Chromosome Res (2013) 21:587–600 DOI 10.1007/s10577-013-9394-4

REVIEW

Small RNAs, big impact: small RNA pathways in transposon control and their effect on the host stress response Bayly S. Wheeler Published online: 20 November 2013 # Springer Science+Business Media Dordrecht 2013

Abstract Transposons are mobile genetic elements that are a major constituent of most genomes. Organisms regulate transposable element expression, transposition, and insertion site preference, mitigating the genome instability caused by uncontrolled transposition. A recent burst of research has demonstrated the critical role of small non-coding RNAs in regulating transposition in fungi, plants, and animals. While mechanistically distinct, these pathways work through a conserved paradigm. The presence of a transposon is communicated by the presence of its RNA or by its integration into specific genomic loci. These signals are then translated into small non-coding RNAs that guide epigenetic modifications and gene silencing back to the transposon. In addition to being regulated by the host, transposable elements are themselves capable of influencing host gene expression. Transposon expression is responsive to environmental signals, and many transposons are activated by various cellular stresses. TEs can confer local gene regulation by acting as enhancers and can also confer global gene regulation through their noncoding RNAs. Thus, transposable elements can act as stress-responsive regulators that control host gene expression in cis and trans. Keywords transposable element . siRNA . piRNA . non-coding RNA . stress

Responsible Editors: Brian P. Chadwick, Kristin C. Scott, and Beth A. Sullivan B. S. Wheeler (*) Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA e-mail: [email protected]

Abbreviations dsDNA dsRNA HDAC HMT LINE LTR ncRNA ORF piRNA Pol II, IV, V RdDM RDRC RITS RNAi SC SINE siRNA TE TIR VN

Double-stranded DNA Double-stranded RNA Histone deacetylase Histone methyltransferase Long interspersed element Long terminal repeat Non-coding RNA Open reading frame PIWI-interacting RNA RNA polymerase II, IV, V RNA-dependent DNA methylation RNA-directed RNA polymerase complex RNA-induced transcriptional silencing RNA interference Sperm cells Short interspersed element Short interfering RNA Transposable element Terminal inverted repeat Vegetative nucleus

Introduction Transposons are selfish genetic elements capable of moving within genomes. Most organisms have been colonized by transposable elements (TE) to varying extents; transposons make up 45 % of the human genome, 3 % of the yeast genome, and 80 % of the maize genome (Kim et al. 1998; Lander et al. 2001; Schnable et al. 2009). Insertion of TEs within host genomes can disrupt gene function, and illegitimate recombination between TEs

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leads to genome rearrangements (Beck et al. 2011; Solyom and Kazazian 2012). Organisms control transposon expression, mobility, and insertion site preference, alleviating their mutagenic potential. A common theme shared among many transposon defense systems is the essential role played by small non-coding RNAs in silencing TEs and preventing transposition. These non-coding RNAs are critical components of pathways that recruit epigenetic modifications and result in heritable gene silencing. In addition to TE regulation by the host defense system, evolutionarily diverse TEs activated in response to cellular stress can act in trans through non-coding RNAs, as well as in cis, to regulate host genes. In this review, I will discuss the mechanisms by which small non-coding RNAs regulate TEs and the role of TEs and their noncoding RNAs in regulating the host stress response. TEs can be divided into two classes based on their method of transposition (classified in Capy 1997). Class I transposons are retrotransposons, which move through a copy and paste mechanism. Class II transposons, or DNA transposons, move through a cut and paste mechanism. Like retroviruses, retrotransposons move through an RNA intermediate that is reverse transcribed into a double-stranded DNA (dsDNA) that integrates within the genome (reviewed in Finnegan 2012). These retrotransposons can be subdivided based on whether they are flanked by long-terminal repeats (LTRs). LTRs are direct repeats that contain essential information for transcription of the intervening open reading frames (ORFs). Between their LTRs, most retrotransposons encode two polyproteins that are sufficient to catalyze reverse transcription and integration. Recombination between LTRs can occur, leaving solo LTRs in the genome. Non-LTR retrotransposons utilize an internal promoter and a Poly(A) sequence to direct expression of their ORFs. Their mechanism of reverse transcription is also distinct from that of LTR retrotransposons (Luan et al. 1993). While many TEs encode the machinery necessary to catalyze their own movement, some are nonautonomous, requiring other TEs for transposition. For example, the non-autonomous retrotransposon SINE (short interspersed element) requires the transposition machinery of the LINE (long interspersed element) retrotransposon. SINEs contain an internal RNA polymerase III promoter but do not encode proteins. DNA transposons encode a transposase that binds to flanking terminal inverted repeats (TIR) and catalyzes TE excision and insertion elsewhere in the genome (Levin and Moran 2011). DNA transposons are

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generally less abundant than retrotransposons and are not active in most mammalian genomes.

