Epigenetic history of an Arabidopsis trans-silencer locus and a test for relay of trans-silencing activity
© Qin and von Arnim; licensee BioMed Central Ltd. 2002
Received: 20 August 2002
Accepted: 11 December 2002
Published: 11 December 2002
Meiotically heritable epimutations affecting transgene expression are not well understood, even and in particular in the plant model species, Arabidopsis thaliana. The Arabidopsis trans-silencer locus, C73, which encodes a fusion protein between the repressor of photomorphogenesis, COP1, and green fluorescent protein (GFP-COP1), heritably modifies the expression pattern and cop1-like cosuppression phenotypes of multiple GFP-COP1 target loci by transcriptional gene silencing.
Here we describe three additional features of trans-silencing by the C73 locus. First, the silencing phenotype of C73 and of similar complex loci was acquired epigenetically over the course of no more than two plant generations via a stage resembling posttranscriptional silencing. Second, imprints imposed by the C73 locus were maintained heritably for at least five generations in the absence of the silencer with only sporadic spontaneous reversion. Third, the pairing of two other GFP-COP1 transgene loci, L91 and E82, showed an increased tendency for epigenetic modification when L91 carried an epigenetic imprint from C73, but not when E82 bore the imprint.
The latter data suggest a transfer of trans-silencing activity from one transgene locus, C73, to another, namely L91. These results extend our operational understanding of interactions among transgenes in Arabidopsis.
Certain genetic loci are known to modify the expression of other allelic or non-allelic partner loci in a meiotically heritable fashion. If allelic, such non-Mendelian interactions are referred to as paramutation. In the non-allelic case, the term 'heritable trans-silencing' may be used. Paramutation has been studied extensively for four maize loci that encode transcriptional regulators of pigment biosynthesis . Paramutation has also been investigated in the Arabidopsis PAI gene family, at the a1 and chalcone synthase (CHS) transgene loci in petunia, and in a number of other cases [2–4]. The Arabidopsis resistance gene BAL/CPR1 displays a related form of epigenetic instability [5, 6]. Interactions resembling paramutation also occur among non-allelic transgene loci with DNA sequence homology. In these cases one master locus tends to suppress the expression of its target locus ('trans-silencing') [7–11]. Paramutation and trans-silencing are related processes. For example, an inverted repeat allele of the tryptophan biosynthetic gene PAI resident in the WS ecotype of Arabidopsis silences homologous PAI alleles, as well as unlinked PAI genes, from the Columbia ecotype . Likewise, complex synthetic transgenes composed of PAI inverted repeats were able to trans-methylate homologous, yet non-allelic, target loci . Similar events have been observed in other species [e.g. ].
Epigenetic activity, defined here as partnership in heritable trans-silencing or paramutation, is not predictable from the DNA sequence alone, and its molecular basis is incompletely understood. Arguably the most widely implicated factor is the presence of complex DNA sequence repeats. These repeats are often part of the trans-silencer locus or paramutagenic locus or its target [10, 14–17]. However, the repeat can sometimes be located at a considerable distance from the affected locus itself, as has recently been shown for the single-copy maize b1 gene . Other than transcriptional gene silencing, paramutation is only weakly correlated with DNA methylation. The methylation status of the maize r1 genes and Arabidopsis PAI genes is altered upon paramutation [12, 19], while that of maize b1 is not , except, in the non-conventional way, in the distal paramutation-control region of b1 . Conversely, extensive exposure of a wild-type Arabidopsis SUPERMAN allele to a heavily methylated and transcriptionally silenced epiallele did not reveal any trans-silencing .
The stochastic nature of gene silencing has been documented on numerous occasions, e.g. [9, 21], including in one of the initial descriptions of cosuppression . However, with specific regards to epigenetically active transgene loci, it is often not transparent whether their silencing behavior was stable over successive generations, nor when and how individual loci acquired their epigenetic activity. Certain loci are known to acquire their epigenetic activity spontaneously , suggesting an epigenetic control mechanism. For instance, at the b1 locus, one specific allele can switch spontaneously from a non-paramutagenic, transcriptionally active, state to a paramutagenic, transcriptionally silenced, state . Whether the same is true for trans-silencing is not well established.
