Mobilization of a plant transposon by expression of the transposon-encoded anti-silencing factor
2013; Springer Nature; Volume: 32; Issue: 17 Linguagem: Inglês
10.1038/emboj.2013.169
ISSN1460-2075
AutoresYu Fu, Akira Kawabe, Mathilde Etcheverry, Tasuku Ito, Atsushi Toyoda, Asao Fujiyama, Vincent Colot, Yoshiaki Tarutani, Tetsuji Kakutani,
Tópico(s)RNA and protein synthesis mechanisms
ResumoArticle30 July 2013free access Mobilization of a plant transposon by expression of the transposon-encoded anti-silencing factor Yu Fu Yu Fu Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan Department of Genetics, School of Life science, The Graduate University for Advanced Studies (SOKENDAI), Shizuoka, Japan Search for more papers by this author Akira Kawabe Akira Kawabe Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan Search for more papers by this author Mathilde Etcheverry Mathilde Etcheverry Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Centre National de la Recherche Scientifique (CNRS), UMR 8197, Institut national de la santé et de la recherche médicale (INSERM), U1024, Paris, France Search for more papers by this author Tasuku Ito Tasuku Ito Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Tokyo, Japan Search for more papers by this author Atsushi Toyoda Atsushi Toyoda Center for Genetic Resource Information, National Institute of Genetics, Shizuoka, Japan Search for more papers by this author Asao Fujiyama Asao Fujiyama Center for Genetic Resource Information, National Institute of Genetics, Shizuoka, Japan Search for more papers by this author Vincent Colot Vincent Colot Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Centre National de la Recherche Scientifique (CNRS), UMR 8197, Institut national de la santé et de la recherche médicale (INSERM), U1024, Paris, France Search for more papers by this author Yoshiaki Tarutani Yoshiaki Tarutani Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan Department of Genetics, School of Life science, The Graduate University for Advanced Studies (SOKENDAI), Shizuoka, Japan Search for more papers by this author Tetsuji Kakutani Corresponding Author Tetsuji Kakutani Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan Department of Genetics, School of Life science, The Graduate University for Advanced Studies (SOKENDAI), Shizuoka, Japan Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Tokyo, Japan Search for more papers by this author Yu Fu Yu Fu Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan Department of Genetics, School of Life science, The Graduate University for Advanced Studies (SOKENDAI), Shizuoka, Japan Search for more papers by this author Akira Kawabe Akira Kawabe Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan Search for more papers by this author Mathilde Etcheverry Mathilde Etcheverry Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Centre National de la Recherche Scientifique (CNRS), UMR 8197, Institut national de la santé et de la recherche médicale (INSERM), U1024, Paris, France Search for more papers by this author Tasuku Ito Tasuku Ito Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Tokyo, Japan Search for more papers by this author Atsushi Toyoda Atsushi Toyoda Center for Genetic Resource Information, National Institute of Genetics, Shizuoka, Japan Search for more papers by this author Asao Fujiyama Asao Fujiyama Center for Genetic Resource Information, National Institute of Genetics, Shizuoka, Japan Search for more papers by this author Vincent Colot Vincent Colot Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Centre National de la Recherche Scientifique (CNRS), UMR 8197, Institut national de la santé et de la recherche médicale (INSERM), U1024, Paris, France Search for more papers by this author Yoshiaki Tarutani Yoshiaki Tarutani Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan Department of Genetics, School of Life science, The Graduate University for Advanced Studies (SOKENDAI), Shizuoka, Japan Search for more papers by this author Tetsuji Kakutani Corresponding Author Tetsuji Kakutani Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan Department of Genetics, School of Life science, The Graduate University for Advanced Studies (SOKENDAI), Shizuoka, Japan Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Tokyo, Japan Search for more papers by this author Author Information Yu Fu1,2, Akira Kawabe3, Mathilde Etcheverry4, Tasuku Ito1,5, Atsushi Toyoda6, Asao Fujiyama6, Vincent Colot4, Yoshiaki Tarutani1,2 and Tetsuji Kakutani 1,2,5 1Department of Integrated Genetics, National Institute of Genetics, Shizuoka, Japan 2Department of Genetics, School of Life science, The Graduate University for Advanced Studies (SOKENDAI), Shizuoka, Japan 3Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan 4Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Centre National de la Recherche Scientifique (CNRS), UMR 8197, Institut national de la santé et de la recherche médicale (INSERM), U1024, Paris, France 5Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Tokyo, Japan 6Center for Genetic Resource Information, National Institute of Genetics, Shizuoka, Japan *Corresponding author. Department of Integrated Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka, Japan. Tel.:+81 55 981 6801; Fax:+81 55 981 6804; E-mail: [email protected] The EMBO Journal (2013)32:2407-2417https://doi.org/10.1038/emboj.2013.169 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Transposable elements (TEs) have a major impact on genome evolution, but they are potentially deleterious, and most of them are silenced by epigenetic mechanisms, such as DNA methylation. Here, we report the characterization of a TE encoding an activity to counteract epigenetic silencing by the host. In Arabidopsis thaliana, we identified a mobile copy of the Mutator-like element (MULE) with degenerated terminal inverted repeats (TIRs). This TE, named Hiun (Hi), is silent in wild-type plants, but it transposes when DNA methylation is abolished. When a Hi transgene was introduced into the wild-type background, it induced excision of the endogenous Hi copy, suggesting that Hi is the autonomously mobile copy. In addition, the transgene induced loss of DNA methylation and transcriptional activation of the endogenous Hi. Most importantly, the trans-activation of Hi depends on a Hi-encoded protein different from the conserved transposase. Proteins related to this anti-silencing factor, which we named VANC, are widespread in the non-TIR MULEs and may have contributed to the recent success of these TEs in natural Arabidopsis populations. Introduction Control of transposable elements (TEs) has been extensively studied in plants. A pioneering early observation in maize is that TE activity often changes between active and inactive states in heritable but reversible manners (McClintock, 1951, 1958). The changes in the TE activity are generally correlated with the DNA methylation status; inactive TEs tend to be more methylated than active TEs (Chandler and Walbot, 1986; Brettell and Dennis, 1991; Fedoroff, 1996; Martienssen, 1996). The importance of DNA methylation in TE control has also been demonstrated using mutants of Arabidopsis; in Arabidopsis mutants with reduced genomic DNA methylation, a variety of silent TEs are de-repressed and mobilized (Miura et al, 2001; Singer et al, 2001; Kato et al, 2003; Lippman et al, 2004; Mirouze et al, 2009; Tsukahara et al, 2009). Intriguingly, some TEs have mechanisms to counteract DNA methylation and silencing by the host. For example, McClintock's Suppressor-mutator (Spm) element in maize encodes a protein TnpA, which induces loss of DNA methylation in regions controlling transcript formation in Spm (Schläppi et al, 1994, 1996; Cui and Fedoroff, 2002). An active Spm transiently activates silent Spm copies in trans, a process likely to be mediated by TnpA (Cui and Fedoroff, 2002). Robertson's Mutator, another well-characterized TE in maize, spontaneously changes its activity and DNA methylation in a coordinated manner (Chandler and Walbot, 1986; Martienssen and Baron, 1994; Martienssen, 1996). Like Spm, a silent Mutator element loses DNA methylation when an active Mutator is present in the same genome (Brown and Sundaresan, 1992; Lisch et al, 1995, 1999). MuDR, an autonomously mobile copy of maize Mutator family, contains two genes, mudrA and mudrB. The mudrA encodes the MURA protein, which is structurally similar to known transposases of other TEs (Eisen et al, 1994; Lisch, 2002). In addition, mudrA is sufficient for excision of Mutator, further suggesting that MURA functions as a transposase (Lisch et al, 1999). TEs similar to the maize Mutator are widespread in eukaryotes and they are referred to as Mutator-like elements (MULEs) (Jiang et al, 2004). ORFs related to mudrA are generally found in autonomous MULEs. Some of the autonomous MULEs also have additional ORF(s), such as mudrB in MuDR, but the structures of the proteins encoded in these ORFs are diverse and their functions remain largely unknown. In addition, some of the MULEs carry fragments of cellular genes, but their impacts on the TE dynamics and host fitness are still elusive (Talbert and Chandler, 1988; Yu et al, 2000; Jiang et al, 2004; Hoen et al, 2006). Most of the class II (DNA-type) TEs have long terminal inverted repeat (TIR), but there are a few exceptions (Wicker, 2007). Although the maize Mutator elements have relatively long TIRs of almost identical sequences, subgroups of MULEs with extensively degenerated TIR have been found in the Arabidopsis genome and they are