Hosts control the expression, transposition, and integration site preference of TEs Organisms maintain control over TEs, in part, by regulating transposon RNA levels both transcriptionally and post-transcriptionally. Common themes have emerged among mechanisms of TE transcriptional control in various organisms. Most have developed multiple layers of regulation, one of which is silencing enacted by small RNAs and small RNA-guided epigenetic modifications. Two major systems of TE control will be discussed here: short interfering RNA (siRNA) silencing in plants and fission yeast (also referred to as the RNAi pathway), and silencing via the PIWI-interacting RNA (piRNA) pathway, which controls TEs in animals. Importantly, while transcription is essential for transposition, increased TE RNA levels are not sufficient for transposition; therefore, mechanisms must exist to regulate subsequent steps in transposition. Although these pathways are less well understood, in Arabidopsis mutations in specific siRNA components can alleviate silencing without allowing transposition (Ito et al. 2011; Mirouze et al. 2009). This demonstrates an uncoupling of TE expression levels and transposition. As a final level of control over TEs, organisms can regulate the site of TE insertion. In S. cerevisiae, the transposon Ty5 integrates predominantly within heterochromatin, possibly to avoid disrupting gene function (Zou et al. 1996; Zou and Voytas 1997). However, S. cerevisiae can abolish this preference in response to environmental cues (Dai et al. 2007), providing a remarkable paradigm for dynamic control of insertion site preference by the host. Preferential insertion of Ty5 within heterochromatin is caused by the interaction of the Ty5 integrase with a yeast heterochromatin protein (Gai and Voytas 1998; Xie et al. 2001; Zhu et al. 2003). This interaction is dependent on phosphorylation of the integrase, which is regulated by nutrient conditions (Dai et al. 2007). In many other organisms, TEs preferentially insert within specific genomic regions (Behrens et al. 2000; Bellen et al. 2011; Chatterjee et al. 2009; Guo and Levin 2010; Miyao et al. 2003; Naito et al. 2009; Singleton and Levin 2002; Spradling et al. 2011; van Luenen and Plasterk 1994), although it is not yet understood whether the host regulates these preferences.

Small RNAs and transposon control

Transposon control in Schizosaccharomyces pombe Multiple pathways regulate TE expression in S. pombe The genome of the fission yeast Schizosaccharomyces pombe contains 13 copies of an LTR retrotransposon, Tf2, as well as TE fragments and solo LTRs that in total compose 1.7 % of its genome (Bowen et al. 2003; Wood et al. 2002). Tf2 activation can lead to transposition. However, most novel Tf2 insertions arise from recombination between Tf2 cDNA and preexisting copies of Tf2 (Hoff et al. 1998). In pombe, TEs are controlled by multiple mechanisms that commonly involve the recruitment of histone deacetylases (HDACs), which are associated with transcriptional repression (Fig. 1a). Multiple HDACs cooperate to silence TEs in fission yeast (Anderson et al. 2009; Durand-Dubief et al. 2007; Hansen et al. 2005; Lorenz et al. 2012; Sugiyama et al. 2007). Control of TE expression is achieved, in part, by CENP-B homologs that bind transposons and solo LTRs (Cam et al. 2008). One of these homologues, Abp1, mediates TE clustering within the nucleus, recruits HDACs, and silences both sense and antisense TE transcripts (Cam et al. 2008; Lorenz et al. 2012). In addition, Abp1 promotes genome stability by preventing replication fork stalling at TEs as well as recombination between TEs (Cam et al. 2008; Zaratiegui et al. 2011). Abp1 is recruited to TEs by a 10 bp motif that is contained within most fission yeast LTRs (Lorenz et al. 2012). CENP-B may have evolved from a domesticated transposase (Tudor et al. 1992), which could explain the ability of Abp1 to recognize and bind TEs within the fission yeast genome. As a final bulwark against transposition, the fission yeast RNAi pathway is also involved in TE control. While RNAi has a modest contribution to TE silencing under normal conditions (Hansen et al. 2005; Woolcock et al. 2011), it is critical for the TE control in the absence of a functional exosome or in wild-type cells grown in low glucose (Fig. 1b) (Yamanaka et al. 2013). The fission yeast siRNA pathway controls gene expression and formation of heterochromatin The siRNA pathway controls heterochromatin formation at the major heterochromatic regions of the S. pombe genome, including the outer centromere repeats

