Dynamic interactions between transposable elements and their hosts

doi:10.1038/nrg3030

Nat Rev Genet. Author manuscript; available in PMC 2011 October 13.

Published in final edited form as:

Nat Rev Genet. 2011 August 18; 12(9): 615–627.

doi: 10.1038/nrg3030

PMCID: PMC3192332

NIHMSID: NIHMS323559

Dynamic interactions between transposable elements and their hosts

Henry L. Levin¹ and John V. Moran²

¹Section on Eukaryotic Transposable Elements, Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892, USA, Tel. 301-402-4281, Fax. 301-496-4491, Email: henry_levin/at/nih.gov

²Departments of Human Genetics and Internal Medicine, and Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, MI, 48109-6518, USA, Tel. 734-615-4046, Fax. 734-763-3784, Email: moranj/at/umich.edu

Corresponding author.

The publisher's final edited version of this article is available at Nat Rev Genet

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Abstract

Transposable elements (TEs) have a unique ability to mobilize to new genomic locations and the major advance of next-generation DNA sequencing has provided insights into the dynamic relationship between TEs and their hosts. It now is clear that TEs have adopted diverse strategies – such as specific integration sites or patterns of activity - to thrive in host environments that are replete with mechanisms – such as small RNAs or epigenetic marks - to combat their amplification. Emerging evidence suggests that TE mobilization might sometimes benefit host genomes by enhancing genetic diversity, but TEs are also implicated in diseases such as cancer. Here, we discuss recent findings about how, where, and when TEs insert in diverse organisms.

Subject categories: Evolutionary Biology, Genomics, Epigenetics, genomic instability, small RNAs

Introduction

Through her pioneering work in maize, Barbara McClintock was the first to realize that eukaryotic genomes are not static entities and contain transposable elements (TEs) that have the ability to move from one chromosomal location to another¹. It now is clear that virtually all organisms harbor TEs that have amplified in copy number over evolutionary time via DNA or RNA intermediates. On occasion, TEs sporadically have been co-opted by the host to perform critical cellular functions^{(e.g., 2–5)}. However, most TEs likely are finely tuned genomic parasites that mobilize to ensure their own survival^6–9. The genomic revolution, coupled with new DNA sequencing technologies, now provides an unprecedented wealth of data documenting TE content and mobility in a broad array of organisms.

In multi-cellular eukaryotes, TEs must mobilize within gametes or during early development to be transmitted to future generations. In humans, there are at least 65 documented cases of diseases resulting from de novo TE insertions; these events account for approximately 1/1000 spontaneous cases of disease in humans^{5, 10}. Indeed, new genomic technologies combined with cell culture based experiments have demonstrated that active TEs are more prevalent in the human population than previously appreciated^11–18. A growing body of evidence further suggests that mammalian TE integration occurs during early development^19–21. In addition, studies of neurogenesis and some forms of cancer have raised the intriguing possibility that TE activity may impact the biology of certain somatic cells^{12, 22–24}. It is likely that we only have observed the tip of the iceberg and still are underestimating the contribution of TE-mediated events to inter-and intra-individual structural variation in mammalian genomes.

TE mobility poses a serious challenge to host fitness. Paradoxically, TE insertions that are harmful to the host jeopardize TE survival. Thus, many TEs have evolved highly specific targeting mechanisms that direct their integration to genomic “safe havens,” thereby minimizing their damage to the host^{(e.g., 25–29, and references mentioned below)}. Nevertheless, host genomes have evolved potent restriction mechanisms, such as the methylation of TE DNA sequences and the expression of small RNAs or cytidine deaminases, to restrict TE activity in the germline and perhaps somatic cells^{(e.g., 30–33, and references mentioned below)}.

Interestingly, a growing number of examples suggest that TEs may become activated under certain environmental conditions, such as stress. Stress has been shown to induce TE transcription or integration, or redirect TE integration to alternative target sites^34–38. These findings are consistent with Barbara McClintock’s hypothesis that environmental challenges may induce transposition, and that transposition, in turn, may create genetic diversity to overcome threats to host survival³⁹.

We begin this review with a brief description of the types of TEs and their modes of mobility. We then describe the latest understanding of TE integration mechanisms and how the host defends against these attacks. Finally, we discuss exciting new research that suggests TE mobility may impact the biology of somatic cells. From the growing understanding of target site selection to the discovery of new active TE copies in human populations, it is clear that the field of transposon biology continues to yield new insights about genome biology.

The diversity and abundance of transposons

Mobilization mechanisms

TEs mobilize by remarkably diverse replication strategies (Table 1 and Figure 1)⁴⁰. Many DNA transposons mobilize by a non-replicative “cut and paste” mechanism, whereby an element-encoded enzyme, the transposase, recognizes sequences at or near TE inverted terminal repeats to “cut” the TE from its existing genomic location and then acts to “paste” the excised DNA into a new genomic location (Fig. 1a)⁴¹.

Table 1

Classes of transposable elements and their mobility mechanisms

Figure 1

The diverse mechanisms of transposon mobilization

Retrotransposons mobilize via the reverse transcription of an RNA intermediate; however, different types of retrotransposons carry out this process by distinct mechanisms (see Fig. 1b and c). Long terminal repeat (LTR) retrotransposons^42–44 (Fig. 1b) and non-LTR retrotransposons⁴⁵ (Fig. 1c) use element-encoded enzymes to mediate their mobility. In addition, the endonuclease and reverse transcriptase activities of non-LTR retrotransposons also play a central role in mobilizing non-autonomous Short INterspersed Elements (SINEs)^46–48, certain classes of non-coding RNAs^49–52, and messenger RNAs, which can result in the formation of processed pseudogenes^{53, 54}.

