Chromosome Res DOI 10.1007/s10577-014-9417-9
REVIEW
Control of transposable elements in Arabidopsis thaliana Hidetaka Ito & Tetsuji Kakutani
# Springer Science+Business Media Dordrecht 2014
Abstract Arabidopsis thaliana serves as a very good model organism to investigate the control of transposable elements (TEs) by genetic and genomic approaches. As TE movements are potentially deleterious to the hosts, hosts silence TEs by epigenetic mechanisms, such as DNA methylation. DNA methylation is controlled by DNA methyltransferases and other regulators, including histone modifiers and chromatin remodelers. RNAi machinery directs DNA methylation to euchromatic TEs, which is under developmental control. In addition to the epigenetic controls, some TEs are controlled by environmental factors. TEs often affect expression of nearby genes, providing evolutionary sources for epigenetic, developmental, and environmental gene controls, which could even be beneficial for the host.
CMT DRM DRD
Keywords Epigenetics . Genomics . DNA methylation . Heterochromatin
Introduction: Arabidopsis thaliana as an ideal model organism to investigate controls of TEs
Abbreviations TE Transposable element RNAi RNA interference MET Methyltransferase DDM Decrease in DNA methylation
Since the first discovery in maize, TEs and their controls have been extensively studied in plants. Among the plant species, Arabidopsis thaliana is a very good material to study the control of TEs. Compared to the genomes of typical plant species, the proportion of TEs in the A. thaliana genome is low—about 10 % (The Arabidopsis Genome Initiative 2000). The low proportion is not due to lack of TE families; A. thaliana genome contains essentially every TE family known in plants (for details, see the accompanying review by Joly-Lopez and Bureau). The low TE proportion is mainly due to low copy number for each of these diverse TE families. The low copy number of each of TE members make their molecular analyses less
Responsible editors: Martin Lysak and Paul Fransz. H. Ito Faculty of Science, Hokkaido University, Kita10 Nishi 8, Kitaku, Sapporo, Hokkaido 060-0810, Japan T. Kakutani (*) Department of Integrated Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan e-mail: [email protected]
RDR RdDM DCL FWA SDC siRNA SINE tasiRNA TIR LTR
Chromomethylase Domain-rearranged methyltransferase Defective in RNA-directed DNA methylation RNA-dependent RNA polymerase RNA-directed DNA methylation Dicer like Flowering Wageningen Suppressor of DRM1 DRM2 CMT3 Small-interfering RNA Short interspersed nuclear element Trans-acting small-interfering RNA Terminal inverted repeat Long terminal repeat
H. Ito, T. Kakutani
complicated. In addition, many of the TE sequences are precisely mapped in the available completed genome sequences of A. thaliana because the genome sequences have been determined after generating the BAC contigs (The Arabidopsis Genome Initiative 2000). These features are the big advantages of A. thaliana for the genome-wide study of TE properties, such as expression, epigenetic modifications, and transposition (The Arabidopsis Genome Initiative 2000; Le et al. 2000; Peterson-Burch et al. 2004). In addition to genomics, genetics is also a very powerful tool to study TE control. Host mutations affecting TE activity are available and have been extensively studied. Approaches using these mutations are especially powerful when combined with the genome-wide approaches.
DNA methylation as a major factor controlling TEs TEs can be silenced by epigenetic marks, such as DNA methylation. Plant genome contains methylation of cytosine at both CpG and non-CpG sites. Most of the TEs, although not all, are heavily methylated for both CpG and non-CpG sites (Cokus et al. 2008; Lister et al. 2008). The CpG methylation is kept by the maintenance DNA methyltransferase MET1 (Finnegan et al. 1996). Methylation at nonCpG sites is catalyzed by chromomethylases, which target regions with methylation in lysine 9 of histone H3 (H3K9me; Chan et al. 2005). H3K9me is an epigenetic mark of silent and condensed chromatin, called heterochromatin (Grewal and Elgin 2007). The methylation of heterochromatic TE depends on a chromatin remodeler DDM1. DDM1 helps the methylation of TEs for both CpG and non-CpG sites, most likely by facilitating the access of DNA methylation machinery to heterochromatin (Jeddeloh et al. 1999; Zemach et al. 2013). The importance of DNA methylation in TE control has been directly shown using mutants of these genes; in Arabidopsis mutants with reduced genomic DNA methylation, a variety of silent TEs are de-repressed and mobilized (Singer et al. 2001; Miura et al. 2001; Kato et al. 2003; Lippman et al. 2004; Tsukahara et al. 2009; Mirouze et al. 2009). The ddm1 mutation, which affects DNA methylation at both CpG and non-CpG sites, mobilizes diverse TEs including mutators, CACTAs, and LTR elements. Mutation in the CpG methyltransferase gene