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(Fig. 1b). These repeats share organizational similarity with TEs and may be ancient TEs themselves (Chikashige et al. 1989; Clarke and Baum 1990). Bi-directional transcripts are embedded within the outer repeats of all three S. pombe centromeres (Djupedal et al. 2005; Kato et al. 2005; Volpe et al. 2002). These transcripts are silenced by two activities of the siRNA pathway: post-transcriptional silencing through cleavage of the centromeric transcripts and transcriptional silencing though the formation of repressive heterochromatin. This section will describe the mechanism by which this multi-level silencing occurs. Centromeric double-stranded RNAs (dsRNA) are cleaved into siRNAs by the S. pombe Dicer Dcr1 (Bühler et al. 2007; Reinhart and Bartel 2002; Sigova et al. 2004; Verdel et al. 2004; Volpe et al. 2002). siRNAs are loaded into the fission yeast RNA-induced transcriptional silencing complex (RITS), which includes the Argonaute Ago1 and the chromodomain protein Chp1 (Verdel et al. 2004). The slicer activity of Ago1 converts duplex siRNA into single-stranded siRNAs (Buker et al. 2007). RITS localizes to centromeric repeats - perhaps through base pairing of the single-stranded siRNAs and the centromeric transcripts - where it recruits the RDRC complex (Motamedi et al. 2004; Verdel et al. 2004). RDRC contains an RNAdependent RNA polymerase that acts on centromeric transcripts, converting them to dsRNA and Dcr1 substrates (Motamedi et al. 2004; Sugiyama et al. 2005). RITS also recruits the H3K9 histone methyltransferase (HMT) Clr4 (Zhang et al. 2008). H3K9me2 stabilizes the association of Chp1 with the centromeres, causing a self-propagating loop whereby the presence of H3K9me2 reinforces the siRNA pathway, which in turn recruits histone methyltransferases and generates additional H3K9me2 (Noma et al. 2004; Schalch et al. 2009; Sugiyama et al. 2005). Additionally, H3K9me2 is recognized by the chromodomain proteins Swi6 and Chp2, which recruit HDACs (Fischer et al. 2009; Sadaie et al. 2008; Sugiyama et al. 2007). Together these complexes silence centromeric transcripts by limiting Pol II occupancy throughout most of the cell cycle (Chen et al. 2008). Although the mechanism of TE silencing by the siRNA pathway differs in some aspects from the centromeric siRNA pathway, TE control in low glucose conditions requires many of the same components and results in the local formation of siRNAs and heterochromatin (Yamanaka et al. 2013).

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Fig. 1 Pathways that silence TEs in fission yeast and plants. a The CENP-B homolog Abp1 (in addition to other CENP-B homologs not depicted here) binds LTRs and recruits the HDACs Clr3 and Clr6. The HIRA histone chaperone complex is required for TE silencing in S. pombe (Anderson et al. 2009). HIRA and Clr6 regulate overlapping sets of genes, which may suggest that they work in collaboration to silence TEs. The HDAC Hst4 also silences Tf2 and is required for normal processing of the 5' end of Tf2 mRNAs (Durand-Dubief et al. 2007). Set1, the H3K4 HMT also regulates TE expression, independently of H3K4me (Lorenz et al. 2012). b The S. pombe RNAi pathway controls heterochromatin formation at the centromere and at TEs in specific environmental conditions. RNA polymerase II transcribes centromeric repeats. Rdp1 converts these transcripts to dsRNA. Dcr1 cleaves dsRNA, resulting in siRNAs that are loaded onto Ago1. Ago1 acts as part of the RITS complex, which includes the chromodomain

protein Chp1, to recruit the H3K9 HMT Clr4 to the centromeres. The chromodomain proteins Swi6 and Chp2 bind H3K9me2 at the centromeres and recruit the HDACs Clr3 and Clr6. c Dcr1 degrades RNAs from LTRs both within Tf2 and solo-LTRs. This pathway does not result in the formation of heterochromatin or generation of siRNAs corresponding to LTRs. d RdDM controls de novo DNA methylation at Arabidopsis TEs. TEs are transcribed by the plant-specific polymerase, Pol IV. SHH1 binds H3K9me and is required for Pol IV recruitment. RDR2 converts Pol IV transcripts to dsRNAs that are then cleaved into siRNAs by DCR3. siRNAs are loaded onto AGO4 (or other Argonaute proteins not depicted here). AGO4 interacts with TE transcripts produced by the second plant-specific polymerase, Pol V. RDM1 interacts with both AGO4 and the DNA methyltransferase, DRM2, which catalyzes de novo DNA methylation

LTRs are silenced by Dcr1 independent of RNAi

Environmental conditions trigger RNAi-mediated TE control

Under normal conditions, silencing of LTRs, but not transposon ORFs, involves Dcr1 (Fig. 1c) (Woolcock et al. 2011). This pathway is distinct from RNAi as it does not require RdRP, result in the formation of heterochromatin, or produce detectible levels of TE-derived siRNAs (Woolcock et al. 2011).