Transposon activity across species

Examples of DNA TEs include Tn5 and Tn7 of E. coli^{55, 56}, P elements of Drosophila⁵⁷, and Tc1 elements of C. elegans⁵⁸. Though they thrive in prokaryotes and simpler eukaryotes, DNA TE activity appears to be extinct in most mammals, which fuelled speculation that DNA TEs play a limited role in the ongoing evolution of mammalian genomes⁵⁹. However, recent studies suggest that DNA TEs, namely non-autonomous hobo/Activator/TAM (nhAT transposons and helitrons), are active in certain bat species^60–62. Thus, these studies highlight how new DNA sequencing technologies can facilitate fundamental discoveries about the impact of different TE families on genome evolution and serve as a cautionary note against deriving general conclusions regarding TE activity from relatively few “reference” sequences.

LTR-retrotransposons are particularly abundant in eukaryotes. For example, Drosophila contains approximately 20 distinct families of LTR-retrotransposons that comprise ~1% of the genome⁶³, while maize contains ~400 families of LTR-retrotransposons that comprise ~ 75% of the genome^{64, 65}. In addition, the mouse genome contains multiple active LTR-retrotransposon families. Indeed, the ongoing retrotransposition of both autonomous LTR-retrotransposons and their non-autonomous derivatives is estimated to account for approximately ~10–12 percent of sporadic mutations in mouse⁶⁶. By comparison, there appears to be little LTR-retrotransposon activity in human genomes⁵⁹; however, a small number of human endogenous retroviruses are polymorphic with respect to presence/absence at a given genomic location, suggesting that they have retrotransposed relatively recently in human evolution⁶⁷.

Non-LTR retrotransposons are widespread among eukaryotes, but have been especially prolific in mammalian genomes. For example, L1 elements and the non-autonomous SINEs that they mobilize (e.g., Alu and SINE-R/VNTR/Alu (SVA) sequences)^{47, 48}, comprise approximately 30% of human genomic DNA sequence⁵⁹. Furthermore, recent research using a combination of transposon display^{68, 69}, second-generation DNA sequencing^{12, 15–17} and analyses of genomic DNA sequences from the Human Structural Variation project^{13, 70–72}, the 1000 genomes project^{18, 73, 74}, and clinical cohorts¹⁴, have revealed that L1 presence/absence dimorphisms, as well as non-allelic recombination between L1 and Alu elements, account for an appreciable proportion of the inter-individual structural variation observed among humans and continue to have a profound effect on the human genome (see ref. ⁵ for a detailed review).

The diverse patterns of integration sites

Transposons exhibit a remarkable diversity of integration behaviors. Some TEs preferentially integrate into gene-dense regions of the genome, others target regions such as heterochromatin, telomeres, or ribosomal DNA arrays, and some appear to insert throughout the genome. Below, we describe several examples of TE integration and what is known about how TEs target specific sites in genomic DNA.

Integration into gene rich regions

Many TEs integrate into gene rich regions although they use mechanisms that prevent the disruption of open reading frames (ORFs). An extreme example is the E. coli Tn7 DNA TE. Tn7 encodes a sequence specific DNA binding protein, TnsD, which mediates integration into a specific position in the host chromosome, termed attnTn7, and thereby avoids damaging the host genome^{75, 76}. A second targeting protein, TnsE, can alter Tn7 target preference by directing integration to plasmid DNAs that are transferred between E. coli by conjugation⁷⁷ or to double strand breaks and DNA structures formed during DNA replication⁷⁸. By comparison, the Drosophila P element avoids disrupting ORFs by integrating within the 500 bp upstream of transcription start sites of genes⁷⁹. However, the mechanism by which P-elements target these sites requires elucidation.

Certain non-LTR retrotransposons encode endonucleases that target specific sites in genomic DNA. For example, the R1 and R2 elements of insects encode sequence-specific endonucleases that cleave at specific positions within the 28S rDNA locus to initiate target-primed reverse transcription (TPRT)^{28, 45}. However, these endonucleases operate by distinct mechanisms. R1 encodes an endonuclease that shares sequence similarity to an apurinic/apyrimidinic (APE) DNA repair endonuclease^{80, 81}, whereas R2 encodes a type II-S restriction endonuclease⁸². Thus, these elements apparently have evolved convergent mechanisms to integrate into ribosomal DNA arrays.

LTR-retrotransposons also have evolved strategies to integrate into gene rich regions, while ensuring minimal damage to their hosts. For example, the Ty1 and Ty3 retrotransposons of S. cerevisiae specifically target gene-free windows located immediately upstream of RNA polymerase III transcribed genes, such as tRNAs^{25, 27, 83, 84}; Ty3 is directed to integration sites 2 or 3 bp upstream of such genes by transcription factors TFIIIB and TFIIIC (Fig. 2a)^{85, 86}. However, in the case of the SNR6 gene, which does not depend on TFIIIC for its expression, the TFIIIB factors Brf1 and TBP are sufficient to direct Ty3 integration^{87, 88}.

Figure 2

Mechanisms that position integration

Ty1 integrates into a ~700 bp window upstream of tRNA genes with a periodicity of 80 bp (Fig. 2b)^{27, 89}. Although the factors that direct Ty1 to tRNA genes remain unknown, the unusual periodicity of integration depends on the amino-terminal domain of Bdp1, another TFIIIB factor⁹⁰. The ability to integrate upstream of RNA polymerase III transcribed genes also can regulate host and TE gene expression. For example, Ty1 and Ty3 insertions can stimulate the transcription of downstream RNA polymerase III transcribed genes and transcription of the RNA polymerase III target genes can reciprocate by repressing Ty1 transcription^{91, 92}. Clearly, determining how Ty1 target integration sites and exploring how integration alters gene regulation remain areas for future study.