MET1 mobilizes some of these elements, such as an LTR element COPIA93/EVADE and a mutator-like element VANDAL21/Hiun (Mirouze et al. 2009; Tsukahara et al. 2009), suggesting that CpG methylation plays major role for immobilization of these TEs. On the other hand, some of other TEs, such as CACTA and GYPSY3, are not mobilized by met1 single mutation, but mobilized by double mutation of MET1 and non-CpG methyltransferase gene CMT3, suggesting that both CpG and non-CpG methylation are involved in their immobilization (Kato et al. 2003; Tsukahara et al. 2009). DNA methylation is maintained by DNA methyltransferases, MET1 and CMTs. Another class of DNA methyltransferase, DRM2, is involved in DNA methylation de novo (Cao and Jacobsen 2002). DRM2 is the DNA methyltransferase conducting the final reaction of “RNA-directed DNA methylation,” or RdDM. RdDM is an intriguing observation found first in tobacco that double-stranded RNA induces de novo DNA methylation of similar sequences (Wasseneger et al. 1994; Mette et al. 2000). Genetic screening of Arabidopsis has lead to identification of upstream factors involved in RdDM that include chromatin remodeling factor DRD1, RNAi components such as RDR2 and DCL3, and components of novel RNA polymerase complexes. The molecular characterization of these factors make RdDM a very active and important research field (Matzke et al. 2009; Law and Jacobsen 2010; Gao et al. 2010; Wierzbicki et al. 2012; Law et al. 2013). The mutations in RdDM components affect DNA methylation in euchromatin (Tran et al. 2005; Huettel et al. 2006; Zemach et al. 2013). This is in contrast to the effect of ddm1 mutation; ddm1 mutation abolishes DNA methylation in heterochromatin. Genome-wide analysis of DNA methylation in mutants of DDM1 and an RdDM component DRD1 revealed that these two mutations affect genomic DNA methylation in complementary manners (Zemach et al. 2013). The ddm1 mutation affects internal region of long TEs, which is heterochromatic. The drd1 mutation affects short TEs and terminal regions of long TEs, which is more euchromatic than regions affected by ddm1.
Release of TE silencing by demethylation in gametophytes Euchromatic TEs can be targets of RdDM. Intriguingly, the demethylation of these RdDM targets has been
Control of transposable elements in Arabidopsis thaliana
found during development. An extensively studied tissue is endosperm (Lauria et al. 2004; Gehring et al. 2009; Hsieh et al. 2009; Ibarra et al. 2012). Endosperm is a tissue supporting growth of embryo within the seed. The endosperm originates from the fertilization of sperm and central cell. Central cell is a companion cell of the egg in the female gametophyte. It is assumed that the DNA methylation is lost in the central cell. After fertilization, the loss of DNA methylation in the central cell is inherited to endosperm, contributing imprinted expression of genes flanking the euchromatic TEs. The reduction of DNA methylation depends on the DNA demethylase DEMETER (DME). Analogous mechanisms operate in the male side, during pollen maturation. A pollen grain has one vegetative cell nucleus (VN) and two sperm nuclei (SN). The two SN fertilizes with egg and central cells to generate embryo and endosperm lineages, respectively. In the companion nucleus, VN, centromeric chromatin was decondensed (Schoft et al. 2009); and many TEs are transcribed and transposed specifically in the VN, although not in the SN (Slotkin et al. 2009). Interestingly, the demethylation in VN also depends on DME (Schoft et al. 2011; Ibarra et al. 2012). The de-repression of TEs in these companion cells may induce de novo silencing of TEs through small RNA (Slotkin et al. 2009; Ibarra et al. 2012; Calarco et al. 2012). As these companion cells do not contribute to the next generation, mutagenization by TE movements would be less deleterious than those in the embryonic lineage.