Work from the Grewal lab demonstrates that the RNAi pathway regulates TEs in response to specific environmental conditions (Yamanaka et al. 2013). The mechanisms that govern this switch are unknown, but may be caused by TE activation in low glucose, which would

Small RNAs and transposon control

increase the level of TE RNAs that can serve as substrates for the RNAi pathway. The expression of TEs in many organisms, including S. pombe, is activated in response to stress (Chen et al. 2003). Glucose deprivation in S. pombe may activate Tf2 expression as it contributes to oxidative stress (Madrid et al. 2004) and oxidative stresses have been shown to increase Tf2 expression (Chen et al. 2003). Furthermore, stress activation may be a general feature of Abp1-bound genes; in addition to TEs, Abp1 binds protein coding genes activated in response to stress (Lorenz et al. 2012). The exosome is a protein complex that degrades RNAs. In S. pombe, transcript degradation by the exosome prevents abundant RNAs from entering the siRNA pathway (Bühler et al. 2007). Loss of the exosome mimics glucose deprivation; in exosome mutants the RNAi pathway silences TEs, suggesting that the exosome and RNAi collaborate to silence transposons in laboratory conditions (Yamanaka et al. 2013). Like activation in low glucose, TE levels may be increased in the absence of a functional exosome, resulting in TE RNAs entering the siRNA pathway. It seems counterintuitive that a burst of TE activation could result in RNAi-mediated silencing. However, as with all endogenous RNAi pathways, transcription is required to enact silencing. At the centromeres, this paradoxical relationship was resolved by the finding that transcription of centromeric repeats increases during S phase of the cell cycle concomitant with a decrease in heterochromatic histone modifications (Chen et al. 2008; Kloc et al. 2008). Increased expression of centromeric repeats during S phase triggers generation of siRNAs and subsequent heterochromatin formation (Kloc et al. 2008). Thus, a temporary burst of transcription provides the RNA template required to enact silencing. TE activation in response to environmental stress could serve as a similar trigger, directing the subsequent formation of siRNAs and heterochromatin. In another fission yeast, S. japonicus, the RNAi pathway regulates TE expression even in unstressed conditions (Rhind et al. 2011). The S. japonicus genome has more TEs than the S. pombe genome (Rhind et al. 2011), which could result in greater transposon RNA levels and processing of these transcripts by the RNAi pathway. To gain insights into the features that drive siRNA-based transposon control, it may be useful to explore the mechanisms that control TEs in S.

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japonicus along with S. pombe wild isolates with greater numbers of TEs. It is possible that other features of fission yeast TEs contribute to their regulation by the RNAi pathway, alternatively, or in conjunction with increased transcript level in stress conditions. RNAi pathways require a dsRNA intermediate to enact gene silencing. TEs, especially LTRs in S. pombe, have transcripts in both the sense and antisense orientation (Mourier and Willerslev 2010). Overlapping forward and reverse TE transcripts could form dsRNA, producing a dsRNA substrate sufficient for the initiation of RNAi. Consistent with this hypothesis, heterochromatin forms at overlapping convergent transcripts elsewhere in the S. pombe genome (Gullerova and Proudfoot 2012; Gullerova and Proudfoot 2008). Heterochromatin may provide an epigenetic memory of stress The extent to which TE heterochromatin is maintained when yeast are returned to full glucose is currently unknown. However, an ectopic domain of heterochromatin in fission yeast—established by inserting a fragment of the centromeric repeats in a euchromatic locus—is inherited through mitosis and meiosis even when the nucleating sequence is excised (Wheeler et al. 2012). The inheritance of heterochromatin persists for up to 600 mitotic generations, but silencing and heterochromatin cannot be reestablished once lost. By extension, heterochromatin formed at fission yeast TEs in low glucose may be maintained upon return to full glucose, providing an epigenetic memory of stress. This memory could shape TE activation in response to future stresses.

Transposon control in Arabidopsis DNA methylation controls transposition in Arabidopsis In plants, TEs are bound and silenced by DNA methylation (Chandler and Walbot 1986; Chomet et al. 1987; Cokus et al. 2008; Kato et al. 2003; Lister et al. 2008; Zhang et al. 2006). DNA methylation occurs at cytosines in three contexts: CG, CHG, and CHH (H= A,C,T). In Arabidopsis, the DNA methyltransferase MET1 copies CG methylation onto hemimethylated sites of replicated DNA, while CMT3 maintains methylation at CHG sites. MET1 and CMT3 act redundantly

