toaccount for the equivalent frequencies ofallelic and ectopic (in different position inthe chromosome) meiotic recombinationbetween homologous sequences (40). InNeurospora, duplicated sequences are de-tected by a homology searching/sensingprocess in the haploid nuclei of dikaryonsbefore meiosis (41). The duplicated se-quences are modified by cytosine meth-ylation and a high proportion of the mod-ified cytosines are substituted by thymi-dine. This repeat-induced point mutationprocess leads to destruction ofgene func-tion and elimination of such mutant prog-eny from the population. The DNA pair-ing process is not dependent on the mei-otic chromosome pairing mechanisms(42). In Ascobolus immersus artificialgene repeats are also heritably inacti-vated, premeiotically, by cytosine meth-ylation but other mutations are not intro-duced (43). DNA sequences can undergoseveral rounds of pairing so that multiplecopies of a sequence can be inactivatedsequentially (44). Thus, the pairing ma-chinery does not distinguish betweenmethylated and nonmethylated copies.These observations on fungi provide a

useful basis for considering possiblemechanisms of trans-inactivation/cosup-pression in plants. They provide a prec-edent for a process to control the expres-sion of unusual duplicated sequences in-volving an efficient homology searching/sensing system. In these fungi, hybrid orpaired DNA is presumably recognized inpremeiotic cells, directly or indirectly, bya de novo methylase or a maintenancemethylase that operates on hemimethy-lated DNA. Any parallel in plants wouldhave to occur, presumably, in any so-matic cell.

If duplicated sequences involving atransgene recognize each other and formhybrid DNA. even only transiently, howcould different states ofthe transgenes ortransgene and endogenous genes emergeand be inherited? There are two sorts ofpossibilities for this, with precedents inother kinds oforganisms: restructuring ofchromatin and DNA sequence modifica-tion. These processes are reviewed be-low.

(it) Chromatin Restructuring. The casefor modification of interacting loci viachromatin changes is based on observa-tions established in Drosophila. Elevenexamples have been reviewed recently(45) in which the expression of a gene isinfluenced by "sensing" the presence ofanother specific gene after some kind oflocalized somatic chromosome pairing(46-48). A transcription factor associ-ated with the chromatin of one of the

genes is postulated to interact also withthe promoter/enhancer of another chro-mosome and modify the expression ofthis second locus (49). However, anothermodel involving transacting regulatoryRNAs as mediators ofthe effects has alsobeen put forward (45). In a second type ofinteraction, a gene becomes inactivatedby assuming a heterochromatic, repress-ing chromatin structure from its neigh-boring sequences. It then pairs with awild-type allele on another chromosomeand a mutant phenotype results becausethe chromatin structure of the wild-typegene is also converted to an inactive,heterochromatic form. This model isbased on the ability ofchromatin proteinsdetermining heterochromatic condensa-tion to initiate the condensation process,which then proceeds along the chromo-some until some interfering componentsare encountered. When such initiatingproteins are transferred to the wild-typegene via close pairing of chromosomes,the heterochromatic structure is imposedupon it also. Such modes of heterochro-matic chromatin assembly are dominantto those normally determining the chro-matin structure at the wild-type genelocus. When somatic pairing is disruptedin these cases, more normal gene expres-sion ensues. In other cases, mutant phe-notypes are enhanced when pairing isdisrupted. The same is true for the trans-sensing effects on polytene chromosomepuffs. In such cases, a mutant site willpuff and accumulate mRNA when pairedwith its wild-type homologue but notwhen these chromosomes are desyn-apsed or remain homozygously paired.An important issue for cosuppression

and trans-inactivation in plants, as notedabove, is how new states of the loci in-volved, however they are created, arestabilized and inherited. Other geneticstudies on Drosophila have revealed geneproducts that appear to influence the stateofgene activity through organismal devel-opment. Paro (50) has described a modelthat represents a way to imprint on/offtranscription status into the higher-orderchromatin structure surrounding a gene.He envisages chromatin of early embryosin a neutral state, allowing signals to ac-tivate specific gene loci. When the signalsare not received, specific proteins interactwith cis-regulatory elements ofa gene andact as nucleation signals for a kind ofheterochromatization, a local state that isclonally inherited through development.A parallel mechanism is envisagedwhereby specific proteins would bind tospecific loci to keep them open and avail-able to developmentally controlled spe-cific transcription factors.The programming of transcriptional

competence based on organization ofchromatin structure is now receivingmuch attention in yeast and Drosophila,following the discovery of numerous

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Proc. Natl. Acad. Sci. USA 91 (1994)

gene products that influence the expres-sion of many genes and that are likely towork via influencing chromatin proteincomplexes (50-52). In summary, there isgrowing evidence from Drosophila andyeast, where the most detailed moleculargenetic studies have been carried outthat, in certain examples at least, chro-matin states can be transferred betweenhomologously paired chromatin seg-ments, that competence for transcriptionis determined by chromatin structure,and that some chromatin states can beclonally inherited when not disturbed.