TE as evolutionary sources for epigenetic gene control TEs in maize were initially described as “controlling elements,” because they often affect the functions of nearby genes (McClintock 1965) that were substantiated later at the molecular level for maize TEs, such as McClintock’s Suppressor-mutator (Spm) and Robertson’s Mutator (Fedoroff 1996; Martienssen 1996). TEs can be targets of epigenetic silencing, which is often heritable but reversible; the epigenetic states of TEs, in turn, affect expression of host genes nearby. Mutations affecting DNA methylation, such as met1 and ddm1, induce de-repression of cellular genes. One of them, the FWA gene was initially identified as the gene responsible for the “epigenetic mutation” with the
phenotype of delayed developmental transition (Soppe et al. 2000). “Epigenetic mutation” refers to a transgenerationally heritable trait without change in genomic nucleotide sequence. The fwa line does not have change in nucleotide sequence in the responsible gene. The late flowering trait in the fwa line turned out to be due to ectopic expression of the responsible gene, which is associated with the loss of DNA methylation in the promoter region (Soppe et al. 2000). The promoter se qu en ce h as s imil ari ty t o S INE, no n- LT R retrotransposon (Lippman et al. 2004; Fujimoto et al. 2008). The SINE-related promoter is normally methylated, and the gene is silent in the adult tissue. The methylation is removed in central cells in female gametophyte, causing imprinted expression of this gene in endosperm (Kinoshita et al. 2004; Fujimoto et al. 2008). Genome-wide analyses revealed that the expression of many genes is affected when DNA is removed from short TEs in central cells and pollen (Gehring et al. 2009; Hsieh et al. 2009; Slotkin et al. 2009; Ibarra et al. 2012). The loss of DNA methylation in drm1drm2cmt3 or ddm1rdr2 mutations induces developmental abnormalities, which is mediated by SDC gene (Henderson and Jacobsen 2008; Zemach et al. 2013). SDC also has repetitive promoter and shows imprinted expression in endosperm (Henderson and Jacobsen 2008; Hsieh et al. 2011). The ddm1 and met1 mutation induces not only de-repressed epialleles, such as fwa, but also silent epialleles (Jacobsen and Meyerowitz 1997; Jacobsen et al. 2000). Among them, a combination of ddm1induced developmental phenotypes, called bonsai (bns), is due to silencing of Anaphase-Promoting Complex 13 (APC13) gene. The silencing of BNS gene was associated with ectopic DNA methylation at both CpG and non-CpG sites (Saze and Kakutani 2007). The ectopic methylation depends on the presence of a LINE element insertion within the 3′ non-coding region (Saze and Kakutani 2007). The LINE insertion generates a potential trigger for epigenetic variation with strong developmental effects. In these examples, TEs regulate nearby genes in cis. TEs also have potential to control genes in trans. (Arteaga-Vázquez et al. 2006; McCue et al. 2012; McCue et al. 2013). In the A. thaliana genome, the most abundant TE species are gypsy type retroelements, called Athila. When de-repressed, Athila6 generates siRNA854, which affects expression of many host genes at the post-transcriptional level. Although
H. Ito, T. Kakutani
siRNA854 is derived from the TE, the length is 21–22 nt and it is incorporated into AGO1 complex. That is in contrast to small RNA generated during the RdDM process, which is 24 nt length and incorporated into AGO4 complex. In addition, the generation of siRNA854 depends on RDR6 and AGO1, suggesting that this siRNA is not controlled by the RdDM pathway, but controlled by tasiRNA pathway. tasiRNA uses specific members of RNAi components, such RDR6 and AGO1. Genes regulated by siRNA854 include UBP1b, which encodes an RNA-binding protein involved in stress granule formation. As UBP1b acts to repress TEs, the generation of siRNA854 from Athila may have advantage for the TE. The repression of host defense machinery by small RNA generated from a TE is also reported in rice (Nosaka et al. 2012).
Anti-silencing by TEs siRNA854 would affect host defense machinery. Although such mechanism would affect TE dynamics globally, some TEs have anti-silencing mechanisms that functions specifically for that TE. For example, 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; Schläppi et al. 1996; Cui and Fedoroff 2002). An active Spm transiently activates silent Spm copies in trans, a process likely be mediated by TnpA (Cui and Fedoroff 2002). Similarly, one of the Arabidopsis TEs mobilized in the ddm1 background, named Hiun (Hi), encodes a protein with anti-silencing activity (Fu et al. 2013). When that protein, named VANC, is expressed from the transgene, that causes anti-silencing of the endogenous Hi copy; in the transgenic lines, Hi loses DNA methylation, it is transcriptionally activated, and it is excised from the original locus. Hi belongs to non-TIR type of Mutator-like elements (MULEs). MULEs in the A. thaliana genome are composed of TIR type and non-TIR type. TIR MULEs have relatively long terminal inverted repeat (TIR), as is the cases for most of DNA type TE. In non-TIR MULEs, TIR is much degenerated (Le et al. 2000; Yu et al. 2000). Proteins related to VANC are widespread in non-TIR TE families, such as VANDAL and ARNOLD. On the other hand, related factor is not encoded in any of canonical MULEs with TIR (Fu et al. 2013). It would be
interesting to learn if the anti-silencing machinery is related to their mode of transposition with the degenerated TIR.
