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Transposable elements (TEs) that proliferate in a disorderly manner result in repetitive sequences in the genome (Doolittle & Sapienza, 1980; Orgel & Crick, 1980). Host genomes have developed mechanisms to prevent TE activities, which disturb genomic integrity under an antagonistic relationship between TEs and the genome. Such antagonistic relationships may have been elaborated into various modification systems that influence gene expression (Fagard & Vaucheret, 2000; Uchiyama et al., 2009; Lisch & Bennetzen, 2011). Representative modification systems, including DNA methylation, RNA interference (RNAi), and chromatin modification, have been explored using phenomena involved in the suppression of TE activity in plants (Slotkin & Martienssen, 2007; Lisch & Bennetzen, 2011). These mechanisms, described as epigenetic modifications, are mostly mediated by small RNAs, which confer pleiotropic effects involving interference with different stages of transcription, or even translation (Brodersen & Voinnet, 2006; Xie & Qi, 2008; Liu & Paroo, 2010). However, there is limited knowledge regarding the alteration of gene expression caused by mechanisms based on the conflict between TE activity and the activity of the host genome.
The genome of Antirrhinum majus contains c. 50 copies of Tam3 (Kishima et al., 1999), which belongs to the hAT family of transposons (Calvi et al., 1991). Tam3 is considered an autonomous element. Among the Tam3 copies in the genome, active copies are known to have an identical structure of 3.6 kb of sequence encoding the transposase (TPase) enzyme (Kishima et al., 1997, 1999). The transposition of Tam3 is controlled by temperature; low temperatures of c. 15°C permit transposition, but high temperatures of c. 25°C strongly inhibit it (Harrison & Fincham, 1964; Hashida et al., 2003, 2006) (Fig. 1). We have studied the effect of Tam3 on the expression of the nivea (niv) locus, which encodes chalcone synthase (CHS), and is responsible for flower pigmentation (Sommer & Saedler, 1986). The nivearecurrens::Tam3 (nivrec) allele from the HAM2 line carries a Tam3 insertion 70 bp upstream of the niv transcription start site (29 bp upstream of the TATA box) and produces variegated spots because of Tam3 excision at low temperature (Sommer et al., 1985). The nivrec allele shows a pale-red background flower color that was described as a ‘flushed flower’ by Harrison & Carpenter (1973). This leaky phenotype caused by the so-called ‘Tam3-permissible allele’ is a common feature in Antirrhinum strains that contain Tam3 in their promoter regions (Uchiyama et al., 2009). These Tam3-permissible alleles share the same Tam3 orientation (the 3′ end of Tam3 adjacent to the 5′ region of the gene coding sequence), whereas the reverse orientation of Tam3 in the promoter region that is observed in the pallidarecurrens::Tam3 (palrec) allele in Antirrhinum does not permit gene expression (Uchiyama et al., 2009).
Figure 1. Different flower pigmentation patterns in Antirrhinum majus lines HAM2 and HAM5. Both the lines exhibit petal variegation at low temperature (15°C) but not at high temperature (25°C). The variegation was attributed to Tam3 in the niv promoter, which exhibits low-temperature-dependent transposition (LTDT). HAM2 shows a pale-red background flower color in either condition, while HAM5 shows a white petal color at high temperature and faint pigmentation at low temperature.
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The HAM5 line has the same Tam3 insertion as the nivrec allele, although this flower shows no background petal pigmentation in Tam3 inactive conditions. Despite the different background colors of the petals, both the HAM2 and HAM5 lines produce similar variegation in the form of full red-coloured sectors because of Tam3 excision from the niv promoter at low temperature (Fig. 1). We examined the mechanism responsible for the difference in the background colors of the HAM2 and HAM5 lines. During the course of this analysis, we isolated individuals with background petal pigmentation similar to HAM2 in the progeny of HAM5 plants grown at low temperature.
In this paper we report identification of another Tam3 copy present 1.6 kb downstream of the niv poly-A signal, insertion of which is responsible for the lack of background pigmentation in HAM5. The mechanisms by which insertion of the Tam3 element results in the different pigmentation patterns were investigated, and possibilities associated with DNA methylation and RNAi were ruled out. Our results suggest that two repeat sequences separated by a short distance interact with each other and disturb the expression of the gene between the repeats. Possible interactions between the repeats and the alteration of host gene expression are discussed.
