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Transposable elements in the mammalian embryo: pioneers surviving through stealth and service Gerdes, Patricia; Richardson, Sandra R; Mager, Dixie L; Faulkner, Geoffrey J May 9, 2016

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REVIEWTransposable elements inembryo: pioneers survivinncell embryonic stages [1, 2]. Initially, the zygotic genomeis transcriptionally inactive, with maternally inheritedman and mouse genomes, though, importantly, humanpy-and-paste”intermediateas a nascentare essentialhanisms usedGerdes et al. Genome Biology  (2016) 17:100 DOI 10.1186/s13059-016-0965-5Woolloongabba QLD 4102, AustraliaFull list of author information is available at the end of the articletranscription initiation (Fig. 1a). Crucially, most new L1Cancer Agency, University of British Columbia, Vancouver, BC V5Z 1L3,Canada1Mater Research Institute, University of Queensland, TRI Building,by LTR and non-LTR retrotransposons (Fig. 1b, c). L1mRNA transcription relies on an internal 5′ promoter,whereas ERV proviruses utilize a 5′ LTR promoter forment. Embryonic genome activation occurs at aroundthe eight-cell stage in humans and the two-cell stage inmice [3] and is accompanied in each species by* Correspondence: dmager@bccrc.ca; faulknergj@gmail.com2Department of Medical Genetics, Terry Fox Laboratory, British ColumbiaAll retrotransposons mobilize via a “comechanism involving a transcribed RNAthat is reverse transcribed and integratedcDNA into genomic DNA. However, theredifferences in the retrotransposition mecfactors regulating embryonic metabolism and develop-ERVs (HERVs) are all likely now retrotransposition in-competent (Box 3).other potentially deleterious processes can causesporadic disease by disrupting genome integrity orinducing abrupt gene expression changes. Here, wediscuss recent evidence suggesting that TEs maycontribute regulatory innovation to mammalianembryonic and pluripotent states as a means to wardoff complete repression by their host genome.BackgroundMammalian embryonic development is governed by acomplex set of genetic and epigenetic instructions. Thisgenomic blueprint undergoes evolutionary selection and,as such, the fundamental order of development is wellconserved among mammals. At fertilization, sperm andegg unite to form the zygote, which undergoes succes-sive cleavage divisions, yielding two-, four-, and eight-mutagenesis, nonhomologous recombination, and all cell lineages and are able to self-renew. Hence, early de-velopment involves rapid cellular diversification driven byand servicePatricia Gerdes1, Sandra R. Richardson1, Dixie L. Mager2* aAbstractTransposable elements (TEs) are notable drivers ofgenetic innovation. Over evolutionary time, TEinsertions can supply new promoter, enhancer, andinsulator elements to protein-coding genes andestablish novel, species-specific gene regulatorynetworks. Conversely, ongoing TE-driven insertional© 2016 Gerdes et al. Open Access This articleInternational License (http://creativecommonsreproduction in any medium, provided you gthe Creative Commons license, and indicate if(http://creativecommons.org/publicdomain/zeOpen Accessthe mammaliang through stealthd Geoffrey J. Faulkner1,3*epigenome-wide remodeling [4]. The zygote and itsdaughter cells are totipotent; that is, they have the poten-tial to differentiate into all embryonic and extraembryoniccell types. During development, the differentiation poten-tial of embryonic cells becomes progressively more re-stricted. At the blastocyst stage, the cells of the inner cellmass (ICM) are pluripotent, meaning that while they can-not give rise to extraembryonic tissues, they can generatemyriad, and largely still undefined, transcriptional and epi-genetic programs (Box 1).Pluripotent states arising embryonically in vivo, orachieved in vitro by cellular reprogramming, are associ-ated with epigenetic derepression and transcriptional ac-tivation of transposable elements (TEs) [4–6]. Thesemobile genetic elements are found in every eukaryoticgenome sequenced to date and account for at least halfof mammalian DNA [7–9]. In most mammals, retrotran-sposons are the predominant TEs. These can be dividedinto long terminal repeat (LTR) retrotransposons, in-cluding endogenous retroviruses (ERVs), and non-LTRretrotransposons such as long interspersed elements(LINEs) and short interspersed elements (SINEs) (Fig. 1a)[10–12]. LINE-1 (L1; Box 2), and ERV families are theonly autonomous retrotransposons identified in the hu-is distributed under the terms of the Creative Commons Attribution 4.0.org/licenses/by/4.0/), which permits unrestricted use, distribution, andive appropriate credit to the original author(s) and the source, provide a link tochanges were made. The Creative Commons Public Domain Dedication waiverro/1.0/) applies to the data made available in this article, unless otherwise stated.Gerdes et al. Genome Biology  (2016) 17:100 Page 2 of 17insertions are 5′ truncated and therefore lack the coreL1 regulatory sequence. Of 500,000 human L1 copies,only about 7000 retain the canonical 5′ promoter [7, 13].By contrast, about 90 % of HERVs exist in the genome asBox 1. Regulatory networks controlling pluripotencyProgrammed shifts in transcriptional and epigenetic statesduring embryogenesis have been studied primarily usingin vitro systems. Embryonic stem cells (ESCs) are pluripotentcells derived from the blastocyst inner cell mass. Cultured ESCsare intensively used to study pluripotency, particularly in humans.Over the past decade, a core regulatory circuit incorporating thetranscription factors Oct4 (also known as Pou5f1), Sox2, andNanog [126–128] has been revealed to regulate ESC pluripotency[129]. This circuit activates pluripotency-associated factors andrepresses lineage-specific genes [130]. Pluripotent cells can also bederived in vitro via somatic cell reprogramming. Inducedpluripotent stem cells (iPSCs) were initially produced by forcedexpression of Oct4, Sox2, Klf4, and c-Myc using retroviralvectors [131, 132]. Numerous methods have since been developedto improve reprogramming efficiency and iPSC safety [133]. As forESCs, iPSCs provide a powerful system to understand thepluripotent state and can differentiate to all cell types of thebody [131, 132]solitary LTRs due to recombination of proviral 5′ and 3′LTRs [11, 14]. Many of these LTRs maintain, or restorethrough acquired mutations, their natural transcriptionaland regulatory signatures, which can perturb the expres-sion of nearby genes [15]. While the regulatory capacity ofolder LTRs will tend to diminish over time, the approxi-mately 440,000 identifiable LTRs in the human genome[7] still carry enormous potential to regulate genes andgene networks [14–17]. Therefore, compared with L1,ERVs are arguably a much greater source of regulatoryinnovation (Fig. 2).Recent studies have revealed a complex and somewhatparadoxical interplay between retrotransposons and theirhost genome in pluripotent cells. On one hand, retro-transposons have long been regarded as fundamentallyselfish genetic elements [18] that, to ensure their sur-vival, must evade host genome surveillance and mobilizein cells that provide opportunities for germline transmis-sion. Transcriptional reactivation of retrotransposons inthe early mammalian embryo aligns with this