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to control transposition (Kato et al. 2003). DDM1, a chromatin remodeler important for DNA methylation in all three sequence contexts also prevents both TE expression and transposition (Hirochika et al. 2000; Jeddeloh et al. 1999; Miura et al. 2001; Tsukahara et al. 2009; Vongs et al. 1993). In addition to DNA methylation, two histone modifications, H3K9me2 and H3K27me1, also constrain TE expression (reviewed in Rigal and Mathieu 2011). RNA-dependent DNA-methylation targets de novo methylation to TEs Analogous to RNAi in S. pombe, the RNA-dependent DNA methylation pathway (RdDM) targets DNA methylation to TEs in Arabidopsis (Fig. 1d) (Chan et al. 2004; Herr et al. 2005; Zilberman et al. 2003). Loci targeted by the RdDM pathway are transcribed by the plant-specific polymerase, RNA Polymerase IV (Pol IV), which is essential for the production of siRNAs and for RdDM (Herr et al. 2005; Zhang et al. 2007). Pol IV interacts with the RNA-dependent RNA polymerase, RDR2, to create dsRNA (Chan et al. 2004; Haag et al. 2012; Herr et al. 2005). As in fission yeast, dsRNAs are cleaved by Dicer (in this case, DCL3), resulting in the production of 24-nt siRNAs (Kasschau et al. 2007; Qi et al. 2005; Xie et al. 2004). These siRNAs are primarily loaded onto the Argonaute AGO4 (Chan et al. 2004; Qi et al. 2006; Zilberman et al. 2003), which is recruited to genomic loci that are transcribed by the second plantspecific polymerase, Pol V (Wierzbicki et al. 2008; Wierzbicki et al. 2009). RdDM recruits the de novo methyltransferase, DRM2, to target loci (Chan et al. 2004; Herr et al. 2005; Zilberman et al. 2003). The RdDM pathway is necessary for silencing some TEs, however it appears to be redundant with other mechanisms of TE control (Huettel et al. 2006; Xie et al. 2004) Pol IV is required for the production of most siRNAs in the Arabidopsis genome (Zhang et al. 2007), but the features that recruit Pol IV to repetitive loci are largely unknown. Recent work has shown that SHH1 is important for Pol IV recruitment and the generation of siRNAs (Law et al. 2013; Zhang et al. 2013). SHH1 contains a SAWADEE domain that binds methylated H3K9 (Law et al. 2013). Since RdDM components are important for recruiting H3K9me to some genomic loci (Zilberman et al. 2003), the requirement for H3K9me in RdDM may function as a self-reinforcing loop. Similarly, RDM1 interacts both with DRM2 and AGO4, and binds

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single-stranded methyl DNA (Gao et al. 2010). Taken together, these studies suggest that RdDM may be stabilized by binding to epigenetic modifications at TEs, analogous to RITS binding H3K9me2 in S. pombe. RdDM prevents transposition in stressed plants While the RdDM pathway silences some TEs in Arabidopsis, RdDM mutations do not mobilize TEs in germinal cells under normal growth conditions. However, RdDM does have a critical role in preventing transposition in plants that have experienced heat stress (Ito et al. 2011). In these stressed plants, transcription of the retrotransposon ONSEN is activated (Ito et al. 2011). Components of the RdDM pathway are important for reducing the level of ONSEN accumulation in stressed plants, but do not regulate ONSEN levels in unstressed plants. Furthermore, in the absence of the RdDM pathway, transposition occurs during flower development in heat-shocked plants, leading to novel ONSEN insertions that are inherited in the next generation (Ito et al. 2011). This study demonstrates that the RdDM pathway is critical for repressing TE expression and mobility after TE activation by heat stress. Activation of TEs in germline companion cells In addition to transposition during heat stress, transposition occurs in Arabidopsis germline companion cells independent of stress conditions. Male gametogenesis in Arabidopsis involves the formation of a three-celled pollen grain from somatic cells of the adult plant: two sperm cells (SC) that fertilize the zygote and the endosperm, and one large vegetative cell, which is important for delivery of the sperm but does not contribute genetic information to the next generation. The endosperm provides nourishment for the developing embryo but also does not contribute genetic material to embryo. Many proteins involved in TE silencing have differential localization between the vegetative nucleus (VN) and the SC: DDM1 is absent from VN (but not from SC); DME, a DNA glycosylase required for TE demethylation, is expressed only in the VN; and DRM2 is expressed mainly in the VN (Calarco et al. 2012; Ibarra et al. 2012; Slotkin et al. 2009). The specific exclusion or inclusion of these proteins within the VN accompanies a reduction in CG methylation and results in TE transposition (Calarco et al. 2012; Ibarra et al. 2012; Slotkin et al. 2009). One TE activated in the VN,

Small RNAs and transposon control

Athila, is processed into siRNAs that accumulate in both the VN and the SC. These siRNAs can silence transgenes within the SC (Slotkin et al. 2009), suggesting TE reactivation in the VN leads to the production of siRNAs that can silence transposons in the SC. Similar to TE activation in the VN, TEs are demethylated in the seed endosperm resulting in TE activation and production of high levels of TE siRNAs (Gehring et al. 2009; Hsieh et al. 2009; Mosher et al. 2009). These siRNAs may play a role in silencing TEs in the embryo (discussed in Van Ex et al. 2011). The observation that TE activation in companion cells precedes a burst of siRNA biogenesis supports the model in which repeat transcription during development or during stress results in the establishment of silencing by siRNA pathways. Unlike S. pombe, which temporally restricts much of centromere transcription to S phase of the cell cycle, Arabidopsis may spatially restrict TE activation to non-germinal cells.