Whether the imprint imposed by a paramutagenic locus is relayed effectively from the first target locus to a secondary target locus is a variable characteristic of paramutation systems, and this has implications for the speed of epiallele conversion within outcrossing populations. Paramutable maize alleles of b1 and pl1 are highly effective in relaying such an imprint [24, 25] (also see ), whereas other loci, although sensitive to imprinting, are less effective  or apparently ineffective  in relaying the imprint to secondary targets. Likewise, Arabidopsis PAI2 and PAI3 genes that have been trans-methylated by the PAI1/PAI4 locus do not transfer their methylation status to naive singlet genes . In fact, there are few well-documented cases for the non-allelic relay of trans-silencing ability [11, 24]. Does this amount to an operational difference between allelic (paramutation) and non-allelic (trans-silencing) interactions, or does it simply reflect a lack of data?
The experimental strategy of monitoring heritable epialleles via their effect on visible cosuppression patterns has been pioneered in the chalcone synthase (CHS) family of petunia and Arabidopsis [4, 10, 30]. For comparison, CHS trans-silencing offers exquisite sensitivity, in part due to the cell autonomy of its pigmentation phenotype, whereas COP1 essentially yields a whole-plant phenotype. Petunia CHS also lends itself to detect somatic transitions in epialleles due to its indeterminate growth habit, whereas Arabidopsis COP1 is more suitable for monitoring early epigenetic activity in seedlings and rosette plants.
We have begun to define the determinants of epigenetic activity of the GFP-COP1 loci. The trans-silencers as well as their targets reside in single-copy, gene-rich regions that are only sparsely populated with repetitive or transposable elements, features that have been shown to mediate epigenetic activity in other cases [18, 31, 32]. Therefore trans-silencing of GFP-COP1 is probably not mediated by transgene flanking sequences . In contrast, as with other genes cited above, trans-silencing ability is correlated with transgene locus structure, given that the C73 and C97 trans-silencers contain multiple T-DNAs while their targets, E82 and L91, are essentially dimeric and monomeric, respectively.
Here we report that the Arabidopsis C73 and C97 trans-silencer loci displayed TGS and trans-silencing after first passing through a transitory phase of PTGS over the course of the first two transgenic generations. A similar epigenetic instability of the initial PTGS phenotype was typical for a subset of other oligomeric loci, but was never observed with monomeric 35S:GFP-COP1 loci. It is this instability of cosuppression that originally prompted us to categorize certain loci as type C rather than type E. We also characterized the heritability of the imprint imposed by the C73 trans-silencer locus with respect to (a) cosuppression of the COP1 endogene and (b) a characteristic yet unusual expansion of transgene expression at the target locus. Finally, our data suggest that the L91 target locus acquires limited trans-silencing activity of its own after exposure to C73, which is an unusual case for transgenes. However, these data must be interpreted in light of another novel observation, namely that even relatively simple, transcriptionally active, transgene loci can interact with nonlinear gene dosage effects that have characteristics of epigenetic trans-silencing.
The silencing phenotypes of C73 and C97 were acquired epigenetically
Transgene locus structure of the E82 locus
Trans-silencing by C73 results in variable novel transgene expression patterns, including spatial expansion of transgene expression
These results underscore the similarity between trans-silencing and paramutation, because in both cases epialleles are often silenced incompletely and may also adopt novel regulatory patterns [4, 10], for example light inducibility . Perhaps, silencing at the dimeric E82 locus involves a hierarchical relationship between its two intact T-DNAs. The C73 locus may preferentially target a postulated 'master' T-DNA, which in turn loses its ability to post-transcriptionally silence the 'subordinate' T-DNA within the E82 locus.