evolutionaryimperative, despite retrotransposition posing a threat togenome integrity. Indeed, cells employ numerous mecha-nisms to restrict retrotransposition at this stage [19–23].On the other hand, transcription from ERV promotersdrives the expression of cellular genes as well as ERV-derived sequences and appears to be a fundamentalcharacteristic of the pluripotent state [16, 24–31]. LTRsmay be permitted to thrive in this environment due to thematerials they provide to the host genome for regulatorynetwork innovation (Fig. 3). Indeed, as well as providingalternative promoters to pluripotency genes [28], ERVscan serve as long-range enhancers [26], produce regula-tory noncoding RNAs [27, 30], and may, in some cases,express their own viral proteins [29, 31]. Hence, tran-scribed products arising from ERVs may promote, or evenbe required for, the pluripotent state [24–33]. Finally, re-ports of L1 retrotransposition in somatic cells have fueledspeculation that TE-derived mosaicism may lead to func-tional innovation during development [34–37].Here, we review the restraint and activity of TEs inembryonic cells and later in development, as well as theunexpected promotion of pluripotent states by ERVs.We further appraise the convergent contributions to em-bryogenesis made by ERVs in distinct mammalian cladesas evidence of an evolved strategy to avoid, or at leastdelay, host genome repression.ERV-driven transcription in the early embryoERV regulation of protein-coding genesAlthough there are spectacular examples of TE proteinsunderpinning functional innovation, such as in the pla-centa [38], regulatory sequences exapted from TEs argu-ably loom larger in our evolutionary history [15]. Indeed,up to 30 % of human and mouse transcription start sites(TSSs) are situated in TEs and display tissue-specificexpression patterns [33, 39]. Embryonic human tissuesexpress the greatest diversity of TE-associated TSSs ob-served to date [33], highlighting the potential of TEs todrive cell type and developmental stage-specific expres-sion, particularly during early embryogenesis when thegenome becomes demethylated [40]. In mouse, theLTR promoters of MuERV-L elements regulate a net-work of genes critical for totipotency and specific tothe two-cell stage of embryonic development [41]. TE-derived regulatory sequences likewise contribute to theevolution of regulatory networks in pluripotent stemcells. For example, only about 5 % of Oct4 and Nanogtranscription factor (TF) binding sites are shared inmouse and human embryonic stem cells (hESCs). TEscontribute a significant proportion (about 25 %) of theremaining, species-specific, binding sites [42]. More-over, in vitro knockdown of specific ERVs via RNAinterference can lead to a reduction in pluripotencymarkers [24, 26–28, 43–46]. Thus, TE sequences arebroadly and strongly transcribed in the early embryoand can influence pluripotency by being exapted into,or at least adding robustness to, pluripotency net-works. These findings underscore the universality andversatility of TEs in driving the evolution of regulatorynetworks.Fig. 1 Long terminal repeat (LTR) and non-LTR retrotransposition mechanisms. a Mammalian retrotransposon structures. A long interspersed element(LINE; human L1 shown) typically consists of a 5′ untranslated region (UTR; blue box) harboring an internal promoter, two open reading frames(ORF1, ORF2), a 3′ UTR (small blue box), and a poly(A)-tail. A short interspersed element (SINE; mouse B1 shown) does not encode proteinsand is trans-mobilized by LINE proteins. An endogenous retrovirus (ERV), such as mouse intracisternal A-type particle (IAP) and Mus type-Drelated retrovirus (MusD), lacks an Env protein but encodes functional Gag and Pol proteins flanked by a LTR at the 5′ (black box) and 3′ (red box)ends. Arrows indicate transcription start sites. b ERV mobilization starts with mRNA transcription and translation to yield Gag and Gag–Pro–Polfusion proteins. The fusion proteins consist of a Gag protein (Gag), a protease (Pr), an integrase (In), and a reverse transcriptase (RT). Gag proteinsbuild a virus-like particle and encapsulate the fusion proteins, which are processed into separate mature proteins. The ERV mRNA is then reversetranscribed, generating a cDNA. This cDNA and the integrase build a preintegration complex. The integrase then creates a double-strand DNAbreak, followed by genomic integration of a new ERV copy. Target site duplications (TSDs) are indicated by blue triangles. c L1 mobilization beginswith transcription of an L1 mRNA, which is translated to yield ORF1p and ORF2p. ORF1p, ORF2p, and the L1 mRNA form a ribonucleoproteinparticle that re-enters the nucleus. The ORF2p endonuclease cleaves the first genomic DNA strand, while its reverse transcriptase uses a now free3′ OH group as a primer for reverse transcription of the L1 mRNA. Following second-strand DNA cleavage, a new L1 copy is integrated into thegenome and is typically flanked by TSDsGerdes et al. Genome Biology  (2016) 17:100 Page 3 of 17Box 2. L1 retrotransposonsThe non-long terminal repeat retrotransposon long interspersed element-1 (L1) is the only autonomous, mobile human transposableelement [10, 12, 116, 134]. L1 occupies approximately 17 % of the human genome [7]. L1 also mobilizes Alu and SINE–VNTR–Alu (SVA)elements in trans [135, 136]. Mice, by contrast, have three L1 subfamilies (TF, GF, and A) that are autonomous, as well as nonautonomousshort interspersed elements (SINEs) retrotransposed by L1 [10]. L1 accounts for 19 % of the mouse genome [8]. A full-length human L1 isapproximately 6 kb long and initiates mRNA transcription from a 5′ sense promoter active in gametes, stem cells, and various somatictissues [33, 36, 48, 71, 137–139]. The bicistronic L1 mRNA encodes two proteins, ORF1p and ORF2p, which are flanked by 5′ and 3′untranslated regions (Fig. 1a). An L1 antisense peptide (ORF0p) [56] can also be expressed by an adjacent L1 antisense promoter [115].This antisense promoter is expressed in many spatiotemporal contexts, including in stem cells, and can provide alternativepromoters to protein-coding genes [33, 56, 115, 140]. L1 ORF2p presents endonuclease [141] and reverse transcriptase [142] activitiesand, during retrotransposition, L1 ORF1p, ORF2p, and the canonical L1 mRNA associate in cis to form a cytoplasmic ribonucleoproteinparticle (RNP) [143]. The RNP can then enter the nucleus, where the ORF2p endonuclease cleaves genomic DNA, and the ORF2p reversetranscriptase synthesizes a new L1 copy at the cleavage site using the L1 mRNA as a template. This process is called target-site primedreverse transcription (TPRT) [144] (Fig. 1c).The L1 5′ promoter is the major focus of host genome efforts to prevent L1 mobility, through DNA methylation and transcription factorrepression and other pathways [145, 146]. Thus, it appears that L1 in the main persists as a mobile element by avoiding detection of its5′ promoter by host genome surveillance pathways and, where this fails, by harnessing new promoter structures [13]. This couldexplain the exceptional L1 5′ promoter diversity observed even among closely related primates [23]. It should also be noted thatthe vast majority of L1 copies in the genome are 5′ truncated and lack the 5′ promoter [13], meaning that the host factors guardingagainst full-length L1 transcription