classified as non-TIR MULEs (Le et al, 2000; Yu et al, 2000). Although the theoretical sequence analyses of Arabidopsis genome suggest movement of these non-TIR MULEs in the past (Yu et al, 2000), direct evidence for de novo movements is limited (Hoen et al, 2006; Tsukahara et al, 2009). We have previously reported that a group of non-TIR MULEs, called VANDAL21, seem to transpose in a background of reduced genomic DNA methylation (Tsukahara et al, 2009). Here, we identified an autonomously mobile copy of VANDAL21, which we renamed Hiun (Hi). Despite the degeneration of TIRs, Hi is competent to excise and transpose in precise manners. Interestingly, a Hi transgene induced loss of DNA methylation, transcriptional activation, and excision of the endogenous Hi copy. Most importantly, these trans-acting effects of Hi do not depend on the protein related to MURA-type transposase but instead depend on another protein encoded by Hi. The function of this novel anti-silencing protein, which we named VANC, will be discussed in the context of TE evolution. Results Identification of mobile VANDAL21 copies The genome sequence of wild-type Col ( http://www.arabidopsis.org/) suggests seven copies of VANDAL21 elements with relatively similar sequences. Other copies of VANDAL21 are distant in the sequences. Consistent with that, Southern analysis revealed seven bands for that group of VANDAL21 (Tsukahara et al, 2009). We have previously shown that additional bands emerge in Arabidopsis plants derived from several rounds of self-pollinations in ddm1 (decrease in DNA methylation 1) mutant backgrounds, suggesting mobility of one or more copies of the VANDAL21 members (Tsukahara et al, 2009). Arabidopsis ddm1 mutation generally induces loss of DNA methylation in TEs, which causes mobilization of diverse TEs (Miura et al, 2001; Singer et al, 2001; Lippman et al, 2004; Mirouze et al, 2009; Tsukahara et al, 2009). In order to know which of VANDAL21 copies are mobile, we used two methods: suppression PCR and whole-genome re-sequencing (see Materials and methods for details). In total, we identified 72 de novo insertions of VANDAL21s (61 by genome re-sequencing and 14 by suppression PCR with 3 overlaps) in the self-pollinated ddm1 lines (Supplementary Table S1). Of these 72 insertions, 69 correspond to one copy (Figure 1A; AT2TE42810) of VANDAL21 element. The remaining three insertions correspond to another copy (AT4TE15615). For the other five copies of related VANDAL21, no new insertion has been identified. In the following parts, we concentrate on the most active copy, AT2TE42810, which we renamed Hiun (Hi, Japanese for 'a flying cloud'). Figure 1.Mobilization of Hi in ddm1 mutant. (A) Schematic diagram for structure of Hi. Terminal regions, exons, introns, and intergenic regions are shown by white bars, grey bars, grey lines, and black lines, respectively. Regions examined by bisulphite sequencing are shown by L1 and R1 with thick black lines. Region examined by McrBC-qPCR is shown by L2. Region examined for copy number quantification is shown by C1. In most of the transgene constructs, silent mutation is introduced for each ORF, so that the transcripts from the transgene and endogenous copy could be distinguished between. The sites of the silent mutations are shown by vertical bars, with surrounding arrowheads showing regions amplified by RT–PCR. Regions for ORFs deleted in each of the deletion constructs are shown by horizontal bars with two arrowheads. (B) De novo integration sites of Hi in relation to flanking transcription units. The position of integration is normalized by length of the flanking transcription unit. Rightward and leftward arrows indicate insertions with 5′ to 3′ and 3′ to 5′ orientations of Hi, respectively. Insertions flanking pseudogenes and transposon genes are shown by grey arrows, and those flanking canonical genes by black arrows. Sequences of the integration sites are shown in Supplementary Table S1. Four out of the sixty-nine insertions are not included in this figure, because they are further away from transcription units. Genomic locations of all 69 transpositions are shown in Supplementary Figure S2. (C) Excision of Hi in ddm1 plants detected by PCR. Genomic DNA of 11 ddm1 plants (lane numbers from 2 to 12) and 5 wild-type sibling plants (lane numbers from 13 to 17) was used to analyse excision of endogenous Hi by nested PCR. These lines are derived from segregating population in self-pollinated progeny of a DDM1/ddm1-1 heterozygote. Sequences of primers used are shown in Supplementary Table S2.Source data for this figure is available on the online supplementary information page. Source data for Figure 1 [embj2013169-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint Structure of Hi Hi is 8177-bp long