Two target loci of C73 maintain their epigenetic imprint heritably for up to five generations
A diagnostic PCR assay was developed for the L91, E82, and C73 loci to unambiguously identify F2 segregants containing a trans-silenced L91 or E82 target locus but lacking the C73 silencer locus (Fig. 5B; compare lanes 1–4 with lanes 5–8). In addition, hemizygous segregants were distinguished from homozygotes by segregation of the kanamycin resistance gene in their selfed progeny (compare lanes 1–2 with 3–4). Subsequently, the rate of phenotypic reversion from trans-silencing was observed for hemizygous and homozygous lineages of E82 and L91. A legend for scoring the cop1-like cosuppression and GFP-COP1 transgene silencing patterns is shown in Fig. 5A. Unless exposed to C73, E82 plants are always cop1-like and silenced for GFP-COP1, and the same is true for homozygous L91 plants. In contrast, E82 plants and homozygous L91 plants were wild type-like, rather than cosuppressed, in the F2 if the transgene had been exposed to C73 in the F1 (Fig. 5C and 5D, previously summarized in ). Both the reactivation of E82's GFP-COP1 transgene expression as well as suppression of endogene silencing were maintained for up to four additional generations in the absence of C73 in hemizygous and homozygous families, while C73 remained silenced (Fig. 5C). The L91 lineage also maintained its imprint from C73 over up to five generations. Specifically, the cop1-like cosuppression phenotype typical for homozygous L91 plants remained suppressed (Fig. 5D).
Although the imprints on the E82 and L91 loci proved to be fairly stable, reversion was observed in two ways. First, a small fraction (less than 20% expected for full reversion) of cop1-like E82 plants and L91 plants appeared in the F4 generation (not reflected in Fig. 5); and second, the GFP-COP1 expression of E82 gradually reverted back from the reactivated pattern to the silenced pattern especially in hemizygous families (Fig. 5C). The transgene expression data for the imprinted L91 lineage were less informative. However, it appears that the hemizygous L91 lineage gradually recovered its GFP-COP1 expression but without reaching the threshold required for posttranscriptional cosuppression of the COP1 gene.
Imprinting relay: Do trans-silenced E82 and L91 loci acquire trans-silencing ability?
Exposure to C73 differentially modifies the trans-silencing activity of the L91 and E82 loci – Gene expression data from individually genotyped F1 hybrid plants. See Fig. 6 for details.
GFP-COP1 transgene [% of plants]
L91'/-; E82 /-
E82'/-; L91 /-
L91 /-; E82 /-
The PTGS-mediated endogene cosuppression phenotype of the E82 locus is reduced by trans-silenced L91' as well as by naïve L91.
F2 family endogene silencing phenotype
L91' × E82
E82' × L91
L91 × E82
L91' × E82'
one cop plant
Both L91' and L91 can heritably suppress the cop-like endogene silencing by the E82 locus.
Transgene locus present
L91 & E82
L91' × E82
L91 × E82
In contrast to the epigenetic activity of trans-silenced L91', E82' had no such activity. When E82' was combined with L91 both loci cooperated to cause endogene silencing, as seen with unmodified E82 and L91 (Table 1). This was unexpected given that E82' caused little or no endogene cosuppression with L91' or on its own. Cooperative cosuppression between bona fide imprinted E82' and a naive L91 locus was again detected in the F2 progeny (Table 2). Thus, E82' was clearly unable to trans-silence a naive L91 locus and in fact seemed to lose its imprint when exposed to L91; therefore these families were not analyzed further. E82' also did not trans-silence a naive E83 locus (not shown).
Taken together, these data allow three conclusions. First, experiments with naive L91 and E82 demonstrated that even relatively simple transgene loci associated with PTGS can display non-additive gene dosage effects, as apparent by heritable suppression of PTGS. Second, the imprint left on the L91 locus by the C73 trans-silencer did modify L91's epistatic interaction with a non-allelic target locus, E82, as seen in the F1 generation. However, it remains to be determined whether this trans-silencing effect is more likely to be heritable when L91 is in an imprinted state (L91') than in the naive state (L91). Third, the imprint on the E82 locus (E82') was more labile than the imprint on L91' and showed no evidence of being transferable.
Epigenetic imprints affecting the expression of nuclear genes differ in the efficiency with which they are relayed onto homologous sequences. In the classical case of paramutation at the maize b1 locus, the imprint is relayed with 100% efficiency . That is, a paramutated allele is turned into a paramutagenic allele. In contrast, to our knowledge, no trans-silenced transgene has been shown to acquire heritable trans-silencing ability suggesting that perhaps trans-silencing ability is more difficult to relay between non-allelic transgenes than between allelic loci. However, the endogenous p1-rr allele of maize did acquire paramutagenicity after exposure to a specific P1-enhancer transgene . Against this background we investigated the possible relay of an imprint triggered by the Arabidopsis C73 trans-silencer locus, asking specifically whether targets trans-silenced by the C73 locus (a) acquired trans-silencing activity, and (b) were able to pass their imprint on to a naive target locus in a heritable fashion.