(ij) DNA Sequence Modification byMethylation. The second mechanism thatcan be envisaged to account for the her-itable modifications of the duplicatedDNA sequences involved in cosuppres-sion and trans-inactivation is methylationof different cytosine residues. This pro-cess could occur either in the hybridDNA or after establishment of a chroma-tin structure by any route.

It is attractive to consider this processfor many reasons: (i) the process occursin all plant nuclei; (ii) cytosine methyl-ation changes have been observed tocorrelate with at least some cases ofcosuppression or trans-inactivation (e.g.,see refs. 16, 23, and 30); (iii) it results inimprinting of DNA, and imprinted pat-terns are inherited due to the, addition ofmethyl groups to new DNA strandsbased on the pattern in the old strand(53); (iv) enhanced cytosine methylationat key sites is known to correlate withmodified chromatin structure and geneexpression (54); and (v) it is known to bepart of the mechanism used to silenceartificially duplicated genes in fungi (41-43).

If hybrid DNA is formed between se-quences of duplicate transgenes or be-tween transgenes and endogenous se-quences, then the DNA strands will behemimethylated at many sites because thecytosine methylation pattern will differbetween the two parental sequences. Thisis because when transgenes are intro-duced from Agrobacterium they are un-methylated and without a plant-deter-mined chromatin structure. The processof cytosine methylation in CpG andCpXpG motifs must involve de novomethylation. It is likely that the stabilizedpattern will reflect the chromosomal en-vironment where it is inserted and randomprocesses (30). The pattern may takemany cell generations to stabilize. Forthese reasons, different insertions of thesame transgene are likely to have differentmethylation patterns.There is a very active methylase that

recognizes hemimethylated DNA inplants (55) since 80%o of the CpG and ofthe CpXpG sites are methylated andthese have to be methylated after everyround of replication. Therefore, givenhybrid DNA and accessibility of these

sequences to the appropriate methylase,the number of methylated sites in thehybrid DNA is likely to increase. Uponseparation of the hybrid DNA strandsand their assimilation back into their par-ent duplexes, hemimethylated parenttemplates would exist and would bemethylated. Thus, after the interaction,both parental genes would be modified inthe regions that formed hybrid DNA-generally in the direction of increasedcytosine methylation. Ifthe altered meth-ylation pattern affected transcription di-rectly or indirectly by the altered bindingof regulatory proteins or by affecting thecondensation pattern into a differentchromatin conformation, then new heri-table states of gene expression wouldhave been created. The paired DNA se-quences would have similar changes im-printed and thus might share similarchanges in gene expression in many butnot all cases. The process is usefully seenas a dynamic cyclical pathway (see Fig.1) and multiple rounds of the cycle couldoccur in each cell cycle. To attain a stablestate for all copies of a sequence, manycycles would probably be required.The observed outcome would depend

on the original state(s) of the trans-gene(s), and of the endogenous loci, andthe role of specific cytosine residues incontrolling levels of transcription, pro-tein binding, and chromatin structure.Thus, gene expression could be reduced,increased, or unchanged.A prediction of methylation of hemim-

ethylated hybrid DNA is that homolo-gous sequences should accumulate thesame cytosine methylation pattern in thesame cell lineage. This is testable. How-ever, the pattern of methylated cytosinesestablished could differ following the res-olution of hybrid DNA duplexes in dif-ferent cells, and thus chimeras would beproduced, as has been observed (10, 11).The requirement for a transgene to pro-voke easily recognized cosuppressionfrequently may be because only newlyinserted genes are in different, unregu-lated states of cytosine methylation andare therefore capable of giving rise to avariation in cytosine methylation andchromatin patterns via hybrid DNA for-mation.

(iv) Inhibition of mRNA Processing,Transport, Export, or Translation. Thefirst product ofRNA transcription under-goes a series ofcomplex processing stepsincluding removal of intervening se-quences, ifpresent, capping at the 5' end,and polyadenylylation at the 3' end toproduce the RNA that is transportedthrough the nuclear pore for translation.How these processes occur within thenuclear architecture is not well docu-mented. It has been proposed that RNAsare transported from the site of synthesisto the pore across a solid nuclear sub-structure distinct from chromatin (56).