The ability to target RNA polymerase III transcribed genes is not peculiar to LTR-retrotransposons. For example, the Dictyostelium discoideum non-LTR retrotransposon DRE (also known as TRE-5A) preferentially inserts ~48bp upstream of tRNA genes, whereas the retrotransposon Tdd3 (also known as TRE-3A) inserts downstream of tRNA genes^{29, 93}. Indeed, experimental evidence suggests that the TRE-5A ORF1-encoded protein directly interacts with subunits of TFIIIB to direct its integration to tRNA genes⁹⁴.

The Schizosaccharomyces pombe retrotransposon, Tf1, preferentially integrates into the promoters of RNA polymerase II transcribed genes and provides another example of how TEs target gene-rich regions^95–97 (Fig. 2c). Tf1 integration has been studied by examining integration into promoters contained within extrachromosomal replicating plasmids²⁶. For example, the fbp1 promoter is induced when the activating transcription factor Atf1p binds to an eight base pair upstream activating sequence (UAS1)⁹⁸. Tf1 integration generally occurs 30 bp and 40 bp downstream of UAS1; however, mutating six nucleotides of UAS1 or deleting the Atf1p gene²⁶ disrupts Tf1 integration specificity, causing integration to occur throughout the plasmid. Although cells lacking Atf1p show little reduction in the overall Tf1 retrotransposition frequency⁹⁹, the above data, as well as the finding that Atf1p forms a complex with Tf1 IN, indicate that specific transcription factors such as Atf1p can play a critical role in directing Tf1 integration to a specific target site. Notably, experiments conducted with a synthetic promoter revealed that RNA polymerase II transcription is not sufficient to target Tf1 integration⁹⁹.

The development of second-generation sequencing technology recently has allowed the in vivo examination of Tf1 integration sites en masse. Characterization of 73,125 Tf1 integration events from four independent experiments revealed a highly reproducible pattern — approximately 95% of integration events are clustered upstream of ORFs⁹⁶. Interestingly, the most frequently targeted promoters are associated with genes that are induced by environmental stressors. The targeting of genes that respond to stress, coupled with the ability of Tf1 to induce the expression of adjacent genes²⁶, suggests that Tf1 integration has the potential to improve survival of specific cells that are exposed to environmental stress. Likewise, the transcription of Tf2, another LTR-retrotransposon in S. pombe, is induced by oxidative and osmotic stress or by growth in low oxygen^{34, 100}. Clearly, understanding the consequences of stress-induced retrotransposition will yield insights about how TE mobility can lead to genetic diversity, which may affect the ability of an organism to cope with stress.

Finally, certain retroviruses, which are descended from LTR-retrotransposons¹⁰¹, also exhibit preferential integration in gene rich regions. For example, human immunodeficiency virus-1 (HIV-1) preferentially integrates into RNA polymerase II transcribed genes, whereas murine leukemia virus shows a strong integration preference near transcriptional start sites^102–104. Structural and biochemical data demonstrate that HIV-1 IN interacts with the cellular lens epithelium-derived growth factor (LEDGF/p75) host factor, and there is evidence that this interaction plays an important role in proviral DNA integration^{105, 106}.

Integration into heterochromatin

Some TEs target heterochromatic sequences that contain relatively few genes. For example, chromoviruses, which are related to Ty3/Gypsy LTR-retrotransposons, reside in heterochromatin of eukaryotes from fungi to vertebrates¹⁰⁷. They contain a chromodomain near the carboxyl-terminus of IN that is related to HP1, a heterochromatin protein that binds to histone H3 methylated at lysine 9¹⁰⁷. Furthermore, chromovirus chromodomains fused to green fluorescent protein (GFP) co-localize with heterochromatin¹⁰⁸, suggesting that the chromodomain plays a principal role in directing integration. Indeed, fusion of one such chromodomain to the carboxyl-terminus of Tf1 IN directs integration of this TE to heterochromatin¹⁰⁸.

The Ty5 LTR-retrotransposon also targets gene poor regions in S. cerevisiae. Approximately 90% of Ty5 integration events occur within the silent mating type loci or near silent heterochromatin at telomeres (Fig. 2d)^109–111. Genetic and biochemical experiments indicate that a nine amino acid targeting domain (TD) in the Ty5 integrase (IN) carboxyl-terminus directly binds to a structural component of heterochromatin, Sir4p, to target integration^{35, 112, 113}. Moreover, fusing Sir4p to the DNA binding domain of the LexA repressor protein causes Ty5 integration to be redirected to Lex A binding sites¹¹⁴. Thus, the Ty5 TD, by interacting with Sir4p, directs integration to heterochromatin. Interestingly, genetic and biochemical evidence indicate that the Ty5 TD evolved its interaction with Sir4p by mimicking residues in a host factor, Esc1p, that binds to the same amino acids of Sir4p¹¹⁵.

Although the ability of Ty5 IN to target heterochromatin suggests that the TE dictates target site integration, there also are indications that TE-host interactions can alter Ty5 target-site preference. Mass spectroscopy revealed that phosphorylation of the integrase TD at S1095 is critical for binding Sir4p³⁵ and mutating S1095 redirects integration to expressed regions of the genome. Although the host-encoded kinase has not been identified, studies using a phospho-specific antibody indicate that stressors, such as nitrogen deprivation, can down-regulate S1095 phosphorylation³⁵. Thus, stress conditions may alter the phosphorylation state of Ty5 IN, thereby redirecting Ty5 integration specificity. This elegant example provides a plausible mechanism for how stress can alter transposon mobilization in a manner that might provide an advantage for the host. It remains to be determined whether retargeting Ty5 to gene rich regions benefits Ty5 by allowing newly retrotransposed copies to reside in permissive expression contexts or benefits the host by generating genetic diversity, offering the potential to adapt to stress.