are not necessarily able to recognize truncated L1s.Box 3. Endogenous retrovirusesEndogenous retroviruses (ERVs) are derived from exogenous retroviruses that, at some point, infected an individual organism’s germcells, integrated into their genome, and were subsequently inherited by their offspring. ERVs are divided into class I, class II, and class IIIelements, based on the exogenous virus class that they are most similar to [11]. Full-length ERVs are 5–10 kb in length, encode proteinsimportant for mobilization, and are flanked by two identical long terminal repeats (LTRs; 300–1000 bp) that regulate ERV transcription.Loss of the env gene, found in exogenous retroviruses, is a common feature of ERVs as they adopt an intracellular life cycle as aretrotransposon [11, 147, 148]. ERV retrotransposition is initiated by the transcription of the 5′ LTR and terminates in the 3′ LTR,generating a terminally redundant mRNA that is translated into Gag and Gag–Pro–Pol fusion proteins. Gag proteins encapsulatethe mRNA and fusion protein. Pro has protease activity whereas Pol possesses reverse transcriptase, ribonuclease, and integrasedomains that generate independent proteins by proteolytic maturation. Together they produce a double-stranded cDNA copy of theERV and flanking LTRs. This cDNA is then integrated into the genome by the ERV integrase [149] (Fig. 1b).Human endogenous retroviruses (HERVs) comprise about 8 % of the human genome [7]. All HERVs are considered to be nowretrotransposition incompetent [150, 151]. The HERV-K (HML-2) family is exceptional, with several members arising after the divergence ofhumans and chimpanzees (approximately 6 million years ago) and a handful of polymorphic HERV-K insertions found in human populations[152–155]. Although a mobile HERV-K element has yet to be identified in humans, it is possible that rare, as yet undiscovered polymorphicelements could retain retrotransposition competence [152]. In contrast to humans, ERVs account for approximately 10 % of the mousegenome [8]. Several mouse ERV families are still autonomously active, including intracisternal A-type particle elements [106], Moloneymurine leukemia virus [156], and Mus type-D related retrovirus (MusD) [147] elements, as well as the MusD-dependent early retrotransposonfamily [157]. Together, new mouse ERV insertions are responsible for about 10 % of documented germline mutations in inbred strains [106].Clade-specific ERVs also occur in other mammals, although genomic ERV content varies significantly between species [11]. Numerousinstances of mammalian ERVs contributing regulatory sequences to genes, including examples of convergent evolution [158], are found inpluripotent cells and elsewhere [15, 159, 160].Gerdes et al. Genome Biology  (2016) 17:100 Page 4 of 17Gerdes et al. Genome Biology  (2016) 17:100 Page 5 of 17Independent ERV expression as a hallmark of thepluripotent stateERV transcription independent of protein-coding geneshas also been linked to pluripotency. Despite an appar-L1~90% of new L1 copies are5′ truncated and lack the promoterSolitary LTRs and proviral LTRsdeliver promoter/enhancer functionERVsRetrotranspositionTimeFig. 2 Long interspersed element 1 (L1) and endogenous retrovirus(ERV) regulatory impact post-integration. Most L1 copies are 5′truncated (left) and lack the sense and antisense L1 promoterslocated in the 5′ untranslated region (large blue box). As a result,these L1 insertions have less capacity to drive chimeric transcriptionwith neighboring genes. ERV insertions (right) remain either full-length,with flanking 5′ (black box) and 3′ long terminal repeats (LTRs; red box)that potentially retain promoter function, or, more commonly,recombine between the LTRs to form a solitary LTR, which retainsthe promoter/enhancer region. Arrows indicate putative transcriptionstart sitesent lack of retrotransposition activity, specific HERVsare actively transcribed in hESCs and are thought to in-fluence pluripotency maintenance [24, 25, 27–32, 47].The HERV families HERV-H and HERV-K (HML-2) inparticular appear to be connected to early human em-bryonic development [25, 31]. While stochastic tran-scriptional derepression of various HERVs [47] as well asnon-LTR retrotransposons [48] in pluripotent cells canprobably be attributed to a general relaxation of TE si-lencing [40], specific classes of elements are consistentlyreactivated across hESC lines, indicating that their ex-pression can serve as a marker for an undifferentiatedstate [28, 29], further raising the possibility that these el-ements have a functional link to pluripotency. DistinctHERV families also denote specific embryonic stages,suggesting HERV expression profiles may signify cellidentity [25]. It is important to note, however, that, inmany cases, only a small fraction of HERVs from a spe-cific family are transcribed [25] and that their genomiccontext likely plays a pivotal role in their expression.The reasons for HERV families presenting distinct ex-pression patterns during early embryogenesis are cur-rently unclear. To speculate, such patterns could be areflection of the optimal “ecological niche” of their an-cestral exogenous counterparts and may mimic the par-allel expression patterns of LTR-binding TFs.Human oocytes and zygotes (to the cell–cell stage)contain the highest percentages of HERV transcripts ob-served during development; these are almost certainlydeposited maternally prior to embryonic genome activa-tion [25]. Abundant transcription emanating from MaLRand ERVK LTRs has also been documented for mouseoocytes [5, 49]. The provision of ERV transcripts by thematernal genome supports ERV functionality in the earlyembryo, as these RNAs already seem to be necessary be-fore the embryonic genome is able to generate its owntranscripts [31]. However, it is also possible that ERVtranscripts do not have a specific function at this earlystage but their maternal deposition is permitted becausethey do not harm the developing embryo. Nevertheless,stage-specific expression from ERV promoters, and ofprotein-coding genes, LTR-driven chimeric transcripts,and ERV transcripts proper, is a defining feature of earlymammalian development.Regulation of HERV-K and HERV-H bypluripotency factorsAs well as gene regulation transacted by ERVs, manystudies have revealed how ERVs are in turn regulated bypluripotency genes. For instance, the core pluripotencyTFs Oct4 and Nanog (Box 1) bind specific HERV fam-ilies (Fig. 3) [26, 42]. HERV-K is the most recently activeHERV family and many HERV-K copies retain theirprotein-coding potential [50]. Notably, transcriptionfrom the youngest subclass of HERV-K is induced fromits LTR, known as LTR5HS (for “human-specific”), atthe eight-cell stage, during embryonic genome activa-tion, and continues through to the blastocyst stage(Fig. 4a). LTR5HS contains an Oct4-binding motif that isnot present in older LTRs such as LTR5a or LTR5b [31].DNA hypomethylation and transactivation by Oct4 atLTR5HS synergistically stimulate HERV-K expressionand lead to the presence of retroviral and viral-like parti-cles in human preimplantation embryos [31]. HERV-Ktype 2 proviruses encode the protein Rec, which derivesfrom alternative splicing of the env gene and is respon-sible for nuclear export and translation of viral RNAs[51]. Rec can be found in pluripotent cells and may