Activation of TEs by environmental stress McClintock found mobile elements in conditions of chromosome breaks and proposed that TEs can be activated as “responses of the genome to challenge” (McClintock 1984). Consistent with that, several examples have been found for TEs activated under environmental stresses. TEs activated and mobilized in tissue culture are known in tobacco, pea, and rice. Wellcharacterized examples are Tnt1 and Tto1, copia-type retrotransposons in tobacco. They are expressed by protoplast isolation, tissue culture, wounding, pathogen infections, microbial elicitors, and salicylic acid (Hirochika 1993; Mhiri et al. 1997; Pouteau et al. 1991; Takeda et al. 1998, 1999). Tissue culture induces mobilization of diverse TEs, such as LORE1 (LTR retrotransposon) in pea (Fukai et al. 2010), Tos17 (LTR retrotransposon; Hirochika et al. 1996), Karma (non-LTR retrotransposon; Komatsu et al. 2003), and PING/PONG (DNA transposon; Jiang et al. 2003; Kikuchi et al. 2003) in rice. Tam3, a DNATE in snapdragon, is mobilized at low temperature. The mobilization is associated with loss of DNA methylation, which is mediated by activation of transposase (Hashida et al. 2006). In A. thaliana, heat induces activation of a copia-type retrotransposon named ONSEN (Ito et al. 2011). ONSEN is transcriptionally activated, and transgenerational transpositions are observed in a mutant impaired in RdDM pathway (Matsunaga et al. 2012). Interestingly, ONSEN is not activated in mutants deficient in DNA methylation such as met1 or ddm1. TE sequences related to ONSEN are found in many species of Brassicaceae. These Brassicaceae elements are also activated by heat stress, suggesting conservation of the control (Ito et al. 2013).
TE, genome evolution, and reproductive strategy Within the A. thaliana genome, the proportion of TEs is much higher in pericentromeric regions than in the chromosomal arms (The Arabidopsis Genome Initiative 2000). The accumulation of TEs and repeats
Control of transposable elements in Arabidopsis thaliana
is a feature commonly found in diverse organisms, including the fruit fly, puffer fish, and fission yeast. In the A. thaliana genome, the bias toward pericentromeric regions differs among TE species; short TEs, such as SINEs and MITEs, show weak bias, while LTR retroelements show strong bias. Among the LTR retroelements, gypsy-type retroelement Athila shows very strong bias toward pericentromeric regions. The bias of TE distribution could be explained by targeted integration, natural selection against insertion into generich regions, or suppression of recombination in the pericentromeric regions. The theoretical analyses suggest that the bias is mainly due to targeted integration (Peterson-Burch et al. 2004). Targeted integration of an LTR-retroelement into the gene-poor centromeric regions is directly shown for Tal1 (transposon of Arabidopsis lyrata 1). Tal1 and related copies distribute within centromeric satellite repeat in the genome of A. lyrata, a closely related species of A. thaliana (Tsukahara et al. 2009). When this A. lyrata element is introduced into A. thaliana genome by transformation, it shows de novo integrations with very strong bias toward centromeric satellite regions of A. thaliana genome (Tsukahara et al. 2012). Interestingly, a very closely related A. thaliana TE, COPIA93 (also called EVADE), does not show the integration bias toward centromere. Phylogenetic analyses of TEs related to Tal1 and COPIA93/EVADE revealed that A. lyrata genome contains at least four clusters of related TEs, which proliferated recently. Such extensive proliferations are not found within A. thaliana genome. In addition, most of the proliferated A. lyrata copies are distributed within centromeric satellite, but A. thaliana copies are not (Tsukahara et al. 2012). The preferential integration into centromere may be less harmful to the host, which would be good for proliferation and survival of TE itself. Such A. lyrata-specific proliferations are also found for other types of LTR elements (Hu et al. 2011). In addition, the comparison of genome sequences of A. thaliana and A. lyrata showed that in average, A. lyrata contains more young TE insertions in the genome than A. thaliana (Hu et al. 2011). In addition, the comparison suggests a recent reduction in TE copy number in the A. thaliana lineage (de la Chaux et al. 2012). These observations are consistent with the theoretical prediction that active TEs propagate more efficiently in out-crossing A. lyrata than in self-pollinating A. thaliana (Hickey 1982).
Perspectives Here, we summarized and discussed control of TEs, mainly in the context of epigenetics. DNA methylation of TEs is important for silencing them. A remaining question would be how a host distinguishes between genes and TEs and specifically methylates TEs (further discussion in Inagaki and Kakutani 2012). The TE–host interactions could provide evolutionary sources for higher-order biological phenomena, such as imprinting, development, and environmental responses. Biological significance of epigenetic variations associated with TEs would also be an important field to investigate (Becker et al. 2011; Schmitz et al. 2011). A. thaliana will remain a powerful model organism to understand control and biological significance of TEs through genetic and genomic approaches. Examination of intra-specific and inter-specific variations, as well as cytogenetic approaches, would also broaden the research field in future.