Transposon control in animals via the piRNA pathway While the siRNA pathway controls TEs in plants and fission yeast, animals use the piRNA pathway to silence TEs. In addition to requiring different protein machinery, a major distinction between the siRNA and piRNA pathways is the event that causes small RNA production. While siRNA biogenesis is initiated by transcription of a target RNA, piRNA biogenesis is initiated by transposition into piRNA loci distributed throughout the genome. These piRNA loci are then transcribed and processed into piRNAs through a mechanism described in more detail below. piRNAs control TEs in Drosophila oocytes In Drosophila, two distinct mechanisms of piRNA production are used to control TEs in the somatic ovarian follicle and the oocyte (Li et al. 2009; Vagin et al. 2006). In addition, the endogenous-siRNA pathway also targets TEs in Drosophila (reviewed in Golden et al. 2008). Here, the focus will be on the oocyte piRNA pathway in which piRNAs are produced from piRNA loci, amplified by a process called the ping-pong cycle, and recruit epigenetic modifications to TEs (Fig. 2a). Most piRNAs originate from clustered piRNA loci, which contain the remnants of many TEs (Brennecke

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et al. 2007; Khurana et al. 2011). Each locus is transcribed as a single transcript that is processed, possibly in part by the 5' endoribonuclease Zucchini (Ipsaro et al. 2012; Nishimasu et al. 2012), into primary piRNAs. Primary piRNAs then enter the ping-pong cycle. Primary piRNAs are bound by the PIWI proteins AUB or PIWI and direct cleavage of cognate transposon RNAs (Brennecke et al. 2007; Vagin et al. 2006). This cleavage silences the TE post-transcriptionally and produces a secondary piRNA. The secondary piRNA is bound by the Argonaute AGO3 and can cleave the original piRNA cluster transcript, producing yet another piRNA (Brennecke et al. 2007; Gunawardane et al. 2007). piRNAs guide PIWI proteins and the heterochromatin machinery, including H3K9me2/3, heterochromatin protein 1, and the H3K9 HMT Su(var)3-9, to their targets (Huang et al. 2013; Le Thomas et al. 2013; Sienski et al. 2012). Together, these proteins reduce Pol II occupancy at piRNA targets (Huang et al. 2013; Le Thomas et al. 2013; Sienski et al. 2012). The existence of piRNA loci in Drosophila provides a very compelling mechanism for how TEs are detected and silenced. Creating a dynamic library of encountered TEs allows regulation by the piRNA pathway to be both heritable and remarkably flexible. Mouse piRNAs reestablish DNA methylation at TEs in male germ cells In murine male germ cells, TEs are silenced by DNA methylation. During embryogenesis, TEs in the male germline are demethylated and subsequent failure to reestablish DNA methylation causes transposition and male sterility (Bourc'his and Bestor 2004; Kato et al. 2007). TE silencing and de novo DNA methylation require two PIWI proteins MILI and MIWI2 (Aravin et al. 2008; Aravin et al. 2007; Kuramochi-Miyagawa et al. 2008), suggesting that the murine piRNA pathway silences TEs by reestablishing DNA methylation in embryonic male germ cells. C. elegans piRNAs collaborate with siRNAs to silence TEs In C. elegans, piRNAs (also called 21U-RNAs, based on their 21 nt length and 5' uracil) play an important role in silencing and preventing transposition of the DNA transposon Tc3 (Fig. 2b)(Das et al. 2008). Primary piRNAs regulate transposition through the endogenous