In summary, the imprinted E82 locus (E82') displayed no significant ability to trans-silence a naive L91 locus. In contrast, L91' was initially able to trans-silence E82, as judged by suppression of the endogene cosuppression phenotype associated with naive E82; importantly, the unmodified L91 control locus did not have this ability (Table 1). Thus, we demonstrated aspect (a) of the imprinting relay. Moreover, the suppression of silencing at the E82 locus was partially but not fully heritable given that some but not all E82 segregants lacking L91' were wild-type (Table 3). Yet, these data fall short of proving aspect (b) of the imprinting relay because, surprisingly, the combination of naive L91 and E82 loci also caused non-linear gene dosage effects that could result in the heritable trans-silencing of the E82 locus in at least one instance. Whether L91' might paramutate an allelic L91 locus remains to be tested. The non-linear gene dosage effects between L91 and E82 were surprising to us because in tobacco, similar experiments conducted with 35S:GUS transgenes did not raise the suspicion of epigenetically heritable effects .
It is informative to compare the epigenetic interactions of the 35S:GFP-COP1 transgenes with those of the PAI (trans)genes, the only other well-characterized system in Arabidopsis . Both gene sets consist of relatively simple repeat structures and singlet loci, which reside in essentially single-copy environments of Arabidopsis ( and unpublished observations). The first comparison relates to the speed of epigenetic change. Here, combining a master locus (C73) with a non-allelic singlet locus possessing 100% sequence identity (L91) resulted in immediate trans-silencing, as seen by a reduction of GFP-COP1 expression and block of cosuppression, which was stably maintained in the presence or absence of the master locus. In contrast, in the PAI gene family, combining the master locus (WS ecotype PAI1/PAI4) with a non-allelic singlet target locus possessing 100% sequence identity (Columbia PAI2) resulted in methylation after two generations of heterozygous contact, and methylation became more pronounced after another two generations . Likewise, in the petunia CHS gene family, interaction between an epiallele of the direct-repeat locus, CHS41, with two types of target loci, either an unlinked inverted repeat locus or a more naïve allele of CHS41, was initially additive and only became suppressive in the second generation [4, 10].
The second comparison relates to the relay of epigenetic activity. In our system, a trans-silenced singlet gene (L91') was able to trans-silence a naive homolog (E82) within a single generation. In contrast, a trans-methylated singlet PAI2 gene did not trans-methylate a naive allelic PAI2 gene within three generations . Additional experimentation may eventually provide an answer to the question of what controls the efficiency of such epigenetic interactions.
Cosuppression of the endogenous COP1 gene is a consequence of posttranscriptional silencing, while transcriptionally trans-silenced loci do not cosuppress COP1 . In our hands, a subset of four 35S:GFP-COP1 transgene loci shifted from a cosuppressing state, bona fide PTGS, to a non-cosuppressing state, i.e. TGS, within two transgenic generations. These loci are therefore referred to as 'complex' loci. Note that type L and type E loci did not lose their PTGS phenotype (Fig. 2B). This includes the L72 locus, which has a reduced penetrance of cosuppression that may be attributable to its pericentromeric location . Intriguingly, both trans-silencer loci, C73 and C97, belong to the 'complex' group, adding weight to the proposition that, like paramutagenicity, trans-silencing activity is encoded epigenetically. Vice versa, this transition raises the question whether the originally strong transcription of the GFP-COP1 genes, or perhaps the ensuing PTGS phase, somehow sets the stage for the subsequent trans-silencing activity, especially if multimeric T-DNA loci are involved. PTGS and trans-silencing are similarly intertwined in other cases, for example in the epimutable petunia CHS41 locus . PTGS often leads to DNA methylation, although primarily in coding regions . However, DNA methylation of promoters, more easily aligned with transcriptional silencing, can be RNA mediated if suitable double stranded RNA versions of the promoter sequence are transcribed, either fortuitously or by design [36–40]. Therefore, there may be a natural tendency for PTGS loci to mature to TGS and associated trans-silencing, which in turn could prove problematic in applying PTGS in commercial plant breeding programs. Our data are certainly consistent with this notion. Such hypotheses are testable with the model system we have established.