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REVIEW
Control of transposable elements in Arabidopsis thaliana Hidetaka Ito & Tetsuji Kakutani
# Springer Science+Business Media Dordrecht 2014
Abstract Arabidopsis thaliana serves as a very good model organism to investigate the control of transposable elements (TEs) by genetic and genomic approaches. As TE movements are potentially deleterious to the hosts, hosts silence TEs by epigenetic mechanisms, such as DNA methylation. DNA methylation is controlled by DNA methyltransferases and other regulators, including histone modifiers and chromatin remodelers. RNAi machinery directs DNA methylation to euchromatic TEs, which is under developmental control. In addition to the epigenetic controls, some TEs are controlled by environmental factors. TEs often affect expression of nearby genes, providing evolutionary sources for epigenetic, developmental, and environmental gene controls, which could even be beneficial for the host.
CMT DRM DRD
Keywords Epigenetics . Genomics . DNA methylation . Heterochromatin
Introduction: Arabidopsis thaliana as an ideal model organism to investigate controls of TEs
Abbreviations TE Transposable element RNAi RNA interference MET Methyltransferase DDM Decrease in DNA methylation
Since the first discovery in maize, TEs and their controls have been extensively studied in plants. Among the plant species, Arabidopsis thaliana is a very good material to study the control of TEs. Compared to the genomes of typical plant species, the proportion of TEs in the A. thaliana genome is low—about 10 % (The Arabidopsis Genome Initiative 2000). The low proportion is not due to lack of TE families; A. thaliana genome contains essentially every TE family known in plants (for details, see the accompanying review by Joly-Lopez and Bureau). The low TE proportion is mainly due to low copy number for each of these diverse TE families. The low copy number of each of TE members make their molecular analyses less
Responsible editors: Martin Lysak and Paul Fransz. H. Ito Faculty of Science, Hokkaido University, Kita10 Nishi 8, Kitaku, Sapporo, Hokkaido 060-0810, Japan T. Kakutani (*) Department of Integrated Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan e-mail: [email protected]
RDR RdDM DCL FWA SDC siRNA SINE tasiRNA TIR LTR
Chromomethylase Domain-rearranged methyltransferase Defective in RNA-directed DNA methylation RNA-dependent RNA polymerase RNA-directed DNA methylation Dicer like Flowering Wageningen Suppressor of DRM1 DRM2 CMT3 Small-interfering RNA Short interspersed nuclear element Trans-acting small-interfering RNA Terminal inverted repeat Long terminal repeat
H. Ito, T. Kakutani
complicated. In addition, many of the TE sequences are precisely mapped in the available completed genome sequences of A. thaliana because the genome sequences have been determined after generating the BAC contigs (The Arabidopsis Genome Initiative 2000). These features are the big advantages of A. thaliana for the genome-wide study of TE properties, such as expression, epigenetic modifications, and transposition (The Arabidopsis Genome Initiative 2000; Le et al. 2000; Peterson-Burch et al. 2004). In addition to genomics, genetics is also a very powerful tool to study TE control. Host mutations affecting TE activity are available and have been extensively studied. Approaches using these mutations are especially powerful when combined with the genome-wide approaches.
DNA methylation as a major factor controlling TEs TEs can be silenced by epigenetic marks, such as DNA methylation. Plant genome contains methylation of cytosine at both CpG and non-CpG sites. Most of the TEs, although not all, are heavily methylated for both CpG and non-CpG sites (Cokus et al. 2008; Lister et al. 2008). The CpG methylation is kept by the maintenance DNA methyltransferase MET1 (Finnegan et al. 1996). Methylation at nonCpG sites is catalyzed by chromomethylases, which target regions with methylation in lysine 9 of histone H3 (H3K9me; Chan et al. 2005). H3K9me is an epigenetic mark of silent and condensed chromatin, called heterochromatin (Grewal and Elgin 2007). The methylation of heterochromatic TE depends on a chromatin remodeler DDM1. DDM1 helps the methylation of TEs for both CpG and non-CpG sites, most likely by facilitating the access of DNA methylation machinery to heterochromatin (Jeddeloh et al. 1999; Zemach et al. 2013). The importance of DNA methylation in TE control has been directly shown using mutants of these genes; in Arabidopsis mutants with reduced genomic DNA methylation, a variety of silent TEs are de-repressed and mobilized (Singer et al. 2001; Miura et al. 2001; Kato et al. 2003; Lippman et al. 2004; Tsukahara et al. 2009; Mirouze et al. 2009). The ddm1 mutation, which affects DNA methylation at both CpG and non-CpG sites, mobilizes diverse TEs including mutators, CACTAs, and LTR elements. Mutation in the CpG methyltransferase gene