in-fluence expression of the interferon-induced viral re-striction factor IFITM1 in epiblast cells [31, 52].Consequently, Grow et al. [31] suggested that antiviralresponses might be induced by HERV-K proteins, pro-tecting the human embryo against new retroviral infec-tions. Similarly, HERV-K type 1 proviruses encode theprotein Np9, which is the product of a new alternativesplicing event and coincides with a deletion in the envregion [53, 54]. Interestingly, Rec and Np9 are notencoded in rodent ERVs, making them a distinguishingfeature of primate ERVs and, moreover, hESCs specific-ally express Rec, Np9, and Gag [29]. It is tempting,tercessRNGerdes et al. Genome Biology  (2016) 17:100 Page 6 of 17LTRLTRAlternative promoHost geneLong-range enhanLTRERV protein expreAAAGagRecTFsTSSTranscriptERV proteinsLTRLong non-coding RegulatoryRPtherefore, to speculate, as per Grow et al. [31], thathESCs allow expression of these HERV-K proteins tofulfill a protective function via, for example, Rec-inducedinhibition of viral infection. It is also possible that someHERV-K elements fortuitously escape silencing andmanufacture viral proteins as innocuous byproducts ofHERV-K transcription in hESCs (Fig. 3).HERV-H is another primate-specific retrotransposon[55] with a potentially important role in the maintenanceof hESC identity and pluripotency (Table 1). HERV-Htranscripts are expressed in pluripotent cells at levelsmuch higher than those seen in differentiated cells and,as a result, HERV-H expression is a proposed marker forpluripotency [28]. Interestingly, HERV-H is expressed insome induced pluripotent stem cell (iPSC) lines (Box 1)at higher levels than for other iPSC lines and embryonicstem cells (ESCs) [47]. Developmental HERV-H expres-sion also appears to be cell type and stage specificin vivo (Fig. 4a). For instance, HERV-H and its flankingLTR element LTR7 can only be detected in epiblast cells[25], whereas other related LTR variants that flankproteinLTRGeGene cRewiring of geneFig. 3 Examples of endogenous retrovirus (ERV) contributions to pluripotencytranscription factors (TFs) and can serve as a transcription start site (TSS). LTRsidentity by: (1) serving as alternative promoters for pluripotency genes, (2) procell-specific long noncoding RNAs that can bind to proteins regulating the plERV protein expression, and (5) rewiring gene regulatory networks by controlPluripotency geneAAALTRrGagRecProvirusAAAionAAAAAAARPPluripotencyDifferentiationHERV-H (LTR7B and LTR7Y) are detectable at theeight-cell stage and morula [25]. LTR7 incorporatesOct4, Nanog, Klf4, and Lbp9 TF binding sites, which to-gether appear to mediate HERV-H transcriptional activa-tion [28]. Once activated, individual LTR7 copies cangenerate noncoding RNAs [43] and form chimeric tran-scripts with protein-coding genes, in some cases supply-ing multiple promoters to the same gene (Fig. 3) [27, 28,56]. LTR7 may also be bound by factors central to so-called naïve, or ground state, pluripotency where cellsare predisposed to self-renew and lack differentiationmarkers, showing that ERVs may be involved in fine tun-ing stem cell phenotype [28, 57]. In sum, HERV-K andHERV-H are clearly activated by pluripotency TFs andtheir expressed products are, at the very least, markersof pluripotency.HERV-derived long noncoding RNAs regulatepluripotency networksLong noncoding RNAs (lncRNAs) are RNA transcriptsgreater than 200 nucleotides long that possess no, orGene ane b regulatory networks. A long terminal repeat (LTR) possesses binding sites for pluripotencybound by pluripotency TFs can thereby impact embryonic stem cellviding long-range enhancers to specific host genes, (3) generating stem-uripotent state, (4) transcribing proviral DNA elements as precursors toling several pluripotency genesGerdes et al. Genome Biology  (2016) 17:100 Page 7 of 17HighLowOocyte Zygote 2-cell(a)HERV-HHERV-KExpressionvery little, protein-coding potential [58–60]. MostlncRNAs are transcribed antisense to protein-codinggenes or are intergenic [58, 59]. More than two-thirds oflncRNAs incorporate TE sequences (Fig. 3) and, in casessuch as Xist, a prototypical lncRNA involved in Xchromosome inactivation, TEs are a core component oflncRNA biogenesis [60, 61]. Other than Xist, and a fewadditional examples, lncRNAs have proven difficult toevaluate functionally because, as well as containing TEs,lncRNAs are often expressed at very low levels [30].However, one of the best established lncRNA functionsis to regulate pluripotency, particularly by mediatingchanges to chromatin [62, 63]. Interestingly, Au et al.[64] reported more than 2000 additional long intergenicnoncoding RNA (lincRNA) isoforms, of which 146 wereexpressed in hESCs. These human pluripotency-associated transcripts (HPATs) typically incorporatedERVs, especially HERV-H [30], and in that regardHighLowSomatic cell InHERV-HHERV-K(b)ExpressionFig. 4 Human endogenous retrovirus (HERV) expression patterns in pluripoduring embryonic genome activation at the eight-cell stage and remains uHERV-K [31]. HERV-H can only be detected in epiblast cells of the late blastreprogramming, HERV-K and HERV-H are derepressed with distinct dynamicreprogrammed. HERV-K expression subsequently decreases in reprogrammduring reprogramming compared with HERV-K [24]. Note: the time points4-cell 8-cell Morula Blastocystwere similar to many other hESC-specific lncRNAs[27, 43, 44, 47]. HPATs appear to contribute to for-mation of the blastocyst ICM, suggesting an essentialrole for HERV-derived lncRNAs in human embryo-genesis [30].One particularly interesting lincRNA, HPAT5, is hy-pothesized to be involved in post-transcriptional generegulation: HPAT5 binds AGO2, a core protein catalyz-ing microRNA (miRNA) processing [65], and the let-7miRNA family, which modulates hESC pluripotency[66]. Durruthy-Durruthy et al. [30] have suggested thatHPAT5 controls the balance between pluripotency anddifferentiation by negatively regulating let-7 expression.However, HPAT5 is promoted by the so-called HUERS-P1 ERV, a low copy number TE that has not been in-vestigated very deeply in this context. Interestingly, theHPAT5 promoter is located in the internal Gag se-quence of the HUERS-P1 ERV, rather than in an LTR.termediate iPSC colonytent cells. a HERV-K transcription in human embryogenesis is initiatedntil the blastocyst stage. Dashed lines indicate proposed expression ofocyst [25]. b After induction of induced pluripotent stem cell (iPSC)s. HERV-K transcription reaches its peak shortly before cells are fullyed cells and is silenced in iPSCs [32]. HERV-H is highly expressed earliershown are approximate due to technical differences between studieslsificSCssesESCcyipoGerdes et al. Genome Biology  (2016) 17:100 Page 8 of 17Therefore, this promoter likely developed by geneticdrift or selection, rather than by harnessing the “readyto use” regulatory motifs found within an LTR. Inaddition, the let-7 binding site within HPAT5 occurswithin an imbedded Alu element. HPAT5 is thus an un-usual, and yet fascinating, example of retrotransposon-driven regulatory innovation.More broadly, HERV-driven transcripts contributingto pluripotency networks unique to humans or primatesare of particular interest. lincRNA-RoR, with its TSSTable 1 Summary of HERV-H findings to date in human stem celFindingsBinding of pluripotency TFs in ESCs