Evidence has been obtained that newlysynthesized RNAs are associated withthe nuclear matrix and pass along definedpaths (refs. 57 and 58 but see ref. 59). Thecomplexes that effect splicing may belocalized in specific foci so RNAs requir-ing splicing may pass through such foci(60, 61). Association of mRNA with asplicing complex inhibits export; exportis dependent on release from a spliceo-some, and data have been reported toshow that 5' capping and a correct 3' endare important for export (62).Knowledge of these mechanisms im-

plies that, for correct gene expression,RNA transcription may need to takeplace in an appropriate place in the nu-cleus and RNAs might traverse specificdomains to be properly processed forexport. If these requirements are notsatisfied due to the relocalization ofgenes after their interaction and modifi-cation, then there could be delays inRNA transport and processing that resultin degradation of the RNA.

Alternatively, if inefficient processingand transport were to occur, antisenseRNA could be generated by using theaccumulated RNA as template, becauseRNA-dependent RNA polymerase oc-curs in plant cells (63, 64). Its role andcellular location are unknown. The activ-ity is induced after infection with virusesand after wounding. It is capable of mak-ing antisense RNA fragments from plantRNA and appears to display little tem-plate specificity. This activity, if nuclear,could synthesize antisense RNA in thenucleus. Alternatively, if cytoplasmic, itcould synthesize antisense RNAs on ex-ported RNAs present in excess in rela-tion to regulated translation capacity oron RNAs inefficiently translated due tostructural defects through aberrant pro-cessing. The antisense RNA productscould remain cytoplasmic and preventtranslation or enter the nucleus to inter-fere with RNA maturation.The inactivation of sense mRNA to the

transgene by unintended antisense RNAas an explanation for cosuppression hasbeen debated by several authors (10, 11,33-35). The authors envisaged that theantisense RNA would originate eitherfrom an active promoter in the host chro-mosome initiating transcription in the op-posite direction to the transgene or fromanother gene promoter in the oppositeorientation on the inserted T-DNA.

Efficient production of antisense RNAfrom a chromosome promoter would leadto down-regulation of sequence-specificgene expression and some antisenseRNA has been detected in cosuppressedpetunia plants (6, 7). However, there arespecific pieces ofevidence in examples ofcosuppression and trans-inactivation thatargue strongly against a chromosomalpromoter being the source of antisenseRNA and the sole component of a model

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Proc. Natl. Acad. Sci. USA 91 (1994) 3495

to explain all the phenomena associatedwith loss of gene expression. The evi-dence has been reviewed by others (10,11, 34).What is needed to evaluate the applica-

bility of antisense RNA formation to thecause of down-regulation of homologousgene expression, in at least some exam-ples oftrans-inactivation by transgenes, isa much better understanding of how an-tisense RNA effects down-regulation ofgene expression, measurements of an-tisense and sense RNA levels in the rele-vant cells and their nuclei before as well asafterRNA degradation, and knowledge ofthe role of RNA-dependent RNA poly-merase and of the ability of accumulatedRNA products to feed back and interferewith transcription. It will also be impor-tant to discover the relationship betweenthe mRNA turnover revealed by trans-genes and endogenous posttranscriptionalcontrol systems that regulate mRNA turn-over.

Implications of the Hypotheses

I have reviewed how chromatin interac-tions, nuclear control systems of chro-matin structure, and DNA methylationmechanisms might produce changes instate in duplicated loci that interact phys-ically or contain homologous sequences.The two key processes of chromatin andDNA modifications are not mutually ex-clusive but are inextricably linked. It isknown that the distribution ofmethylatedcytosine can affect the complement ofchromatin proteins, chromatin condensa-tion, and accessibility of the chromatin totranscription complexes and specifictranscription factors. Similarly, the com-plement of chromatin proteins can influ-ence the accessibility of DNA to meth-ylase and hence the cytosine methylationpattern.The occurrence and outcome of these

potential processes in a transgenic plantwould depend on the following: (i)whether two homologous sequences areable to pair (this might depend on theirphysical positions in the nucleus and onwhether the DNA is accessible to allowhomology-based recognition); (ii) thekinds ofproteins bound to the loci and thenature ofthe nuclear processes that couldmodify the proteins at the loci; and (iii)the influence of the new chromatin stateon its competence to influence chromatinbinding to the nuclear matrix, active tran-scription, and efficient mRNA process-ing and export from the nucleus.