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Fig. 2 piRNA pathways. a In Drosophila, the piRNA pathway proteins silence TEs. piRNA loci in the Drosophila genome contain anti-sense TE fragments and are transcribed as a single transcript. Zuc is thought to cleave the 5' end of these transcripts into primary piRNAs that are bound by PIWI and AUB. Primary piRNAs target PIWI proteins to TE transcripts where they recruit the heterochromatin proteins Su(var)3-9 and HP1, excluding Pol II from TEs. In addition, primary piRNAs initiate the ping-pong cycle by cleaving TE transcripts, resulting in the formation of secondary piRNAs that are antisense to the TE. These secondary

piRNAs are loaded into AGO3 and target the initial piRNA locus transcript for cleavage, resulting in the production of more piRNAs. b In C. elegans, piRNAs are transcribed from individual loci and are bound by the PIWI Argonaute, PRG-1. PRG-1 localizes to transcripts and may recruit RdRP to convert dsRNA into 22-G RNAs that are loaded onto the Argonaute WAGO-9. Heritable silencing of piRNA loci is dependent on the putative H3K9 HMTs SET-25 and SET-32 as well as the heterochromatin protein, HPL-2

siRNA pathway (also called 22-G; 22 nt length and 5' guanosine), and do not appear to participate in pingpong amplification (Bagijn et al. 2012; Batista et al. 2008; Das et al. 2008; Lee et al. 2012). PRG-1 (the PIWI-specific Argonaute) is thought to recruit RdRP and initiate production of 22G-RNAs from piRNA target loci. While piRNAs are responsible for establishing silencing, they are dispensable for maintenance, which instead depends on the siRNA pathway (Lee et al. 2012; Shirayama et al. 2012). Silencing of a transgene that is targeted by the piRNA pathway requires the putative H3K9 HMTs SET-25 and SET-32 as well as a C. elegans HP1 homologue (Ashe et al. 2012). Unlike

piRNAs in Drosophila, C. elegans 21U-RNAs have their own promoter and are expressed as individual units (Billi et al. 2013; Cecere et al. 2012). However, TE integration near piRNA promoters can still give rise to transposon piRNAs (Bagijn et al. 2012).

Non-coding RNA regulation of transposon integration in S. cerevisiae Although S. cerevisiae has lost its capacity for RNAi (Drinnenberg et al. 2009), they maintain the ability to regulate their TEs. S. cerevisiae exhibit copy number

Small RNAs and transposon control

control over their Ty1 retrotransposons, in which increased Ty1 copy number leads to TE silencing (Jiang 2002). An antisense RNA contained within the 5' LTR can enact copy number control in trans (Berretta et al. 2008; Matsuda and Garfinkel 2009). As in fission yeast, TE silencing is associated with histone deacetylation (Berretta et al. 2008).

TEs regulate the host stress response TE expression is especially sensitive to environmental factors. It has been hypothesized that in stressful conditions genome rearrangements, like those induced by TE activation, may benefit the organism (McClintock 1984). Indeed, transcriptional activation during cellular stress is a hallmark of TEs. The S. pombe retrotransposon Tf2 is activated in response to various stresses, including exposure to heat and oxidative stress (Chen et al. 2003). In plants, TEs are regulated in response to temperature and bacterial infection (Dowen et al. 2012; Tittel-Elmer et al. 2010; Zeller et al. 2009). In mammalian genomes, SINE expression increases in response to stress (Li et al. 1999; Liu et al. 1995). Activation of TEs during stressful conditions requires that TEs overcome the mechanisms that maintain their silence. This could be achieved by a temporary attenuation of host defenses and/or by enhancing TE transcription to exceed host control, perhaps by recruiting stressrelated transcription factors to the TE. The distinction between these two possibilities is subtle, and in most cases we do not know their relative contribution to TE activation in stress. However, there are examples of gene activation by both mechanisms. In Arabidopsis, an increase in temperature reduces the abundance of SGS3, a protein involved in siRNA formation, and releases siRNA-mediated silencing of a transgene (Zhong et al. 2013); it is possible that this mechanism also results in TE activation. In low oxygen, the S. pombe transposon Tf2 is bound by the transcription factor Sre1, which induces both transcriptional activation and transposition (Sehgal et al. 2007). Recent work has demonstrated this TE regulation in response to stress can impart stress-specific regulation on neighboring host genes. Furthermore, evidence suggests that transposon-derived ncRNAs can regulate the host stress response at multiple loci throughout the genome. The studies that follow will highlight instances where TEs regulate the host stress response both in cis and trans.