MET1 mobilizes some of these elements, such as an LTR element COPIA93/EVADE and a mutator-like element VANDAL21/Hiun (Mirouze et al. 2009; Tsukahara et al. 2009), suggesting that CpG methylation plays major role for immobilization of these TEs. On the other hand, some of other TEs, such as CACTA and GYPSY3, are not mobilized by met1 single mutation, but mobilized by double mutation of MET1 and non-CpG methyltransferase gene CMT3, suggesting that both CpG and non-CpG methylation are involved in their immobilization (Kato et al. 2003; Tsukahara et al. 2009). DNA methylation is maintained by DNA methyltransferases, MET1 and CMTs. Another class of DNA methyltransferase, DRM2, is involved in DNA methylation de novo (Cao and Jacobsen 2002). DRM2 is the DNA methyltransferase conducting the final reaction of “RNA-directed DNA methylation,” or RdDM. RdDM is an intriguing observation found first in tobacco that double-stranded RNA induces de novo DNA methylation of similar sequences (Wasseneger et al. 1994; Mette et al. 2000). Genetic screening of Arabidopsis has lead to identification of upstream factors involved in RdDM that include chromatin remodeling factor DRD1, RNAi components such as RDR2 and DCL3, and components of novel RNA polymerase complexes. The molecular characterization of these factors make RdDM a very active and important research field (Matzke et al. 2009; Law and Jacobsen 2010; Gao et al. 2010; Wierzbicki et al. 2012; Law et al. 2013). The mutations in RdDM components affect DNA methylation in euchromatin (Tran et al. 2005; Huettel et al. 2006; Zemach et al. 2013). This is in contrast to the effect of ddm1 mutation; ddm1 mutation abolishes DNA methylation in heterochromatin. Genome-wide analysis of DNA methylation in mutants of DDM1 and an RdDM component DRD1 revealed that these two mutations affect genomic DNA methylation in complementary manners (Zemach et al. 2013). The ddm1 mutation affects internal region of long TEs, which is heterochromatic. The drd1 mutation affects short TEs and terminal regions of long TEs, which is more euchromatic than regions affected by ddm1.
Release of TE silencing by demethylation in gametophytes Euchromatic TEs can be targets of RdDM. Intriguingly, the demethylation of these RdDM targets has been
Control of transposable elements in Arabidopsis thaliana
found during development. An extensively studied tissue is endosperm (Lauria et al. 2004; Gehring et al. 2009; Hsieh et al. 2009; Ibarra et al. 2012). Endosperm is a tissue supporting growth of embryo within the seed. The endosperm originates from the fertilization of sperm and central cell. Central cell is a companion cell of the egg in the female gametophyte. It is assumed that the DNA methylation is lost in the central cell. After fertilization, the loss of DNA methylation in the central cell is inherited to endosperm, contributing imprinted expression of genes flanking the euchromatic TEs. The reduction of DNA methylation depends on the DNA demethylase DEMETER (DME). Analogous mechanisms operate in the male side, during pollen maturation. A pollen grain has one vegetative cell nucleus (VN) and two sperm nuclei (SN). The two SN fertilizes with egg and central cells to generate embryo and endosperm lineages, respectively. In the companion nucleus, VN, centromeric chromatin was decondensed (Schoft et al. 2009); and many TEs are transcribed and transposed specifically in the VN, although not in the SN (Slotkin et al. 2009). Interestingly, the demethylation in VN also depends on DME (Schoft et al. 2011; Ibarra et al. 2012). The de-repression of TEs in these companion cells may induce de novo silencing of TEs through small RNA (Slotkin et al. 2009; Ibarra et al. 2012; Calarco et al. 2012). As these companion cells do not contribute to the next generation, mutagenization by TE movements would be less deleterious than those in the embryonic lineage.