Integration into Telomeres

Some TEs exclusively integrate at or near telomeric ends. For example, the Het-A, TART, and TAHRE non-LTR retrotransposons comprise the ends of Drosophila chromosomes and likely substitute for the function of telomerase in maintaining chromosome end integrity^{2, 116, 117}. The SART1 and TRAS1 non-LTR retrotransposons may have a similar role in Bombyx mori¹¹⁸. The proteins encoded by TART, TAHRE, SART1, and TRAS1 have a Apurinic/apyrimidinic (AP)-like endonuclease domain^{118, 119} and it is likely that the SART1 and TRAS1 endonuclease proteins direct their integration into telomeric repeats¹¹⁸; however, the functional role of the putative endonuclease domain in TART and TAHRE remains unknown.

Excitingly, recent studies have revealed that certain retrotransposons can target telomeric sequences for integration. For example, by an alternative endonuclease-independent retrotransposition mechanism, human L1 retrotransposons containing missense mutations in the L1 EN active site can integrate at endogenous DNA lesions and dysfunctional telomeres in Chinese Hamster Ovary cell lines that are deficient for factors important in the non-homologous end-joining pathway of DNA repair as well as p53 function^{120, 121}. Similarly, members of the Penelope clade of retrotransposons, which encode an RT that lacks an obvious endonuclease domain, reside at telomeres in organisms from four eukaryotic kingdoms¹²². The RNAs encoded by these terminal Penelope elements also contain sequences that are complementary to telomeric DNA sequences, suggesting that base pairing between the TE RNA and single stranded telomeric DNA is critical for integration. Interestingly, both of the above cases can be considered as a type of RNA-mediated DNA repair that appears curiously similar to the mechanism used by telomerase^{120, 122}. Future studies should elucidate whether host factors are critical for the localization of these retrotransposons to DNA lesions and/or chromosomal termini.

Dispersed patterns of integration

In contrast to elegant mechanisms that target integration of some TEs into specific regions of the genome, other TEs appear to lack target site specificity. For example, L1s and the non-autonomous elements they mobilize are interspersed throughout the genome⁵⁹. Indeed, ~30% of engineered human L1 retrotransposition events in cultured cells, and a similar proportion of recently discovered full-length, dimorphic human-specific L1s, are near or within the introns of genes^{13, 50, 123, 124}. Since protein-coding genes constitute ~40% of the human genome^{125, 126}, these findings suggest a lack of robust mechanisms employed by L1s or the host to prevent L1 retrotransposition into genes.

The interspersed nature of L1 and Alu sequences probably reflects the fact that the L1 endonuclease has relatively weak target-site specificity, preferentially cleaving the sequence 5’-TTTT/A-3’ (and variants of that sequence), to initiate TPRT^{80, 121, 127, 128}. Interestingly, while “young” Alu and L1 insertions exhibit similar interspersed integration patterns, cytogenetic studies and examination of the human genome reference sequence revealed that evolutionarily “older” L1s and Alus show distinct genomic distributions^{59, 129}. Older L1s preferentially reside in gene-poor AT-rich sequences, whereas older Alus are preferentially reside in gene-rich GC-rich regions of the genome⁵⁹.

The distinct distributions of older L1s and Alus likely result from post-integration selective processes that have operated on the genome for millions of years⁵⁹. However, how these skewed distributions arose remains a mystery. Some researchers have suggested that Alus may possibly play an advantageous, albeit undefined, role in gene-rich regions of the genome⁵⁹. Others have suggested that L1 retrotransposition events into genic regions may exert a greater fitness cost to the host than Alu insertions¹³⁰. If so, negative selection would lead to the removal of detrimental L1 alleles from the population. Consistent with this hypothesis, data suggest that evolutionary recent human full-length L1s insertions are detrimental to the host^{131, 132}, whereas in vitro studies have revealed that L1s contain cis-acting sequences that can reduce gene expression^{133, 134}. Clearly, further studies are needed to explain how the distributions of L1 and Alu have diverged over evolutionary time.

Despite their interspersed distribution, a small body of evidence suggests that there may be preferred, albeit rare, L1 integration sites. For example, independent L1-mediated retrotransposon insertions at the same nucleotide position in the BTK gene (i.e., an SVA and an Alu element) have resulted in two sporadic cases of X-linked agammaglobulinemia¹³⁵. Similarly, independent L1 and Alu insertions associated with colorectal and desmoid tumors, respectively, have occurred at the same nucleotide position in the APC gene^{22, 135, 136}, whereas two independent Alu insertions at the same nucleotide position in the Factor IX gene have caused hemophilia B^{135, 137}. Thus, it would not be surprising to find that chromatin structure and accessibility impact L1-mediated retrotransposon target preference¹³⁸.

How the host defends against transposons

Although many TEs have evolved mechanisms to limit genome damage, TE integration still poses a potential threat to the host. Thus, it is not surprising that host organisms have evolved a diverse array of mechanisms to combat TE activity. However, the host must be able to discriminate TE sequences from host genes to accomplish this feat. Below, we discuss mechanistic strategies employed by the host to restrict TE mobilization.

DNA methylation

Cytosine methylation (5-methylcytosine) is an important DNA modification in eukaryotes with genomes larger than 5×10⁸ bp, which includes vertebrates, flowering plants, and some fungi. The majority of cytosine methylation in plants and mammals, and almost all cytosine methylation in Neurospora crassa, occurs within repetitive elements and is correlated with the transcriptional repression of retrotransposons in somatic and germline cells^{139, 140}.

Experiments in mammals and plants demonstrate that global demethylation of genomic DNA strongly reactivates TE transcription^141–144. For example, deletion of DNA cytosine-5-methyltransferase 3-like gene (Dnmt3L) in mice leads to loss of de novo cytosine methylation of both LTR and non-LTR retrotransposons, reactivation of transposable element expression in spermocytes and spermatogonia, and meiotic catastrophe in male germ cells¹⁴⁵. Determining whether TE mobilization directly is responsible for the meiotic defects requires further study. Moreover, recent data demonstrates that inactivation of cytosine methylation in Arabidopsis thaliana causes a burst of retrotransposon and DNA TE activity and results in substantial increases in TE copy number¹⁴⁴. Thus, epigenetic mechanisms act to control the expression, and perhaps mobility of various TEs.