and iPSCs within or near LTRs or specInduction of HERV-H, or LTR7-driven lncRNA/chimeric RNA expression, in EActive chromatin marks on specific LTRs in ESCs or iPSCsDNA hypomethylation at specific LTRs in ESCsLTR enhancer activity in ESCsChanges in HERV-H associated with expression changes of genes near LTRInduction of HERV-H in iPSCs, declining upon differentiationDifferentiation-defective iPSC clones retain high levels of HERV-H RNAKnockdown of general HERV-H expression inhibits iPSC formation and cauKnockdown of specific LTR-driven RNAs inhibits iPSC formation or causesLTR-driven lncRNA acts as miRNA sponge to positively regulate pluripotenHERV-H RNA associates with coactivators and LTR loci in ESCsHERV-H expression marks for naïve-like stem cellsHERV-H LTR subtypes expressed sequentially in early developmentESC embryonic stem cell, HERV human endogenous retrovirus, iPSC induced plurmicroRNA, TF transcription factorlocated in a HERV-H element, represents an excellentexample of a primate-specific TE found to modulatepluripotency [43]. Notably, lincRNA-RoR is expressedmore highly in iPSCs than in ESCs and can promoteiPSC reprogramming [44], perhaps by serving as anmiRNA sponge protecting Sox2 and Nanog frommiRNA-mediated degradation [45]. In another example,the gene ESRG, which uses a domesticated HERV-Hpromoter, plays a role unique to human pluripotency[28]. Unusually, ESRG encodes an intact open readingframe (ORF) in humans, but possibly not in other pri-mates, and is expressed exclusively in the human ICMand cultured pluripotent cells [67]. ESRG knockdowncompromises stem cell self-renewal and promotes differ-entiation, while ESRG overexpression aids reprogram-ming [28]. These case studies demonstrate recurrentincorporation of annotated HERV-derived transcriptsinto pluripotency networks.To discover new lncRNAs regulating pluripotency,Fort et al. [26] surveyed in depth the noncoding tran-scriptomes of mouse and human stem cells. The result-ing pluripotency lncRNA catalog included numerouspreviously unreported antisense, intergenic, and intronictranscripts that initiate in ERVs. Consistent with an earl-ier report [33], Fort et al. found an exceptional variety ofstem cell-specific TSSs that are not directly associatedwith protein-coding genes. These TSSs often overlapwith TEs, especially with ERVK and MaLR LTR subfam-ilies in mice and ERV1 in humans, and frequently flankenhancer elements. In addition to bidirectional tran-scription denoting enhancer activity [68, 69], TE-derivedenhancer sequences are enriched for bound Nanog,Sox2, Oct4, and the enhancer-related protein p300 [26].Reference(s)LTR-driven lncRNAs [24, 28, 42–45, 47]s, declining upon differentiation [26–28, 43–45, 47, 60, 161][28, 32, 43, 44, 47, 162][28, 161][27][27, 28, 32, 42][24, 27, 28, 32, 46][46]ESC differentiation [24, 27, 28]differentiation [24, 28, 44, 45]TFs [45][27][28][25, 31]tent stem cell, lncRNA long noncoding RNA, LTR long terminal repeat, miRNAAs such, regulation of TE-derived enhancers andlncRNAs by pluripotency TFs can result in the forma-tion of positive-feedback loops, potentially bolsteringpluripotency networks [25, 26, 62]. Thus, in agreementwith other studies, Fort et al. demonstrated that specificERVs are major contributors to the stem cell transcriptomeand found a plethora of novel stem cell-associated ERV-derived transcripts that await functional characterization,in line with expectation that some of these lncRNAs willbe involved in the establishment and maintenance of pluri-potency [70].ERV expression dynamics during somatic cellreprogrammingDomesticated TEs clearly play important functional rolesin stem cell biology. However, TE repression can shift ascells transition through pluripotent states, as encoun-tered during reprogramming. As a result, opportunisticTEs may mobilize, cause insertional mutagenesis and,potentially, compromise the integrity of reprogrammedcells [32, 48, 71]. TE activity in stem cells therefore car-ries risk as well as benefits for the host genome, alongwith major incentives for TEs, given potential for earlyembryonic retrotransposition events to be germlinetransmitted. It follows that, although reprogrammingcan broadly reactivate TEs, particularly those controlledby TFs expressed dynamically during reprogramming[16, 42], silencing is selectively re-established in theresulting pluripotent cells, potentially ameliorating riskto the host genome. For instance, although HERV-H andHERV-K are both transcriptionally active during repro-gramming, HERV-H is expressed in cultured iPSCs,whereas the more recently mobile HERV-K family is si-lenced [28] (Fig. 4b). This contrast is also found formouse iPSCs, where Mus type-D related retrovirus(MusD) expression contrasts with intracisternal A-typeparticle (IAP) silencing [32]. Importantly, more experi-ments are required to confirm the generality of these ob-servations, as technical considerations in iPSC generation(e.g., reprogramming and culture conditions) can lead todifferences in TE expression between iPSC lines [71].TE repression is dynamic during reprogramming. In ahigh-resolution analysis of mouse and human iPSC lines,Friedli et al. [32] found that most ERVs peaked in ex-pression shortly before reprogramming was completelevels seen in hESCs and, notably, ectopic LTR7 hyper-activity in iPSCs resulted in a differentiation-defectivephenotype [24]. Similarly, cumulative HPAT expres-sion rises markedly during reprogramming and is di-minished in iPSCs and, as for HPAT5, may influencereprogramming efficiency [30]. Taken together, thesedata indicate that TE hyperactivity is potentially dele-terious to the host genome due to an elevated risk ofretrotransposition but may also be a requirement ofinduced reprogramming.ERV silencing in pluripotent statesThe machineries responsible for ERV regulation in ESCsare evidence of the complex relationships that can formbetween TEs and their host genome. Broadly speaking,to reduce the probability of retrotransposon-derived mu-tagenesis, mammalian genomes target ERVs with DNAmethylation, heterochromatin-forming factors, transcrip-tional repressor complexes, proviral silencing factors,and post-transcriptional arrest or degradation of viralRNAs (Table 2) [19, 20, 72]. Prominently, histone modi-fications silence ERVs in ESCs [73–75] by making chro-rebin/Es hweK9dnigchingints hsitGerdes et al. Genome Biology  (2016) 17:100 Page 9 of 17and were then repressed in pluripotent cells. Broad TEexpression during somatic cell reprogramming may bein itself important for the induction of the pluripotentstate. Ohnuki et al. [24] reported, for example, thatLTR7 elements (associated with HERV-H) are hyperacti-vated by Oct4, Sox2, and Klf4 during reprogramming. Inthe resultant iPSCs, however, LTR7 activity decreased toTable 2 Selected factors silencing ERVs in embryonic stem cellsFactor Relevant function in ESCsZfp809 KRAB zinc finger protein, recognizes PBS Pro andYY1 Yin-Yang 1 (YY1) is a zinc finger protein, initiallyERV LTRs (U3 region) and helps assemble Trim28complexSHIN Short heterochromatin inducing sequence initiateand transcriptional repressionTrim28 (Kap1, Tif1-β) Transcriptional co-repressor, acts as a bridge betand other transcriptional repressors, mediates H3Eset (Setdb1, Kmt1e) Histone methyltransferase, trimethylates H3K9 anfor HP1 bindingSumo2 