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The different background petal pigmentation phenotypes of HAM2 and HAM5 were the result of different abundances of niv (CHS gene) transcripts (Figs 1,5). Both lines shared the same promoter sequence containing Tam3 (upstream Tam3), while transposon tagging identified a structural difference 1.6 kb downstream region of the 3′ end of the niv gene, where HAM5 had an additional Tam3 insertion (downstream Tam3) (Fig. 4). The insertion of the upstream and downstream Tam3 copies both resulted in the reduction of niv expression to one-seventh and one-half, respectively (Fig. 7). The simultaneous insertion of both the upstream and downstream Tam3 copies induced a synergistic fall in the level of niv expression, by c. 360-fold, reflected by a petal phenotype with no background pigmentation observed in HAM5. Therefore, it was certain that the two Tam3 copies spanning the niv gene coordinately suppressed niv gene expression.
The nivrec allele carrying the upstream Tam3 exhibited a leaky phenotype, because the 3′ end region of Tam3 adapts any promoter sequence for limited expression of the gene (Uchiyama et al., 2009). In fact, leaky phenotypes in Antirrhinum have been described in several loci where Tam3 was inserted in the same orientation as the downstream gene; a reporter gene with Tam3 in the promoter sequence validated this finding in yeast (Uchiyama et al., 2009). These alleles, called ‘Tam3-permissible alleles’, seem to be ubiquitous for the expression of any genes (Uchiyama et al., 2009).
In the nivbeni allele, where the presence of two Tam3 copies reduced with niv transcript abundance, epigenetic controls were examined. However, we found no evidence that differences in DNA methylation or RNAi were associated with the reduction in the niv expression between HAM2 and HAM5 (Figs 8, 9). The tail-to-tail orientation of the two Tam3 copies either side of the niv gene is a structural feature that typically can induce production of double-stranded RNA if both Tam3 sequences were transcribed through the niv gene. Double-stranded RNA is eventually digested to generate siRNA that triggers DNA methylation or RNAi (Brodersen & Voinnet, 2006; Liu & Paroo, 2010). As a possible explanation, niv transcription might be suppressed by DNA methylation or the transcript might be degraded by RNAi. However, this scenario was not supported by our analysis: a relatively low concentration of methylated cytosine was detected in the nivbeni promoter region compared with that in other niv alleles, and no corresponding siRNA was detected using the niv coding sequence as a probe (Figs 8, 9). This interpretation was supported by the fact that F1 plants between HAM2 and HAM5 gave rise to intermediate background petal pigmentation (Fig. 2), indicating that the silencing effect of nivbeni was not trans-acting. The intermediate phenotype in the F1 plants suggested that transcription of the nivrec allele was independent of interference by the nivbeni allele, and that the reduced pigmentation and silencing of nivbeni was cis-acting. The significance of the inhibition of gene expression might be related to homology between the Tam3 sequences in inverted orientation. The situation in the nivbeni allele could be likened to being trapped between the two Tam3 copies.
Insulators are chromatin boundary elements that can block communication between enhancer and promoter elements, resulting in the suppression of gene expression (Valenzuela & Kamakaka, 2006). The enhancer-blocking activity of the insulator prevents enhancer action on a promoter when the insulator is placed between the enhancer and the promoter (Wei et al., 2005). We do not know whether the downstream Tam3 exerted an insulator-like function to inhibit niv expression, although the presence of only the downstream Tam3 copy (i.e. in the absence of the upstream Tam3 copy in the nivbeni allele) reduced the transcript abundance of the wildtype allele by half, resulting in nearly full red petal pigmentation (Fig. 6). The further inhibition of niv expression could occur when two Tam3 sequences are located either side of the gene. In the nivbeni allele, the downstream Tam3 negated the function of permissible expression in the nivrec allele (Figs 4, 5).