Multiple lines of evidence indicate that DNA methylation inhibits TE transcription. Patterns of DNA methylation are established during gametogenesis and are mediated by Dnmt3a and the non-catalytic paralog, Dnmt3L, in mammals, but how TEs are recognized as methylation substrates requires further study¹⁴⁶. By comparison, during plant development, small 24 nt RNAs target paralogous DNA sequences that share high levels of homology (such as TEs) for cytosine methylation. The mechanism of RNA-directed DNA methylation (RdDM) is not fully understood, but it appears to require the canonical RNA interference (RNAi) machinery (see below and Fig. 3), the DNA methyltransferase DRM2, and two plant specific RNA polymerases, Pol IV and Pol V¹⁴⁶.

Figure 3

The degradation of transposon mRNA by RNAi

Small RNAs inhibit TEs

Small RNA-based mechanisms (including, endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs)), also act to defend eukaryotic cells against TEs. The mechanisms by which these small RNAs are generated and how they inhibit TEs remain an active area of investigation in various model organisms. Mechanistic details regarding these processes can be found in many outstanding reviews on this topic^{(e.g., 30, 147–152)}; here we briefly summarize common themes that have emerged from the above studies.

Endo-siRNAs have the potential to inhibit TE mobility through the post-transcriptional disruption of transposon mRNA. For example, double-strand “trigger” RNAs (ds-RNAs) can be derived from the complementary inverted terminal repeats in DNA transposons, from structured mRNA transcripts, or from overlapping regions contained within convergent transcription units^{30, 147, 148, 153}. The resultant ds-RNAs then can be processed into ~21–24 nt endo-siRNAs by members of the Dicer family of proteins (Fig. 3a)^{30, 154}. These endo-siRNAs are loaded onto an Argonaute protein, and the “passenger” RNA strand (typically the sense strand of a TE) is degraded. The remaining complex of a single stranded RNA and Argonaute is called the RNA-Induced Silencing Complex (RISC); the RNA directs RISC to complementary sequences in target mRNAs, leading to their post-transcriptional degradation. Importantly, the RNAi machinery has the capability to inhibit any TE that generates a ds-RNA “trigger” that can serve as a substrate for the RNAi machinery.

By a different mechanism, piRNAs can be generated from genomic loci encoding long precursor RNAs that contain the remnants of different families of TEs¹⁵¹. In general, processing of these precursor RNAs leads to the production of a mature ~24–35 nt piRNAs (Fig 3b). A subfamily of Argonaute proteins, known as the Piwi clade of proteins, predominantly binds mature antisense piRNAs and directs them to complementary sequences in TE mRNA. An endonuclease activity associated with the Piwi protein cleaves the TE mRNA to release a sense strand piRNA, which can interact with other Piwi clade proteins. Binding of this complex to the original piRNA precursor RNA then reiterates this amplification cycle by a “ping-pong” mechanism^{151, 155}. In addition to this type of mechanism that restricts TE mobility in the germline, recent studies suggest that specialized piRNA pathways, which do not operate via a ping-pong mechanism, might restrict somatic TE activity^{32, 155–157}.

Examples of TEs that are controlled by small RNA-based mechanisms include Tc1 transposons in C. elegans, and P elements in Drosophila^{153, 158}. Also, 21nt small interfering RNAs (siRNA) derived from the Athila family LTR-retrotransposons in the vegetative nucleus of the pollen grains in Arabidopsis thaliana are delivered to the sperm cells to inhibit expression of transposons that, in principle, could mobilize in the germline¹⁵⁹.

Small RNA based mechanisms also may be critical for silencing mammalian L1s. For example, an antisense promoter located within the human L1 5’UTR allows the production of an antisense RNA that, in principle, could base pair with sense strand L1 mRNA to establish a ds-RNA substrate for Dicer¹⁶⁰. Furthermore, mouse mutants lacking the murine Piwi family proteins MILI or MIWI2 exhibit a loss of L1 and intracisternal A particle (IAP) LTR-retrotransposon DNA methylation,; this loss correlates with their transcriptional activation in male germ cells¹⁶¹. Similarly, mice lacking a MILI interacting protein, Tudor containing protein-1, exhibit a similar loss of L1 DNA methylation and a reactivation of L1 expression¹⁶². Finally, mouse mutants lacking the non-canonical Maelstrom protein, a component of the nuage complex that may be important for small RNA biogenesis, exhibit de-repression of L1 transcription, an increase of L1 ribonucleoprotein particle intermediates in spermatids, and a chromosomal synapsis defect during male meiosis¹⁶³. Together, the above examples provide compelling data that small RNA-based pathways likely act to control the expression of certain TEs in the mammalian germline.

Finally, it is noteworthy that other antisense RNA-based mechanisms may be involved in TE silencing. For example, antisense transcripts from S. cerevisiae Ty1 elements reduce Ty1 IN and RT protein levels by a post-translational mechanism; this leads to inhibition of Ty1 mobility and thus controls Ty1 copy number¹⁶⁴. Since S. cerevisiae lacks RNAi machinery, these results suggest that genomes have evolved other RNA-dependent strategies to tame TEs.

Cytosine deaminases and DNA repair factors restrict TEs

Proteins involved in nucleic acid metabolism and/or DNA repair can also restrict TE mobility. For example, members of the APOBEC3 family of cytidine deaminases can restrict the retrotransposition of a various retroviruses and LTR and non-LTR retrotransposons³³. For retroviruses and LTR-retrotransposons, APOBEC3 proteins generally deaminate cytidines during the first strand cDNA synthesis, which leads to either cDNA degradation or the integration of a mutated provirus. The mechanisms by which certain APOBEC3 proteins restrict non-LTR retrotransposons require elucidation. Similarly, over-expression of the 3’-repair exonuclease 1 (Trex1) gene, mutations in which cause Aicardi-Goutieres syndrome, can inhibit L1 and IAP retrotransposition in cultured cell assays^{165, 166}, but the mechanism of Trex1-mediated TE repression requires elucidation.