Sumoylation factor, post-translational sumoylatioenhances recruitment of Trim28 to proviral DNAHP1 Heterochromatin protein 1, binding to Trim28 mfor repression of transcriptionChaf1a Histone chaperone, deposits histone H3/H4 whiDNA for silencing, might execute different silencon different classes of ERVsAsf1a/b Histone chaperones, components of the Chaf1aAtrx ATP-dependent helicase, establishes and maintainDaxx H3.3-specific chaperone, may facilitate H3.3-deposequences, possible role in SHIN-silencingERV endogenous retrovirus, ESC embryonic stem cell, KRAB Krüppel-associated box,zinc finger proteinmatin inaccessible to polymerases and transcriptionfactors [76], although this silencing in itself carries po-tential for deleterious side effects when nearby genes arealso inadvertently repressed [77]. Moreover, some ERVsare marked by H3K9me3 and H4K20me3 for repressionin ESCs but not in differentiated cells [6], suggestingthat this pathway is used for de novo establishment ofInteracting proteins Reference(s)cruits Trim28 Trim28 [85]ds to mouseset silencingTrim28 [92]eterochromatin KRABZfps (likely) [93]en KRAB-Zfpsme3 recruitmentKRABZfps, Eset, NuRD deacetylasecomplex, HP1[74, 86]H4K20, crucial Trim28, HP1 [73]of Trim28 Trim28 [72]ht be important Trim28, Eset, Atrx [86, 89]marks proviralmechanismsEset, Kdm1a, Hdac1/2, Asf1a/b [72]eractome Chaf1a [72]eterochromatin Eset, Trim28 (both likely) [93]ion on ERV Atrx [93, 95]LTR long terminal repeat, SHIN short heterochromatin inducing sequence, Zfpdue to the high potential of SINEs to impact gene regu-Gerdes et al. Genome Biology  (2016) 17:100 Page 10 of 17heterochromatin around ERV sequences [75, 78] or, al-ternatively, is used to maintain repression already estab-lished in oocytes [79, 80].Even ERVs in accessible chromatin can be decisivelysilenced by DNA methylation. In mice, de novo DNAmethylation is regulated by the canonical Zfp/Trim28/Eset machinery [75]. Krüppel-associated box (KRAB)zinc finger proteins (Zfps) play a major role in the initi-ation of ERV silencing [81, 82]. Indeed, the number ofERVs and Zfp genes in vertebrates are correlated,suggesting coevolution [83]. As an example of the com-plexity of Zfp-mediated retrovirus silencing, Zfp809knockout induces the in vivo expression of Moloneymurine leukemia virus (MMLV)-like 30 (VL30) provirus[84]. Zfp809 also binds to MMLV and initiates silencingby recruiting Trim28 (also known as Kap1) [74, 85, 86].Trim28 activity is enhanced by post-translational sumoy-lation by Sumo2 [72, 87] and binds HP1, which isthought to contribute to the ability of Trim28 to represstranscription in the context of MMLV silencing [86, 88,89]. Another Zfp, YY1, also binds to MMLV [90, 91]and, together with Zfp809, is thought to recruit Trim28to ensure a stably DNA-bound silencing complex [92].In another example, KRAB Zfps have been shown totrigger heterochromatin formation in IAP retrotranspo-sons by binding to a short heterochromatin inducing(SHIN) sequence, dependent on Eset and Trim28 [93],enacting H3K9 and H4K20 trimethylation [73]. Chaf1afacilitates deposition of these H3 and H4 variants andalso interacts with Eset [72]. Eset-mediated ERV silen-cing is also important in mouse primordial germ cellsbefore the onset of de novo DNA methylation [80].Hence, ERV silencing is enacted by a multilayered andinterleaved system that ensures robust and specific re-pression of ERV families, subsets, and individual loci.It follows that models explaining ERV silencing aretypically complex, which, at times, can lead to differingconclusions. For instance, the SNF2-type chromatin re-modeler Atrx is another crucial component for IAP si-lencing that renders Eset-dependent heterochromatinless accessible [93] and is likely to be recruited to IAPsby Trim28 and Eset [93] (Fig. 5a). Interestingly, Atrx hasbeen reported to interact with the H3.3-specificchaperone Daxx to facilitate H3.3 deposition at telo-meric heterochromatin [94]. Yet, it is not clear if H3.3 isrequired for ERV silencing, despite detection of H3.3across ERV flanking regions and solo LTRs [95]. In gen-eral, Sadic et al. [93] and Elsässer et al. [95] reached op-posing conclusions with regards to H3.3 enrichmentaround ERV sequences (Fig. 5b). One possible explan-ation here is that Elsässer et al. used chromatin immu-noprecipitation sequencing (ChIP-seq) to detect H3.3-enriched regions across the entire mouse genome andfound a correlation between H3.3, H3K9me3, and ERVlation [12, 71, 103, 104]. Klawitter et al. estimated thatapproximately one de novo L1 insertion occurred percell in human iPSCs. Strikingly, more than half of thedetected de novo L1 insertions were full length and thuspotentially able to mobilize further. Klawitter et al. alsoobserved extraordinary induction of L1 mRNA and pro-tein expression after reprogramming. To speculate, nu-merous L1 ribonucleoprotein particles (RNPs; Box 2)could form as a result and be carried through iPSC cul-ture and differentiation. This could enable L1-mediatedinsertional mutagenesis in cells descending from thosewhere L1 expression originally occurred, as others havecoordinates. Sadic et al., on the other hand, used anengineered reporter assay to measure ERV silencingwhich, in H3.3 knockout cells, remained intact. Furtherstudy is therefore required to resolve the place of H3.3in models of ERV silencing. Overall, these and other ex-amples of TE repression in pluripotent cells, such as thesilencing of nascent L1 and MMLV insertions in embry-onic carcinoma derived cell lines [96, 97], reflect the extra-ordinary efforts made by the host genome to orchestratesilencing of currently and recently retrotransposition-competent TEs during embryonic development.Endogenous L1 mobilization in mammaliansomatic cellsThe early embryo is a viable niche for the generation ofpotentially heritable retrotransposon insertions. In par-ticular, L1 mobilization in human and rodent embryosmay drive somatic and germline mosaicism [98–101]and, indeed, deleterious human L1 insertions transmit-ted from mosaic parents to offspring have resulted insporadic genetic disease [101]. In vitro experiments havelikewise provided support for L1 mobilization occurringin pluripotent cells [99–101] and, potentially, the pres-ence of the L1 retrotransposition machinery beingrequired for preimplantation mouse embryo develop-ment [102]. Human iPSCs and ESCs allow low-levelmobilization of an engineered L1 reporter [22, 48, 99].Consistently, endogenous L1 promoter hypomethylationand transcriptional activation have been observed iniPSCs [32, 48, 71], as has induction of a primate-specificL1 antisense peptide (ORF0p) that appears to increaseL1 mobility in stem cells [56] (Box 2). Endogenous denovo L1 retrotransposition and mobilization of nonau-tonomous Alu and SINE–VNTR–Alu (SVA) elementshave also been reported by Klawitter et al. [71] in severaliPSC lines, as well as an Alu insertion in a culturedhESC line. L1 may, therefore, trans mobilize Alu andother SINEs during development, an important findingconsidered for L1 RNPs arising in gametes and carryingover into the zygote [100].Zfp809PBSGerdes et al. Genome Biology  (2016) 17:100 Page 11 of 17Trim28LTRChaf1aEsetH3K9me3Histones H3/H4Asf1aYY1HP1Asf1b(a)Although both L1 and ERV retrotransposons are activein the mouse germline [105, 106], their capacity tomobilize during embryogenesis is less clear than for hu-man L1. Quinlan et al., for instance, concluded de novoretrotransposition in mouse iPSCs did not occur, or wasvery rare [107], in contrast to results for human iPSCs[22, 48, 71]. However, an earlier study found that engi-neered L1 reporter genes mobilize efficiently in mouseembryos [100]. Interestingly, the vast majority of engi-neered L1 insertions in these animals were not heritable,perhaps indicating retrotransposition later in embryo-genesis [100]. Targeted and whole-genome sequencingapplied to mouse pedigrees has, conversely, revealed thatendogenous L1 mobilization in early embryogenesis isrelatively common and often leads to heritable L1 inser-tions (SRR and GJF, unpublished data). PolymorphicLTRTrim28Chaf1aTrim28?Histones H3.1/2AtrxDaxxZfp809Eset EsetYY1Zfp?ProvirusHP1 HP1(b)DaxxAtrxPBS SHINFig. 