TE as evolutionary sources for epigenetic gene control TEs in maize were initially described as “controlling elements,” because they often affect the functions of nearby genes (McClintock 1965) that were substantiated later at the molecular level for maize TEs, such as McClintock’s Suppressor-mutator (Spm) and Robertson’s Mutator (Fedoroff 1996; Martienssen 1996). TEs can be targets of epigenetic silencing, which is often heritable but reversible; the epigenetic states of TEs, in turn, affect expression of host genes nearby. Mutations affecting DNA methylation, such as met1 and ddm1, induce de-repression of cellular genes. One of them, the FWA gene was initially identified as the gene responsible for the “epigenetic mutation” with the
phenotype of delayed developmental transition (Soppe et al. 2000). “Epigenetic mutation” refers to a transgenerationally heritable trait without change in genomic nucleotide sequence. The fwa line does not have change in nucleotide sequence in the responsible gene. The late flowering trait in the fwa line turned out to be due to ectopic expression of the responsible gene, which is associated with the loss of DNA methylation in the promoter region (Soppe et al. 2000). The promoter se qu en ce h as s imil ari ty t o S INE, no n- LT R retrotransposon (Lippman et al. 2004; Fujimoto et al. 2008). The SINE-related promoter is normally methylated, and the gene is silent in the adult tissue. The methylation is removed in central cells in female gametophyte, causing imprinted expression of this gene in endosperm (Kinoshita et al. 2004; Fujimoto et al. 2008). Genome-wide analyses revealed that the expression of many genes is affected when DNA is removed from short TEs in central cells and pollen (Gehring et al. 2009; Hsieh et al. 2009; Slotkin et al. 2009; Ibarra et al. 2012). The loss of DNA methylation in drm1drm2cmt3 or ddm1rdr2 mutations induces developmental abnormalities, which is mediated by SDC gene (Henderson and Jacobsen 2008; Zemach et al. 2013). SDC also has repetitive promoter and shows imprinted expression in endosperm (Henderson and Jacobsen 2008; Hsieh et al. 2011). The ddm1 and met1 mutation induces not only de-repressed epialleles, such as fwa, but also silent epialleles (Jacobsen and Meyerowitz 1997; Jacobsen et al. 2000). Among them, a combination of ddm1induced developmental phenotypes, called bonsai (bns), is due to silencing of Anaphase-Promoting Complex 13 (APC13) gene. The silencing of BNS gene was associated with ectopic DNA methylation at both CpG and non-CpG sites (Saze and Kakutani 2007). The ectopic methylation depends on the presence of a LINE element insertion within the 3′ non-coding region (Saze and Kakutani 2007). The LINE insertion generates a potential trigger for epigenetic variation with strong developmental effects. In these examples, TEs regulate nearby genes in cis. TEs also have potential to control genes in trans. (Arteaga-Vázquez et al. 2006; McCue et al. 2012; McCue et al. 2013). In the A. thaliana genome, the most abundant TE species are gypsy type retroelements, called Athila. When de-repressed, Athila6 generates siRNA854, which affects expression of many host genes at the post-transcriptional level. Although
H. Ito, T. Kakutani
siRNA854 is derived from the TE, the length is 21–22 nt and it is incorporated into AGO1 complex. That is in contrast to small RNA generated during the RdDM process, which is 24 nt length and incorporated into AGO4 complex. In addition, the generation of siRNA854 depends on RDR6 and AGO1, suggesting that this siRNA is not controlled by the RdDM pathway, but controlled by tasiRNA pathway. tasiRNA uses specific members of RNAi components, such RDR6 and AGO1. Genes regulated by siRNA854 include UBP1b, which encodes an RNA-binding protein involved in stress granule formation. As UBP1b acts to repress TEs, the generation of siRNA854 from Athila may have advantage for the TE. The repression of host defense machinery by small RNA generated from a TE is also reported in rice (Nosaka et al. 2012).
Anti-silencing by TEs siRNA854 would affect host defense machinery. Although such mechanism would affect TE dynamics globally, some TEs have anti-silencing mechanisms that functions specifically for that TE. For example, 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; Schläppi et al. 1996; Cui and Fedoroff 2002). An active Spm transiently activates silent Spm copies in trans, a process likely be mediated by TnpA (Cui and Fedoroff 2002). Similarly, one of the Arabidopsis TEs mobilized in the ddm1 background, named Hiun (Hi), encodes a protein with anti-silencing activity (Fu et al. 2013). When that protein, named VANC, is expressed from the transgene, that causes anti-silencing of the endogenous Hi copy; in the transgenic lines, Hi loses DNA methylation, it is transcriptionally activated, and it is excised from the original locus. Hi belongs to non-TIR type of Mutator-like elements (MULEs). MULEs in the A. thaliana genome are composed of TIR type and non-TIR type. TIR MULEs have relatively long terminal inverted repeat (TIR), as is the cases for most of DNA type TE. In non-TIR MULEs, TIR is much degenerated (Le et al. 2000; Yu et al. 2000). Proteins related to VANC are widespread in non-TIR TE families, such as VANDAL and ARNOLD. On the other hand, related factor is not encoded in any of canonical MULEs with TIR (Fu et al. 2013). It would be
interesting to learn if the anti-silencing machinery is related to their mode of transposition with the degenerated TIR.