Other mechanisms are also likely to restrict the mobility of non-LTR retrotransposons. For example, the overwhelming majority of L1 elements in mammalian genomes are 5’ truncated and are essentially “dead on arrival” because they cannot synthesize proteins critical for retrotransposition¹⁶⁷. It has been proposed that 5’ truncation may be due to the low processivity of the L1-encoded reverse transcriptase. However, recent work on the reverse transcriptase encoded by the R2 non-LTR retrotransposon of Bombyx mori demonstrated that this enzyme is more processive than the reverse transcriptases encoded by retroviruses¹⁶⁸. Alternatively, L1 5’ truncation might result if host factors cause the dissociation of the L1 reverse transcriptase from the nascent cDNA and/or degrade the L1 mRNA during integration. In this scenario, to generate a full-length insertion the L1 RT would need to complete integration before the TPRT intermediate is recognized as DNA damage by the host^{50, 169}. Indeed, proteins involved in the non-homologous end-joining pathway of DNA repair seem to act to restrict the retrotransposition of a zebrafish LINE-2 element in DT40 chicken cells¹⁷⁰, whereas members of DNA excision repair pathway (that is, ERCC1/XPF1) might restrict L1 retrotransposition in cultured human cells¹⁷¹.

Finally, in addition to recognizing the L1 integration intermediate as a form of DNA damage, recent data suggests that retrotransposition indicator cassettes delivered by engineered L1s in human embryonic carcinoma cell lines can be epigenetically silenced during or immediately after their integration into genomic DNA¹⁷². Given that L1 is an ancient “stowaway” in mammalian genomes, it is likely that the host has evolved multiple mechanisms to combat L1 mobility at discrete steps in the retrotransposition pathway, and that some of these mechanisms operate in a context dependent manner. Clearly, continued studies will reveal new and more diverse host mechanisms to restrict TE mobility.

Developmental triggers of transposition

Despite mechanisms to combat TE mobility, TEs continue to thrive in many host genomes. Thus, TEs must have evolved ways to either overwhelm or counteract these host defenses. TEs must mobilize in germ cells or during early development to ensure their survival (Figure 4). However, some TEs can mobilize in somatic cells, providing a potential mechanism to generate intra-individual genetic variation.

Figure 4

Timing of transposition

Transposition in the germ line or during early development

Drosophila P elements provide one of the best-studied cases of cell type specific transposition¹⁷³. P element transposition occurs when females lacking P elements (the M cytotype) mate with males carrying P elements (the P cytotype); P element mobilization can cause hybrid dysgenesis in the offspring. In the reciprocal cross, eggs from P cytotype females produce a repressor protein and piRNAs that inhibit P element transposition^{57, 158}. The repressor is an alternatively spliced truncated form of the transposase. Importantly, the repressor not only controls which crosses produce germline integration, but also inhibits transposition in the soma.

The Drosophila gypsy element is another example of a TE that exhibits tissue specific control^{174, 175}. Gypsy transcription is induced in somatic follicle cells that surround the oocyte. The TE mRNA assembles into virus-like particles that are thought to traffic to the oocyte to carry out transposition. It remains unclear whether the transfer of gypsy virus-like particles to the oocyte occurs via an enveloped particle (similar to retroviruses) or by a form of endocytosis. However, the Flamenco locus encodes piRNAs that silence gypsy elements in follicle cells, thereby preventing the spread of these TEs to the surrounding germ cells¹⁵⁵.

Relatively little is known about the developmental timing of L1 retrotransposition in mammals. The sheer numbers of L1 and Alu retrotransposons that populate mammalian genomes provide prima facie evidence that they mobilize in the germline. Various studies, using endogenous and engineered L1s, provide strong experimental evidence to back this assertion. For example, full-length mouse L1 RNA and the mouse L1 ORF1-encoded protein are co-expressed in leptotene and zygotene spermatocytes during meiotic prophase¹⁷⁶. In addition, the mouse ORF1 protein is expressed in the cytoplasm during specific stages of development in oocytes¹⁷⁷. Similarly, human oocytes express L1 RNA and support the retrotransposition of an engineered human L1¹⁷⁸. Finally, transgenic mouse experiments demonstrated that an engineered human L1 retrotransposon, whose expression is driven from a heterologous pPol II promoter, can retrotranspose in male germ cells¹⁷⁹.

Unexpectedly, a growing body of experimental evidence suggests that L1 retrotransposition also might occur frequently during early development (Figure 4) (also reviewed in ref. ⁵). For example, human embryonic stem cells can express L1 RNA and ORF1 protein, and accommodate the retrotransposition of engineered L1s, albeit at lower levels than in other types of transformed human cells^{19, 180}. In addition, studies of a male patient with X-linked choroideremia revealed that his mother had mosaicism for the mutagenic L1 insertion in both germline and somatic tissues²⁰. Thus, the initial retrotransposition event must have occurred during early embryogenesis in the mother. Finally, recent transgenic experiments conducted in rats and mice led to the conclusion that most L1 retrotransposition occurs during early embryogenesis and that most of the resultant events are not heritable²¹. Intriguingly, these data suggest that L1 ribonucleoprotein particles can be deposited into zygotes by either the sperm or egg to undergo retrotransposition during early development, thereby providing a possible mechanism to generate somatic mosaicism and intra-individual genetic variation (see below).