Our results allowed us to consider an interaction between the two Tam3 sequences. An analogy for the ‘trapping of a gene’ by paired sequences is found in the case of the insulator suHW, which is a 340 bp element of the Drosophila gypsy retrotransposon (Gerasimova & Corces, 2001). When two suHW insulators were inserted between the enhancer and promoter of a gene in Drosophila, enhancer-blocking activity arising from one of the two suHW insulators was modulated probably via interaction of the two insulators (Cai & Shen, 2001; Muravyova et al., 2001), suggesting that bound protein complexes formed a mini-loop (Byrd & Corces, 2003). Insulators are known to facilitate loop conformation mediated with insulator-binding protein (Gaszner & Felsenfeld, 2006; Valenzuela & Kamakaka, 2006). The two identical Tam3 sequences in this study might come in to close proximity or bind to each other to form a loop and prevent the access of transcription factors to the niv promoter, resulting in the loss of transcription (Fig. 10). In the nivbeni allele, the low degree of DNA methylation in the promoter region (Figs 8, S4) might reflect the prevention of access for DNA binding proteins.
Figure 10. Models of transcriptional regulation of the two Antirrhinum niv alleles. Transcription factors can access the promoter containing the Tam3 sequence in the nivrec allele, resulting in a pale-red color in the flower lobe, whereas two Tam3 copies in the nivbeni allele do not permit transcription factors to activate the niv allele, resulting in white flowers.
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Several protein complexes have been identified as insulator-binding proteins required for loop conformations (Wei et al., 2005; Gaszner & Felsenfeld, 2006). A mammalian protein, CTCF, is one of the most extensively studied insulator-binding proteins, which demarcate boundaries between euchromatin and heterochromatin (Bell et al., 1999; Yusufzai et al., 2004). Genome-wide analysis identified CTCF interactions at 1480 intrachromosomal and 336 interchromosomal loci in the mouse genome and showed that chromatin loops organize the boundaries of chromatin domains to demarcate different transcriptional control units (Handoko et al., 2011). In addition to suHW, other TEs and/or repetitive sequences that exist in eukaryote genomes could be employed widely as target sites for insulator-binding proteins. These elements have the potential to form loops that participate in genome architecture or influence gene expression in plant genomes (Louwers et al., 2009). In particular, the loop structures of TEs tend to generate alternative gene expression patterns (West et al., 2002; Sexton et al., 2009). The Tam3 TPase in Antirrhinum does not participate in loop conformation because it does not enter the nucleus when plants are grown at high temperatures (Fujino et al., 2011). The reduced pigmentation phenotype of the nivbeni allele is temperature-independent, so if loop formation between the paired Tam3 copies occurs, proteins other than the TPase must be involved. Alternatively, the two Tam3 copies might interact autonomously in a homology-dependent manner.
Suppressor-mutator (Spm) and Mutator (Mu) elements are the well-known maize DNA-type TEs that can also control the expression of the proximal host genes (Craig, 2002). The expression of maize genes with the insertion of a defective Spm element can be under either positive (Spm-dependent) or negative (Spm-suppressive) control in the presence of an autonomous Spm element (McClintock, 1958, 1961; Masson et al., 1987; Grant et al., 1990). In several maize loci, Mu insertion in the promoter regions interfered with transcription when Mu was active (Martienssen et al., 1990; Martienssen & Baron, 1994; Cui et al., 2003), but the genes were transcribed when Mu was inactive (Greene et al., 1994). This Mu-suppressive system is correlated with the epigenetic state of the Mu element (Barkan & Martienssen, 1991; Robbins et al., 2008).
The occurrence of the different phenotypes in suppressible alleles described for Spm and Mu elements in maize depends on the presence/absence of their TPases (autonomous elements), although the background petal pigmentation driven by the nivrec allele in HAM2 was not related to transcription and translation of the Tam3 TPase gene (Uchiyama et al., 2008, 2009). Thus, niv regulation as a result of Tam3 is apparently distinct from that occurring in the suppressible alleles of Spm and Mu in maize. The nivbeni allele in HAM5 showed no background pigmentation in petals owing to an interaction of the paired Tam3 copies, a phenomenon dissimilar to those described for the modulation of gene expression associated with TEs. This example demonstrates another mechanism whereby TEs may influence gene expression, a mechanism that could be significant in times of ‘genome shock’ (McClintock, 1984), when transposition is frequent and elements may insert in close proximity to each other on the genome.