5 Proposed models of de novo endogenous retrovirus (ERV) silencingbox (KRAB) zinc finger protein (Zfp) Zfp809 interacts with the proline primeleukemia virus) [85] whereas other KRAB-Zfps bind to a short heterochromretrotransposons and other ERV families [93]. Subsequently, Trim28 is recruterminal repeat (LTR) and Trim28 [92]. Interaction with HP1 and sumolyatiomediated by Trim28 [72, 86, 89]. Eset also interacts with Trim28 and enableChaf1a, aided by Asf1a/b, marks proviral DNA for silencing by depositing hERV silencing by H3.3 deposition. The Atrx–Daxx complex is suggested to pindependent. Here, Atrx is thought to promote ERV heterochromatin inaccH3.3 and to interact with Trim28, followed by H3.3 being marked with H3KTrim28Sumo2ProvirusEsetZfp?HP1 SumoylationSHINERV and nonautonomous SINE insertions are also foundin different mouse strains [105, 106]. Although the de-velopmental timing of these events is as yet unresolved,we reason that they can occur in spatiotemporal con-texts supporting L1 retrotransposition. It follows thatboth human and mouse L1s, and probably mouse ERVs,can mobilize in embryonic and pluripotent cells (Fig. 6),as well as gametes. The resultant mosaicism can be dele-terious to the host organism or their offspring [101],again reinforcing the need for TE restraint during earlydevelopment.Somatic L1 retrotransposition can also occur later indevelopment. Over the past decade, it has becomeaccepted that the mammalian brain, particularly cells ofthe neuronal lineage, accommodate mobilization ofengineered and endogenous L1 elements [34–37, 108].LTRTrim28Chaf1aTrim28Histone H3.3Zfp809Eset EsetYY1Zfp?ProvirusHP1 HP1DaxxAtrxPBS SHINin embryonic stem cells. a To initiate silencing, the Krüppel-associatedr binding site (PBS Pro) of some ERV families (e.g., Moloney murineatin-inducing (SHIN) sequence found in intracisternal A-type particleited by the Zfps [74, 86], assisted by binding of YY1 to the longn by Sumo2 are thought to contribute to transcriptional repressions trimethylation of H3K9 and H4K20 [73]. The histone chaperoneistones H3 and H4 and interacts with Eset [72]. b Conflicting models oflay an important role in SHIN-mediated silencing, which is H3.3-essibility (left) [93]. However, Atrx–Daxx is also proposed to deposit9me3 by Eset (right) [95]Gerdes et al. Genome Biology  (2016) 17:100 Page 12 of 17L1 mobilisationEmbryoAdultAlthough the frequency of somatic L1 insertions duringneurogenesis is disputed [35, 36, 108, 109], this is largelydue to differences in the advanced techniques requiredto discriminate genuine de novo L1 insertions and mo-lecular artifacts arising during whole-genome amplifica-tion of individual human neurons. This discriminationcan, broadly, be achieved quantitatively, by assumingtrue-positives will accrue more DNA sequencing readsthan artifacts [108], or qualitatively, by analyzing thejunction DNA sequences between putative L1 insertionsand the flanking genome and excluding examplesinconsistent with target-site primed reverse transcription[35]. Despite this debate, there is agreement that L1mobilization occurs in the brain and can, for the mostpart, be traced to neuronal precursor cells [35, 36, 109].Remarkably, neuronal L1 insertions are distributed un-evenly genome-wide and are enriched in neurobiologicalgenes and transcribed neuronal enhancers [34, 35]. Som-atic L1 insertions oriented in sense to host genes, as theconfiguration most likely to disrupt transcription [110,111], are heavily depleted versus random expectation,providing possible evidence of selection against theseFig. 6 Long interspersed element-1 (L1) contributes to somatic mosaicism.a insert into protein-coding exons; b influence neighboring genes by the sc initiate sense or antisense transcription of neighboring genes, thereby cretranscripts, using host gene provided splice acceptor sites, which are translendonuclease activity of L1 ORF2p; and e lead to premature termination omeORF2pL1meme memeabcdNucleusGe neL1Gene L1ORF0events during neurogenesis [35]. Concordantly, somaticL1 insertions in neurobiological genes carry an elevatedchance of yielding a molecular phenotype in the brain,especially provided the numerous routes by which L1 in-sertions can profoundly modify gene structure and ex-pression (Fig. 6) [12, 33, 77, 110, 112–118].Neuronal L1 insertions impart no obvious evolution-ary benefit as they cannot be transmitted to subsequentgenerations. Thus, it is tempting to speculate that L1activity is derepressed during neuronal commitment toserve a biological purpose for the host organism, analo-gous to the potential exaptation of ERV transcriptionfor pluripotency maintenance and following the ex-ample of the vertebrate adaptive immune system, wheredomesticated TEs mediate V(D)J recombination andfunctional diversification through genomic mosaicism[119]. Similarly, although individual somatic L1 inser-tions in neurons are not inherited, it is plausible thatthe cellular mechanisms and factors enabling their pro-duction may undergo evolutionary selection [109].While L1-mediated somatic mosaicism in neurons mayeventually be shown to have functional or behaviorale AAL1Ge nepolyA-signalL1 mobilizes in the brain and early embryo (left) and may, for example:preading of repressive histone modifications, such as methylation (me);ating new transcripts, including open reading frame 0 (ORF0) fusionated to fusion proteins; d generate DNA double-strand breaks via thef host gene transcripts by providing alternative poly(A) signalsGerdes et al. Genome Biology  (2016) 17:100 Page 13 of 17consequences [109, 118], numerous additional experi-ments are required to assess this hypothesis. Whetherperturbation of L1 regulation and retrotransposition inthe brain is connected to neurological disease is not yetclear [35, 120–122]. The available evidence does, how-ever, show conclusively that TE mobilization occursduring embryogenesis and, in a more restricted fashion,later in life.ConclusionsThe mammalian genome clearly strives to limit TE activ-ity in pluripotent cells. The silencing mechanisms in-volved are collectively complex and broadly potent andyet are also capable of great specificity and dynamism intargeting individual TE copies [17]. In this regard, ERVspresent two contrasting facets: firstly, the control mech-anisms that have evolved to restrict ERV activity and,secondly, the domestication of ERV sequences into pluri-potency maintenance. Specific ERV families, such asHERV-H and HERV-K, can provide