Somatic transposition

Classical experiments in maize revealed that DNA TE activity in somatic tissues could lead to variegated corn color phenotypes^{1, 181}. Since that time, somatic TE events also have been reported in other organisms. For example, it is well established that Tc1 transposition in the Bergerac strain of C. elegans preferentially occurs in somatic cells¹⁸². Similarly, a recent study has revealed that somatic transposition of a DNA TE (Hatvine1-rrm) into the promoter region of the VvTFL1A gene of the grapevine cultivar Carnigan affects the grapevine branching pattern and size of fruit clusters¹⁸³. Also, a mutagenic L1 insertion was identified in the adenomatous polyposis coli (APC) gene in tumor tissue, but not in the surrounding tissue, of a patient with colon cancer, suggesting a role for the insertion in cancer development²². Together with the transgenic L1 experiments (discussed above), these findings establish that somatic TE mobility can lead to phenotypic changes in the host.

Intriguingly, several lines of evidence suggest that somatic L1 retrotransposition may also occur in the mammalian nervous system (also reviewed in ref. ⁵). First, an engineered human L1 can retrotranspose in neurogenic zones of the brain in transgenic mice²⁴ when its expression is driven by a promoter contained within its native 5’ UTR¹⁸⁴. Second, engineered human L1s can retrotranspose in cultured rat neuronal progenitor cells (NPCs), human embryonic stem cell-derived NPCs, and at low levels in human fetal derived NPCs^{23, 24}. Third, sensitive multiplex quantitative PCR experiments suggest a modest increase in L1 copy number in post mortem brain tissue, when compared to heart and liver tissue derived from the same individual²³. Finally, retrotransposition of an engineered human L1 is elevated in a mouse model of Rett syndrome (a neurodevelopmental disorder), and induced pluripotent stem cells derived from Rett syndrome patients exhibit an increase in L1 DNA copy number when compared to normal controls, suggesting a potential increase in endogenous L1 retrotransposition¹⁸⁵.

The above studies strongly suggest that certain neuronal cells may be permissive for L1 retrotransposition. However, additional research is needed to truly understand the impact of L1 retrotransposition in the brain. For example, recent advances in DNA sequencing technology should provide a means to directly test whether L1 DNA copy number changes detected in quantitative PCR experiments represent actual de novo endogenous retrotransposition events or result from other forms of genomic instability reported in neurons^{186, 187}. Similarly, it remains unclear whether endogenous L1 retrotransposition events represent a type of “genomic noise” or whether they have any functional impact on neuronal development. Finally, it remains a mystery why neuronal cells may accommodate L1 retrotransposition at apparently higher levels than other somatic cells. Nonetheless, these studies have unveiled a new area of investigation that surely will be the subject of future work.

Deregulated L1 retrotransposition in cancer cells

A growing body of evidence suggests that L1 retrotransposition may become deregulated in certain cancers. For example, early studies revealed that hypomethylation of the L1 promoter is correlated with increased L1 expression and/or the production of the L1 ORF1-encoded protein in certain tumors^188–190. Moreover, engineered human L1s readily retrotranspose in a variety of transformed human and mouse cell lines, but generally show lower levels of retrotransposition activity in “normal” human cells such as fibroblasts^{(e.g.,11, 191, 192)}. Consistent with this, recent findings using second-generation DNA sequencing revealed a total of 9 de novo L1 retrotransposition events in 6 of 20 examined non-small cell lung tumors¹². Intriguingly, the tumors containing the new L1 insertions also exhibited a specific genome-wide hypomethylation signature, which is consistent with the notion that altering the epigenome can create a permissive environment for L1 expression and/or retrotransposition, and perhaps the retrotransposition of other classes of non-LTR retrotransposons. Clearly, further innovations in DNA sequencing of heterogeneous cell populations will be critical to reveal patterns of TE activity in diverse tumors. The challenge then will be to determine whether all these TE insertions are “passenger” mutations that are a consequence of the altered cellular milieu of cancer cells or whether some act as “drivers” to promote tumorigenesis.

Closing Remarks

It is undeniable that TEs have played important roles in structuring genomes and generating genetic diversity. By understanding how, when, and where TEs integrate, and how the host responds to this ever-present threat, we will unveil the dynamic forces that shape our genomes. Indeed, we are now able to critically evaluate the McClintock doctrine and future experiments should allow valuable insight into whether the increases in TE transcription caused by environmental stress lead to higher levels of TE integration, and whether these insertions impact host phenotypes and/or survival.

It remains a curiosity why sequences without any apparent purpose continue to thrive in genomes. What is clear is that an understanding of TE biology is necessary to understand genome biology. It is intriguing to speculate that some phenotypic differences among organisms and/or between individuals are due to the effects of TEs. These speculations require rigorous experimental tests. However, the coming years should be an exciting time for TE biology.

At a glance summary

Many TEs employ highly specific mechanisms to direct integration to sites in the host genome that lack coding information. This minimizes damage to the host genome that occurs during integration.
High throughput sequencing allows the identification of a saturated map of targeted integration sites in S. pombe.
Host organisms have evolved a diverse array of mechanisms to combat TE activity. Examples include DNA methylation of TEs, siRNA based degradation of TE mRNA, and APOBEC mediated cytosine deamination of TE sequences.
Studies of diverse human populations revealed significantly higher numbers of active L1 elements than exist in the human genome reference sequence.
Recent experiments unexpectedly discovered TE integration in somatic cells. These include insertions of L1 in non-small cell lung tumors. Several lines of evidence suggest that somatic L1 retrotransposition may also occur in the mammalian nervous system.

Acknowledgements

We thank Dr. John Kim, Dr. José Garcia-Perez, and members of the Moran lab for critical reading of the manuscript. H.L. was supported in part by the Intramural Research Program of the NIH from the Eunice Kennedy Shriver National Institute of Child Health and Human Development. H.L. received additional support from the Intramural AIDS Targeted Antiviral Program. J.V.M. was supported in part by grants from the National Institutes of Health (GM060518 and GM082970). J.V.M. also is an Investigator of the Howard Hughes Medical Institute.