Different transgenes, or the sametransgenes at different locations, mightbe expected to differ in i and ii, and henceiii, especially as the molecular environ-ment of a gene is known to influence itsadopted chromatin structure. If there isno significant variation in ii or no effecton iii then cosuppression or trans-

inactivations would not be recognized.Failure to observe effects would not,however, necessarily imply that the cy-cles depicted in Fig. 1 do not occur.Nevertheless, it is important to askwhether the proposed cycles occur reg-ularly to affect genes in all plant cells orwhether they are peculiar to transgenes.It is extremely unlikely that the cellularprocesses are invoked only by trans-genes-indeed, the examples to supportthe model-include paramutation and thedominant alleles of endogenous plantgenes reviewed earlier. However, it ap-pears probable to me that transgenesadopt positions and chromatin structurethat increase the probability that theyparticipate in hybrid DNA formation. Ifthey lack close-by sequences (scaffoldattachment regions; SARs) to aid integra-tion into the nuclear matrix, have strongconstitutive promoters, and sustain anopen chromatin structure, they are morelikely than other genes to become in-volved in homology-searching processes,at least until they are inactivated into aclosed, more silent chromatin structure.Recent results have suggested that if

matrix binding or flanking sequences thatmight contain a SAR are added to trans-genes, then activity is much less sensitiveto position effects (65). These importantobservations suggest that SARs may ei-ther interfere with hybrid DNA forma-tion by enclosing DNA more efficientlyor initiate a chromatin structure that isdominant and not significantly modifiedirregularly after hybrid DNA formation,or both. If the former, then hybrid DNAformation may be a default pathway ofgenes not properly associated with thenuclear matrix. I suspect that adaptedgenes, those evolved as part of the nor-mal genome, are likely to have a muchreduced probability of participating inhybrid DNA formation with their allelesor other members of their family or haveadopted chromatin and DNA sequenceenvironments to produce an outcomethat is neutral with respect to gene ex-pression. Unusual gene structures suchas inverted duplication in complex trans-genes or in mutants could provoke hybridDNA pairing or its aberrant resolution.Any model to explain trans-inactiva-

tions must account for how gene reacti-vation occurs following segregation ofthe duplicated loci into separate progeny.If the inactive state is dependent on con-tinual and repeated interactions (hybridDNA formation), then failure to form ahybrid DNA could rapidly lead to rever-sal. However, when the inactivation isdue to an inherited state of methylationand chromatin configurations that is notreedited at meiosis, then the inactivatedstate might linger until new rounds ofchromatin resetting had occurred. Casesof both types probably occur.

It is now essential to test the varioushypotheses further. This will requirestudying the localization of genes andtranscripts in nuclei, investigating chro-matin structure and cytosine methylationpatterns, searching for antisense RNAs,and exploring the activity of RNA-dependent RNA polymerase. It is alsoimportant to find out why transgenes insome locations do not involve cosuppres-sion or trans-inactivation and find waysto control the phenomena. Uncontrolledinactivation of genes is a serious problemfor the exploitation of plants in com-merce, if not in the laboratory. On theother hand, inactivation of endogenousgenes by insertion of homologous se-quences into the genome opens up theopportunity to create mutants and iden-tify the function of unknown sequences.

Significance of Cosuppression for GeneOrganization and Genome Evolution

Whatever the mechanism(s) of cosup-pression and trans-inactivation, the phe-nomena are likely to have played a sub-stantial part in the evolution of genes,genomes, and mechanisms controllinggene expression. The immediate silenc-ing of duplicated genes is a powerfulevent and is likely to have evolved forgood reasons. This, in plants, is likely tohave been to silence transposable ele-ments and other repeats that can accu-mulate in the genome by mechanismsthat make it difficult for them to be elim-inated without very strong selectionforces. Active transposable elements andretrotransposons are exceedingly muta-genic, and there would be strong selec-tion in favor of mechanisms that cansilence them immediately. New, activecopies of transposable elements are innonallelic positions with respect to othercopies of homologous elements, similarto transgenes, and are likely to have newmethylation and chromatin propertiesupon insertion and to be in accessiblechromatin because of their mode of inte-gration/movement in the chromosome.The genes that are a regular part of the

primary gene catalog, including membersof multigene families, must have evolvedmechanisms that either prevent them in-teracting or have evolved chromatinstructures and mRNA processing andexport strategies that upon interactionwith the cellular processes outlined dis-play a neutral outcome with respect togene expression. This is consistent withgenes evolving in particular environ-ments of SARs and sequences that con-trol chromatin structures, methylationpatterns, nuclear locations, and adaptivelevels of gene expression during plantdevelopment.

I wish to thank Rich Jorgensen, MarjorieMatzke, and Peter Meyer for teaching me so

Rveview: Flavell

Proc. Natl. Acad. Sci. USA 91 (1994)

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