binding sites forpluripotency TFs, produce stem cell-specific protein-coding and noncoding transcripts, and harbor new en-hancers. Over time, these contributions have led to theintegration of ERVs into gene networks governing em-bryogenesis and, surprisingly, independent ERV hyper-activity appears to be a harbinger of pluripotent states.Conversely, notwithstanding a need for more experimen-tal data for murine ERVs, L1 appears to be the most suc-cessful TE to mobilize in mammalian somatic cells and,at the same time, is arguably less likely to impact theirphenotype than ERVs (Fig. 2). During human iPSC re-programming, for example, L1 and ERVs can both bebroadly derepressed, but with divergent repercussionsfor the host genome and providing different opportun-ities to each TE family.Why are TEs active, and apparently essential, in theembryo? The relationship between TEs and the hostgenome is often referred to as an evolutionary arms race[123, 124]. A review specifically addressing the role ofTEs in pluripotency [14] refined this concept to more ofa genetic conflict of interest between ERVs and the hostgenome, where exposure to retrotransposition was a ne-cessary risk of the pluripotent state. The authors, asothers have done [28], also considered the possibilitythat ERVs were active in stem cells by serendipity. Des-pite their merits, each of these alternatives is contra-dicted by several considerations. Firstly, L1 mobilizationappears to be far more common in the embryo thanERV mobilization, despite ERV domestication beingovertly more useful to the host given the many waysERVs can reinforce pluripotency (Fig. 3). The benefits ofunleashing L1 and ERV activity do not seem then, in ei-ther case, to be commensurate with the implied risk ofdoing so. Secondly, ERVs are intrinsic to the pluripotentstate but are now almost, if not fully, immobile inhumans. Thirdly, different ERV families are centrally in-volved in human and mouse pluripotency; convergentevolution driven by the common environmental de-mands of embryonic development, which are conservedamong mammals, is an improbable outcome of chance.Here, time and scale are critical considerations: the vastmajority of new ERV insertions will be immediately si-lenced but, as the retrotranspositional potential of anERV family is eliminated over time via mutations, pres-sure to silence the associated LTRs may also diminish,allowing them to regain their regulatory activity. Hence,with sufficient time, distinct ERV families in differentspecies can ultimately come to occupy similar niches, inpluripotency and elsewhere. TEs pervade mammaliangenomes and, as such, even the low probability of a denovo ERV insertion immediately escaping silencing pre-sents a reasonable overall chance of such events becom-ing important to genome-wide regulation. This remainstrue even if the ERV family is eventually immobilized.Although not rejecting models based on serendipity orconflict, we highlight that ERVs and other successful TEfamilies commonly arise as low copy number familiesand then rapidly expand over generations. This scenariocould lead to TEs acquiring traits of early pioneers in apotentially hostile genomic landscape. Two not necessar-ily exclusive strategies can aid TE survival in this envir-onment. One is stealth. For example, adaptation of theL1 5′ promoter (Box 2) enables evasion of host genomesurveillance, leading to continuing L1 retrotranspositionduring development. That most new L1 copies are 5′truncated, and lack the canonical promoter, also reducestheir visibility to surveillance. Although this self-limitsthe capacity of new L1 insertions to retrotranspose, italso reduces pressure on the host genome to clampdown on L1 activity. The other strategy is gaining ac-ceptance by being useful. ERV promoters are repeatedlyfound in pluripotency regulatory networks and may,therefore, be intrinsic to the pluripotent state. In thissetting, efforts by the host genome to limit ERV activitycould be detrimental to pluripotency. As such, ERVsmay be able to propagate for longer than would be pos-sible should the host engage in resolute inhibition. Im-portantly, these strategies are predicated on embryonicretrotransposition having potential for germline trans-mission, i.e., carrying risk for host genome integrity, asmany studies have now found. Even after ERV familiesare no longer capable of mobilization, their inherentcapacity for regulation, especially by solo LTRs, isretained and provides a long-term evolutionary incentivefor the host genome to maintain at least one active TEfamily, as almost all mammals do. As such, rather thanan arms race, conflict, or even symbiotic relationship, wewould propose that pioneer ERVs adopt peaceful survivalGerdes et al. Genome Biology  (2016) 17:100 Page 14 of 17strategies and that intricate mechanisms for TE repres-sion have evolved to allow the host genome to harnessthose strategies over time, allowing some ERV familiesto expand and, as witnessed in the embryo, securelyembed themselves by becoming indispensable. In advo-cating this model, we emphasize that indispensability ofERV-mediated regulatory effects in natural pluripotencyand embryogenesis in vivo is still an open question.While difficult to pursue in humans, genetic knockoutor deletion of individual mouse ERVs or ERV familiesimplicated in pluripotency is possible [125] and, indeed,ultimately necessary to demonstrate their functional im-portance to the embryo.AbbreviationsERV: endogenous retrovirus; ESC: embryonic stem cell; HERV: humanendogenous retrovirus; hESC: human embryonic stem cell; HPAT: humanpluripotency-associated transcript; IAP: intracisternal A-type particle;ICM: inner cell mass; iPSC: induced pluripotent stem cell; KRAB: Krüppel-associated box; L1: long interspersed element-1; lincRNA: long intergenicnoncoding RNA; LINE: long interspersed element; lncRNA: long noncodingRNA; LTR: long terminal repeat; miRNA: microRNA; MMLV: Moloney murineleukemia virus; ORF: open reading frame; RNP: ribonucleoprotein particle;SINE: short interspersed element; TE: transposable element; TF: transcriptionfactor; TSS: transcription start site; Zfp: zinc finger protein.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsPG, SRR, DLM, and GJF planned the review, read the literature, and draftedthe text. PG drafted the figures and assembled Table 2. DLM assembledTable 1. All authors approved the final manuscript.AcknowledgementsWe thank Matthew C. Lorincz, Adam D. Ewing, Francisco J. Sanchez-Luque,Patricia E. Carreira, and Santiago Morell for critical reading of the manuscript.GJF acknowledges the support of an Australian NHMRC Senior ResearchFellowship (GNT1106214), NHMRC project grants (GNT1045991, GNT1067983,GNT1106206), and the Mater Foundation. DLM acknowledges grant supportfrom the Canadian Institutes of Health Research (MOP-10825) and the NaturalSciences and Engineering Research Council of Canada (RGPIN-170104).Author details1Mater Research Institute, University of Queensland, TRI Building,Woolloongabba QLD 4102, Australia. 2Department of Medical Genetics, TerryFox Laboratory, British Columbia Cancer Agency, University of BritishColumbia, Vancouver, BC V5Z 1L3, Canada. 3School of Biomedical Sciences,University of Queensland, Brisbane QLD 4072, Australia.References1. 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