and includes three ORFs: At2g23500, At2g23490, and At2g23480 (Figure 1A). One ORF (At2g23500; called vanA) encodes a protein with high sequence similarities with MURA-type transposases, which are generally found in MULEs. Proteins encoded by two other ORFs (vanB and vanC) do not have sequence similarity to any characterized proteins. An unorthodox feature is that, unlike other typical mobile DNA-type TEs, the TIRs of this TE are extensively degenerated (Supplementary Figure S1A), showing the characteristics of non-TIR MULEs (Yu et al, 2000). Integration and excision of Hi Hi is transposed throughout the genome, although insertions may be more concentrated near the original locus than in unlinked regions (Supplementary Figure S2). Mutator elements in maize preferentially transpose into 5′ region of genes (Hardeman and Chandler, 1989; Dietrich et al, 2002; Liu et al, 2009). That was also the case for Hi; most of the integration sites are localized around transcription start sites of genes (Figure 1B). Interestingly, integration of Hi there had bias in the orientation (Figure 1B). Such a bias in the orientation has not been reported for the maize Mutator (Brown et al, 1989). The bias in the orientation of Hi integration might be related to the asymmetry in its terminal sequences. We could detect 9 bp of target site duplication (TSD) for most of the insertions examined (Supplementary Figure S1B), as is the case for integrations of other MULEs (Yu et al, 2000). Many DNA-type TEs often transpose with loss of the original copy. Because Hi does not have typical structure of DNA-type TE, an interesting question would be whether Hi excision occurs at defined termini or not. In order to detect somatic excision events, we used PCR with primers for both of the flanking regions of the original Hi locus (details in Materials and methods). Using this assay, we could detect Hi excision in all independent ddm1 lines examined (Figure 1C). We examined the mode of the excisions by sequencing the PCR products (Supplementary Figure S3A). Interestingly, despite the degeneration of TIRs, many of the excision products showed excision around the terminal sites predicted from the integrated copies. Even TSDs are lost in a significant part of the excision product. In summary, these observations suggest that long perfect TIRs are dispensable not only for integration, but also for reasonably precise excision of this element in the defined termini. The transposition of Hi occurred in a manner comparable to that of typical MULEs with long TIR. Mobilization of endogenous Hi by transgene As the ddm1 mutation results in transcriptional de-repression of many repeat sequences (Lippmann et al, 2004; Tsukahara et al, 2009), it is formally possible that mobilization of Hi in ddm1 is triggered by de-repression of other sequence(s). In order to test if expression of proteins encoded by Hi is sufficient for its mobilization, we introduced a cloned Hi copy into wild-type plants by Agrobacterium-mediated transformation. In these transgenic Hi lines, we could detect transcripts corresponding to their three ORFs (Supplementary Figure S4). In these lines, the Hi transgene induced excision of the original copy (Figure 2A). Control transformants with empty vectors did not show excision of the Hi, confirming that Hi transgene triggered mobilization of the endogenous copy. We also examined Hi activity in self-pollinated progeny of one of the Hi transgenic lines. We could detect Hi excision in most of 15 progeny plants that inherited the transgene (Supplementary Figure S6). On the other hand, we could not detect the excision in any of six progeny plants that lost the transgene by segregation, further confirming that the Hi transgene is responsible for the mobilization. Figure 2.Introduction of Hi transgene induces loss of DNA methylation and excision of endogenous Hi copy. (A) Excision of endogenous Hi induced by transgene for Hi (Hi TG: lanes 5–18). Lanes 1 and 2–4 are non-transgenic plant (wt) and transformant lines with empty vector (V) used as negative controls, respectively. Excision of Hi copy in the transgene was also detected in some of the transgenic lines (Supplementary Figure S5). (B) DNA methylation status of Hi termini in the transgenic line and progeny. T1 transformant with Hi transgene showed reduction in DNA methylation in both termini, compared to non-transgenic plant (NT) and transformants with empty vector (V). T2/TG+ and T2/TG− are self-pollinated progeny of the T1 with and without transgene, respectively. In both classes, averages and standard deviations of three segregants are shown. We also obtained essentially the same results for a segregating T3 family (Supplementary Figure S7). Regions L1 (upstream of vanA) and R1 (upstream of vanC) were examined (shown in Figure 1). At least 11 clones were examined for each plant.Source data for this figure is available on the online supplementary information page. Source data for Figure 2 [embj2013169-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint DNA demethylation induced by Hi in trans The observations described above suggest that expression of Hi transgene induces mobilization of the endogenous copy. Interestingly, in the presence of the transgene, DNA methylation levels were reduced in both termini of the endogenous Hi (Figure 2B). The loss of methylation is more extensive in non-CpG sites than in CpG sites. When the transgene was segregated away in the self-pollinated progeny of the transgenic line (T2/TG− in Figure 2B), Hi was remethylated in the terminal regions, which was associated with its loss of excision activity. We also tested the trans-acting demethylation effect for transposed Hi copies, using epi-Recombinant Inbred Lines (epi-RILs) from ddm1 mutant (Johannes et al, 2009). The epi-RILs are originated from ddm1 mutant backcrossed to parental wild-type Col to generate DDM1/DDM1 homozygotes, and subsequent self-pollination to fix the heritable epigenetic defects induced by ddm1. The methylation status of Hi at the original locus was examined by PCR after digestion by methylation-sensitive restriction enzyme. As expected from the crossing scheme used to generate the epi-RILs (Johannes et al, 2009), approximately three quarters of the epi-RILs tested had inherited the original Hi copy from wild-type DDM1 parent. Some of these lines have additional transposed Hi copies in trans (Figure 3A). In those lines, Hi at the original locus showed loss of methylation (Figure 3B), despite its wild-type origin. On the other hand, in epi-RILs that did not carry the additional Hi, Hi at the original locus remained methylated to the level comparable to parental wild type. Together, these results suggest that the stable demethylation of Hi at the original locus is due to trans-acting effect of the transposed Hi copies. In addition to its demethylation, the Hi copies present at the original locus showed excision (Figure 3C), and that happened only when the additional trans-acting copies exist. These trans-acting effects of the transposed Hi are consistent with the trans-acting demethylation and mobilization by the Hi transgene. Figure 3.Transposed Hi induces loss of DNA methylation and excision of Hi in the original locus. (A) Copy number of Hi in each of the epi-RILs. The copy number was estimated by quantitative PCR using region C1 in Figure 1. Average and standard deviation of two technical replicates are shown in this and next panel. Parental origin of the original Hi locus (wt or ddm1) was determined by methylation status of the linked region (Colomé-Tatché et al, 2012). (B) DNA methylation status in the 5′ region (L2 in Figure 1) of the original Hi locus was estimated by McrBC digestion and subsequent qPCR. Details are described in Materials and methods. Hi in the original locus showed loss of DNA methylation when extra copies of Hi exist. (C) Excision analysis of original Hi copy by nested PCR. (D) Two of the epi-RILs showed germinal transmission of the excised Hi allele. Origin of the Hi locus is wild-type DDM1 for line 166 and ddm1 mutant for line 458. Presence of Hi in the original locus was examined by PCR in the 5′ border of Hi (the primer sequences are shown in Supplementary Table S2). Lack of the signal suggests fixation of the empty allele.Source data for this figure is available on the online supplementary information page. Source data for Figure 3 [embj2013169-sup-0003-SourceData-S3.zip] Download figure Download PowerPoint Expression of vanC is sufficient for the trans-activation and mobilization of endogenous Hi The results shown above demonstrate that Hi transgenes induced mobilization of endogenous copy in trans, which is associated with loss of DNA methylation in the terminal regions. In order to further dissect the role for each of the ORFs in Hi, we generated transgenes with deletion in each ORF. Transgene with deletion of the central small ORF (vanB) still caused loss of methylation in both termini of Hi (Figure 4). By contrast, deletion of 3′ ORF (vanC) abolished the demethylation effect for both termini, suggesting that this ORF is essential for the demethylation. Transgene with deletion of 5′ ORF (vanA), which is structurally similar to transposase, still caused loss of methylation in 3′ (vanC side) terminal region of Hi, although the demethylation effect in the 5′ (vanA side) region was less complete than that in the full-length Hi transgenic lines. These results suggest that vanC is important for the demethylation of both of the terminal regions. Figure 4.DNA methylation status of endogenous