Glossary


LTR	A terminal repeated sequence present at the ends of LTR-retrotransposons. The LTR contains cis-acting sequences that allow the transcription and polyadenylation of retrotransposon mRNA. The LTRs also play critical roles in the reverse transcription of LTR-retrotransposon mRNA.

Virus-like particles (VLPs)	A cytoplasmic particle that comprises LTR-retrotransposon mRNA, the LTR-retrotransposon-encoded proteins, and host factors that are required for reverse transcription of LTR-retrotransposon mRNA. LTR-retrotransposon mRNA is reverse transcribed into a double stranded cDNA within VLPs.

Long Interspersed Element (LINE)	A family of autonomous non-LTR retrotransposons that mobilize by retrotransposition.

Short Interspersed Elements	A family of non-autonomous retrotransposons that require functional protein(s) encoded by LINE elements to mediate their retrotransposition.

LINE-1 or L1	An abundant family of autonomous non-LTR retrotransposons in mammalian genomes. In humans, L1 elements comprise roughly 17% of genomic DNA. The vast majority of L1s are inactive; however, it is estimated that an average human genome contains ~80–100 active elements (reviewed in ref. ⁵).

Alu elements (Alus)	An abundant class of SINEs that comprise ~10% of human genomic DNA. Alu elements require the endonuclease and reverse transcriptase activities contained within the L1 ORF2-encoded protein to mediate their mobility. Some Alu elements remain active in the human genome.

SINE-R—VNTR—Alu (SVA) elements	A composite non-autonomous retrotransposon that also requires the L1 encoded proteins to mediate its mobility. SVA elements are less abundant than Alu elements, although certain families of SVA elements remain active in the human genome.

Target-site primed reverse transcription (TPRT)	The mechanism of mobility generally employed by LINEs and SINEs. An endonuclease encoded by the LINE nicks genomic DNA to expose a 3’ hydroxyl residue at the target site that can be used as a primer to initiate the reverse transcription of the retrotransposon RNA by a LINE-encoded reverse transcriptase.

RNA-directed DNA methylation	A pathway in which 24 nt small RNAs interact with a de novo methyltransferase to mediate the methylation and transcriptional silencing of homologous genomic loci in plants.

Dicer	A family of RNAse III proteins that possess an endonuclease activity that can process double strand “trigger” RNAs into small-interfering RNAs (siRNAs) and microRNAs (miRNAs).

Small interfering RNAs (siRNAs)	A family of small (~21–24 nt) RNAs that are generated from double-strand “trigger” RNAs by Dicer-dependent and Dicer-independent mechanisms. These small RNAs bind Argonaute proteins and guide the resultant complex to complementary mRNAs in a cell to mediate their post-transcriptional destruction.

Argonaute proteins	The proteins that mediate the small RNA-induced silencing processes. Argonaute proteins bind small RNAs and are the defining component of the RNA Induced Silencing Complex (RISC). The small RNAs guide Argonaute proteins to target mRNAs to mediate post-transcriptional degradation and/or translational silencing. Argonaute proteins possess a single strand RNA binding domain (PAZ) and a ribonuclease domain (Piwi).

Piwi proteins	A specialized class of Argonaute proteins that interact with piRNAs to mediate transposable element silencing. Members include: Piwi, Aubergine, and Argonaute 3 in Drosophila; MIWI1, MIWI2 and MILI in mice, and HIWI1, HIW12, HIWI3, and HILI in humans.

Piwi-interacting RNAs (piRNA) cluster	A genomic DNA locus that encodes piRNA precursor RNAs. Many piRNA clusters contain sense and anti-sense sequences derived from mobile genetic elements. An example of a piRNA cluster is the Flamenco locus of Drosophila.

Piwi-interacting RNAs (piRNAs)	A family of small (~24–35 nt) RNAs that are processed from piRNA precursor mRNAs. The mature piRNAs interact with specialized Argonaute proteins (from the Piwi clade), to mediate RNA silencing.

Hybrid dysgenesis	A syndrome, which includes sterility, induced by the mobilization of P elements in crosses between females lacking P elements (the M cytotype) and males containing P elements (the P cytotype).

X-linked choroideremia	A recessive degenerative retinal disease.

Desmoid tumor	A soft tissue tumor that can arise in the abdomen as well as other parts of the body. Desmoid tumors generally are benign and grow slowly.

Aicardi-Goutieres Syndrome	A rare, autosomal recessive genetic disorder that leads to brain dysfunction as well as other symptoms. The early onset form of the disease can be caused by mutations in the Trex1 gene and is usually fatal.

Biographies

•

Henry L. Levin heads the Section on Eukaryotic Transposable Elements in the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH. He received his Ph.D. in molecular biology at the University of California, Berkeley with Dr. Howard Schachman and completed postdoctoral research at Johns Hopkins University School of Medicine with Dr. Jef Boeke. Over the past 18 years Levin’s studies of LTR retrotransposons in fission yeast have identified mechanistic details of particle formation, reverse transcription, and integration. Recently, work in the Levin lab has focused on the mechanisms that control the position of integration.

•

John V. Moran currently is the Gilbert S. Omenn Collegiate Professor of Human Genetics at the University of Michigan Medical School in Ann Arbor, Michigan, USA. He also is an Investigator of the Howard Hughes Medical Institute. He received his Ph.D. in biochemistry from the University of Texas Southwestern Medical Center in Dallas, Texas, USA, and conducted his postdoctoral studies in the laboratory of Dr. Haig H. Kazazian Jr. at Johns Hopkins University School of Medicine in Baltimore, MD, USA and at the University of Pennsylvania School of Medicine in Philadelphia, PA, USA. His laboratory uses genetic, molecular biological, biochemical, and genomic approaches to understand the biology of human LINE-1 retrotransposons.

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