Activation of TEs by environmental stress McClintock found mobile elements in conditions of chromosome breaks and proposed that TEs can be activated as “responses of the genome to challenge” (McClintock 1984). Consistent with that, several examples have been found for TEs activated under environmental stresses. TEs activated and mobilized in tissue culture are known in tobacco, pea, and rice. Wellcharacterized examples are Tnt1 and Tto1, copia-type retrotransposons in tobacco. They are expressed by protoplast isolation, tissue culture, wounding, pathogen infections, microbial elicitors, and salicylic acid (Hirochika 1993; Mhiri et al. 1997; Pouteau et al. 1991; Takeda et al. 1998, 1999). Tissue culture induces mobilization of diverse TEs, such as LORE1 (LTR retrotransposon) in pea (Fukai et al. 2010), Tos17 (LTR retrotransposon; Hirochika et al. 1996), Karma (non-LTR retrotransposon; Komatsu et al. 2003), and PING/PONG (DNA transposon; Jiang et al. 2003; Kikuchi et al. 2003) in rice. Tam3, a DNATE in snapdragon, is mobilized at low temperature. The mobilization is associated with loss of DNA methylation, which is mediated by activation of transposase (Hashida et al. 2006). In A. thaliana, heat induces activation of a copia-type retrotransposon named ONSEN (Ito et al. 2011). ONSEN is transcriptionally activated, and transgenerational transpositions are observed in a mutant impaired in RdDM pathway (Matsunaga et al. 2012). Interestingly, ONSEN is not activated in mutants deficient in DNA methylation such as met1 or ddm1. TE sequences related to ONSEN are found in many species of Brassicaceae. These Brassicaceae elements are also activated by heat stress, suggesting conservation of the control (Ito et al. 2013).
TE, genome evolution, and reproductive strategy Within the A. thaliana genome, the proportion of TEs is much higher in pericentromeric regions than in the chromosomal arms (The Arabidopsis Genome Initiative 2000). The accumulation of TEs and repeats
Control of transposable elements in Arabidopsis thaliana
is a feature commonly found in diverse organisms, including the fruit fly, puffer fish, and fission yeast. In the A. thaliana genome, the bias toward pericentromeric regions differs among TE species; short TEs, such as SINEs and MITEs, show weak bias, while LTR retroelements show strong bias. Among the LTR retroelements, gypsy-type retroelement Athila shows very strong bias toward pericentromeric regions. The bias of TE distribution could be explained by targeted integration, natural selection against insertion into generich regions, or suppression of recombination in the pericentromeric regions. The theoretical analyses suggest that the bias is mainly due to targeted integration (Peterson-Burch et al. 2004). Targeted integration of an LTR-retroelement into the gene-poor centromeric regions is directly shown for Tal1 (transposon of Arabidopsis lyrata 1). Tal1 and related copies distribute within centromeric satellite repeat in the genome of A. lyrata, a closely related species of A. thaliana (Tsukahara et al. 2009). When this A. lyrata element is introduced into A. thaliana genome by transformation, it shows de novo integrations with very strong bias toward centromeric satellite regions of A. thaliana genome (Tsukahara et al. 2012). Interestingly, a very closely related A. thaliana TE, COPIA93 (also called EVADE), does not show the integration bias toward centromere. Phylogenetic analyses of TEs related to Tal1 and COPIA93/EVADE revealed that A. lyrata genome contains at least four clusters of related TEs, which proliferated recently. Such extensive proliferations are not found within A. thaliana genome. In addition, most of the proliferated A. lyrata copies are distributed within centromeric satellite, but A. thaliana copies are not (Tsukahara et al. 2012). The preferential integration into centromere may be less harmful to the host, which would be good for proliferation and survival of TE itself. Such A. lyrata-specific proliferations are also found for other types of LTR elements (Hu et al. 2011). In addition, the comparison of genome sequences of A. thaliana and A. lyrata showed that in average, A. lyrata contains more young TE insertions in the genome than A. thaliana (Hu et al. 2011). In addition, the comparison suggests a recent reduction in TE copy number in the A. thaliana lineage (de la Chaux et al. 2012). These observations are consistent with the theoretical prediction that active TEs propagate more efficiently in out-crossing A. lyrata than in self-pollinating A. thaliana (Hickey 1982).
Perspectives Here, we summarized and discussed control of TEs, mainly in the context of epigenetics. DNA methylation of TEs is important for silencing them. A remaining question would be how a host distinguishes between genes and TEs and specifically methylates TEs (further discussion in Inagaki and Kakutani 2012). The TE–host interactions could provide evolutionary sources for higher-order biological phenomena, such as imprinting, development, and environmental responses. Biological significance of epigenetic variations associated with TEs would also be an important field to investigate (Becker et al. 2011; Schmitz et al. 2011). A. thaliana will remain a powerful model organism to understand control and biological significance of TEs through genetic and genomic approaches. Examination of intra-specific and inter-specific variations, as well as cytogenetic approaches, would also broaden the research field in future.
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