Hi after introduction of Hi transgene and its deletion derivatives. For each of the deletion derivatives, averages and standard deviations of four independent transgenic plant lines are shown. WT and V are from Figure 2. Download figure Download PowerPoint We then examined the effect of these deletion constructs on excision of endogenous copies (Figure 5A). Very importantly, transgenic lines with deletion in vanA (ΔA-TG) still induced excision of endogenous Hi (Figure 5A). This was surprising because vanA encodes the putative transposase. We then examined expression of endogenous vanA gene in the presence of ΔA-TG. In most of the ΔA-TG lines, endogenous vanA was de-repressed and transcribed, although the expression level was generally lower and less robust than that of the Hi transgene keeping vanA (Figure 5B; Supplementary Figure S8). Figure 5.Trans-activation by Hi transgene without putative transposase. (A) Excision of endogenous Hi induced by ΔA transgene. Lanes 1 and 2–6 are non-transgenic plant (wt) and transformant lines with empty vector (V) used as negative controls, respectively. Excision of endogenous Hi induced by ΔB and ΔC transgene is also shown below. The results using additional ΔC lines are shown in Supplementary Figure S9. (B) Transcriptional activation of vanA induced by ΔA transgene. Materials for the same lane number in (A) and (B) are from the same plant, although the DNA and RNA are prepared from different leaves.Source data for this figure is available on the online supplementary information page. Source data for Figure 5 [embj2013169-sup-0004-SourceData-S4.zip] Download figure Download PowerPoint The results above suggest that expression of vanB and/or vanC can cause de-repression of vanA. We could also detect expression of vanB in ΔB-TG lines, but vanC transcript was undetectable in ΔC-TG lines (Supplementary Figure S8), suggesting possible role of vanC in the trans-activation. In addition, deletion of vanB from the transgene did not affect excision of endogenous Hi by the transgene, while the excision tended to be less robust in ΔC-TG (Figure 5A; Supplementary Figure S9). In order to know if vanB is dispensable for the trans-activation of Hi, we examined the effect of transgene with deletion of both vanA and vanB (ΔAB-TG). Only the vanC ORF remains in the ΔAB-TG construct. The ΔAB-TG also induced demethylation of 3′ (vanC side) terminal regions to the level comparable to the ΔA-TG (Figure 4). In most of the ΔAB-TG lines, we could detect excision of Hi and transcription of vanA and vanB (Figure 6A and B). In T2 generation that originated from self-pollination of a T1 ΔAB-TG plant, all T2 plants with the transgene showed excision, but none of T2 plants without transgene showed excision, confirming that the ΔAB transgene induces the excisions of endogenous Hi (Figure 6C). Taken together, these results demonstrate the key role of vanC for the trans-acting anti-silencing of Hi. Figure 6.Expression of vanC is sufficient for the trans-activation. (A) Excision of endogenous Hi induced by ΔAB transgene. (B) Transcriptional activation of vanA and vanB genes induced by ΔAB transgene. Materials for the same lane number in (A) and (B) are from the same plant, although the DNA and RNA are prepared from different leaves. (C) Excision of endogenous Hi induced by ΔAB transgene in the T2 generation. T2 plants from self-pollinated progeny of a T1 (the plant shown in lane 9 of A) were examined after determining the presence/absence of the transgene.Source data for this figure is available on the online supplementary information page. Source data for Figure 6 [embj2013169-sup-0005-SourceData-S5.zip] Download figure Download PowerPoint Proteins related to the anti-silencing factor are widespread in non-TIR MULEs but not found in TIR MULEs Non-TIR MULEs in A. thaliana genome are consisted of multiple VANDAL and ARNOLD families (Yu et al, 2000; Figure 7A). Interestingly, the non-TIR MULEs seem to be very successful in the recent proliferation. The phylogenetic analyses revealed recent proliferations in multiple subfamilies of non-TIR MULEs; each of the subfamilies shows terminal proliferations after separation of A. thaliana and A. lyrata lineages. The proliferation rates of the non-TIR MULE clusters are significantly higher than those of TIR-MULE in both A. thaliana and A. lyrata lineages (Figure 7A; Table I). Figure 7.Evolution and proliferation of TIR and non-TIR MULE families. (A) Phylogenetic relationship among MULE families in genomes of A. thaliana and A. lyrata. A. lyrata-specific lineages are shown by red lines. An NJ tree made by p-distance is shown. Scale bar is shown in the centre of the tree. The families containing DUF287 or Ulp1 protease domai
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