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TEs act in cis to control host gene expression TEs can regulate host gene expression by acting as enhancers that control the expression of neighboring genes. In S. pombe, TE integration preferentially occurs within the promoters of genes stress-responsive genes (Chatterjee et al. 2009; Guo and Levin 2010; Singleton and Levin 2002). At many of these insertion sites, TEs function as enhancers, increasing the extent of induction in response to stress (Feng et al. 2013). In rice, the DNA transposon mPing also acts as an enhancer that can increase gene expression of neighboring genes in response to salt and cold stress (Naito et al. 2009). As another mechanism of host gene control in cis, in many organisms the heterochromatin formed at TEs can spread and silence host genes (Eichten et al. 2012; Rebollo et al. 2011; Sienski et al. 2012). Gene expression within heterochromatin is often activated by stress (Allshire et al. 1994; Gowen and Gay 1933; Tittel-Elmer et al. 2010), but whether this mechanism contributes to the host stress response is unknown. Mammalian Alu ncRNAs act in trans to alter host gene expression in response to heat stress In response to heat shock, human cells drastically reshape their transcriptome, repressing many genes and activating heat shock response genes. The human SINE, Alu, is activated upon heat shock and facilitates this stress-dependent repression of genes throughout the genome (Mariner et al. 2008). Alu ncRNAs bind RNA Pol II and inhibit its ability to synthesize RNA, preventing transcription of the genes at which it binds (Mariner et al. 2008). Complexes containing RNA Pol II and Alu ncRNAs localize to genes repressed during heat shock, suggesting that Alu RNAs facilitate gene repression by acting directly at loci throughout the genome. The mouse SINE B2 also regulates gene expression during heat shock through a mechanism similar to that of Alu (Allen et al. 2004; Espinoza et al. 2007). This suggests that mammalian SINEs have conserved roles in regulating host gene expression in times of stress. TE siRNAs confer desiccation tolerance in Craterostigma plantagineum Stress tolerance is especially important in plants that are sessile and must adapt to changing environmental conditions. The resurrection plant Craterostigma plantagineum

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is an extreme example of this flexibility, being able to survive a near complete loss of water (Bartels and Salamini 2001; Hilbricht et al. 2008). In C. plantagineum, a stress-responsive TE and its cognate siRNAs confer stress-responsiveness to host genes and desiccation tolerance to the organism. While the vegetative tissue of C. plantagineum is desiccation-tolerant under normal conditions, the undifferentiated callus is not (Furini et al. 1997). However, the callus does become desiccation tolerant when the gene CDT-1 is activated. CDT-1 is thought to be a retrotransposon as it is found in many copies throughout the genome, lacks introns, and is flanked by direct repeats. CDT-1 expression is activated by desiccation, and small RNAs homologous to CDT-1 are only detected in desiccation-tolerant plants (Hilbricht et al. 2008). CDT-1 homologous siRNAs act in trans to control the expression of desiccation genes, activating the host desiccation response (Hilbricht et al. 2008). A TE-derived siRNA regulates the Arabidopsis stress response Regulation of the stress response by TE-derived ncRNAs does not always benefit the host. In Arabidopsis, activated Athila retrotransposons produce a small RNA that regulates a stress-response gene in trans. This regulation is unusual among the studies cited here in that it confers a stress-sensitive phenotype to Athila-activated plants. When Athila transposons are activated the level of homologous small RNAs increases (Slotkin et al. 2009). One of these siRNAs can repress the stress response gene, UBP1b in trans. Since UBP1b is activated in stress conditions and ubp1b mutants are impaired in ionic and osmotic stress (McCue et al. 2012), repression of UBP1b by Athila negatively impacts the host. UBP1b shares homology with an RNA-binding protein that aggregates in stress granules. The authors of the study suggest that repression of UBP1b by Athila may be a selfish response that blocks sequestration of Athila RNAs into stress granules.

Conclusions We now understand that ncRNAs play a crucial role in TE regulation across taxa. Plants, animals, and some fungi use small RNAs to recognize and silence TE expression, whereas TE silencing in S. cerevisiae requires non-coding antisense RNAs. In fission yeast

B.S. Wheeler

and plants, siRNAs are generated from the processing of TE transcripts resulting in two layers of silencing: posttranscriptional silencing and transcriptional silencing via the recruitment of epigenetic modifications. Interestingly, in both organisms, the production of siRNAs or the establishment of siRNA-mediated silencing is preceded by a burst of transcription (during S phase, in germline companion cells, or in times of stress). Major questions remain about how TEs are targeted for regulation by the siRNA pathways in both organisms. In animals, the piRNA pathway is important for TE control and is highly adaptable, allowing organisms to silence novel transposons. Host genomes have evolved mechanisms to control TEs but, in turn, TEs are able to alter the expression of host genes. TEs contribute to host gene expression by providing stress-responsive regulatory elements that can act in cis and trans to regulate the host stress response. Since TEs often interact with the host stress response through epigenetic mechanisms, it is possible that stressinduced gene regulation may be heritable even after the stressful condition has abated. Both the heritability and the ubiquity of stress-response regulation by TEs remain to be addressed. Acknowledgments BSW is supported by the NIH Ruth L. Kirschstein National Research Service Award F32 GM100647-02 (NIGMS). Thanks to Teresa Lee for critical reading of the review.

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Since this review was written, it has been shown that fission yeast heterochromatin formation at TEs in the absence of the exosome requires recruitment of RNA splicing factors. This work raises the intriguing possibility that heterochromatin formation at TEs in low glucose conditions could result from differential regulation of splicing in response to a changing environment (Lee et al. 2013)