1. Posttranscriptional gene silencing by structurally complex transgenes loci may be unstable and may be supplanted by transcriptional gene silencing over the course of a few plant generations.
2. Heritable modifications of transgene expression caused by exposure to a trans-silencer locus may be stable for five generations or more.
3. Upon exposure to a trans-silencer locus, certain Arabidopsis transgenes display an alteration in their epistatic interaction with other transgenes. These data suggest that the competence for trans-silencing may be transferred between non-allelic transgenes, reminiscent of the allelic transfer of epigenetic activity during paramutation.
4. Cosuppression phenotypes can serve as sensitive indicators of epigenetic interactions between transgenes in Arabidopsis thaliana.
Transgenic Arabidopsis lines and plant growth
The GFP-COP1 expression cassette contains a double 35S enhancer, translational enhancer, and 35S terminator. Most GFP-COP1 lines and the GUS-COP1 line L4 have been described [27, 41, 42]. Unsilenced GUS-COP1 and GFP-COP1 transgenes complement the cop1 mutation [42, 43]. The C11 and C71 loci contain at least three and two linked T-DNAs, respectively, as seen by Southern blotting (not shown). Plants were germinated on agar solidified MS medium containing 1% sucrose in either constant light or constant darkness and subsequently grown in soil in growth chambers at 22°C under constant white light from fluorescent tubes.
Test for acquired trans-silencing activity
Trans-silenced L91' and E82' plants that lacked the C73 silencer locus ('prime'-label) were crossed in pairwise combinations with naive E82 and L91 plants. Because trans-silencing of E82 was observed regardless of whether L91' was the male or the female parent, data from reciprocal crosses were pooled. Individual F1 dihybrid plants were inspected for GFP-COP1 and COP1 endogene silencing phenotypes. Representative F1 dihybrids were checked for the presence of two different transgene loci by diagnostic PCR assay and by segregation analysis. F2 segregants containing only the target locus were identified by PCR based genotyping, analogous to the strategy exemplified in Fig. 5. Plants in which GFP-COP1 expression was restricted to roots and cotyledons were regarded as silenced, whereas plants with GFP-COP1 visible beyond the cotyledons were considered active.
The diagnostic PCR assays for the C73 and E82 loci display unique PCR products arising from fortuitous and distinct junctions between the T-DNA right border and the COP1 cDNA, which we recovered in early attempts to isolate flanking sequences. For E82 the primers are ATATTTGCTAGCTGATAGTGACC-3' and GATCCTAGGGGTCTCGTGATTTCTTGTGAT-3'; for C73 they are TGTCAGTTCCAAACGTAAAACGG-3' and GACACATCACAAGATCTTTGTAGTGC-3'. The assay for the L91 locus was based on oligonucleotides specific for the L91 flanking sequence (AGGCACACAAGCCCAAAAAGAC-3') and the RB of the T-DNA . PCR fragments representing the structure of the E82 locus were made as follows: Fragment 1 in Fig. 3B: 5'-AAACAGGATTTTCGCCTGCTGGG-3' (LB) and 5'-ACATCCAAACAGAACGTGCC-3' (Arabidopsis). Fragment 2: 5'-AAACAGGATTTTCGCCTGCTGGG-3' (LB) and 5'-TGCTGTTCAAACCCCAAAATTC-3' (Arabidopsis). Fragment 3: 5'-ATATTTGCTAGCTGATAGTGACC (RB) and 5'-TGCTGTTCAAACCCCAAAATTC-3' (Arabidopsis). Fragment 4: 5'-GGGGGCCATGGAGTATGAAGAGCACGAA-3' (COP1) and 5'-CGGAGAACCTGCGTGCAATCCATC-3' (RB).
We thank Byung-Hoon Kim and Andreas Nebenführ for critical comments on the manuscript. This work received partial support from Department of Energy Basic Energy Sciences grant DE-FG02-9622023.
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