23.2: DNA Transposable Elements

Search Fundamentals of Biochemistry

Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Classification and Mechanisms of Transposable Elements

• Distinguish between Class I retrotransposons (which transpose via an RNA intermediate using a "copy-and-paste" mechanism involving reverse transcription and reintegration, thereby increasing copy number) and Class II DNA transposons (which use a "cut-and-paste" conservative mechanism involving excision and reinsertion without an RNA intermediate, generally without net copy number increase unless excision gaps are repaired using a sister chromatid).
• Compare the major orders of Class I retrotransposons—LTR retrotransposons (structurally similar to retroviruses, encoding gag and pol; ~8% of the human genome), LINEs (autonomous, encode their own RT and endonuclease; L1 comprises ~21% of the human genome with ~500,000 copies), and SINEs (nonautonomous, derived from tRNA/7SL RNA genes, mobilized by L1 machinery; Alu alone exceeds one million copies)—and explain why 99.9% of L1 copies are no longer mobile due to accumulated mutations and truncations, with only ~100 active elements per person.
• Explain the structural features that distinguish major Class II transposon families: describe how TIR order elements are flanked by terminal inverted repeats (TIRs) and encode a transposase that mediates cut-and-paste mobility, generating target site duplications (TSDs) of characteristic length; contrast with Helitrons (rolling-circle replication, tyrosine recombinase, no TSD formation) and Mavericks (large elements encoding DNA polymerase B and integrase, single-strand excision followed by extrachromosomal replication and reintegration).

Genome Impact: Mutagenesis, Rearrangements, and Host Defense

• Explain how TE insertions within introns, exons, and regulatory regions cause gene inactivation or altered expression, and describe how TE-mediated genome rearrangements arise through homologous recombination between dispersed copies of related TE sequences causing large-scale deletions, duplications, and inversions—and through microhomology-mediated template switching during replication repair—citing evidence that >120 independent TE insertions are associated with human disease, with de novo germline transposition rates of approximately 1 in 21 births for Alu and 1 in 95 for L1.
• Describe the multilayered host defense mechanisms against TE activity—including DNA methylation silencing of TE promoters, piRNA-mediated TE repression in the germline, and KRAB zinc-finger protein sequence-specific repression—and explain the evolutionary tension arising from the fact that genome-wide DNA methylation erasure (required to reset genomic imprinting in primordial germ cells) simultaneously provides a window of opportunity for TE expression, and how plants exploit a companion cell strategy to allow TE activity in a meiotic product that does not contribute genetic material to the next generation.
• Explain how TE-derived proteins, RNA transcripts, and extrachromosomal DNA copies cause harm beyond transposition itself: describe how L1 ORF2p endonuclease activity induces DNA double-strand breaks; how cytosolic TE-derived DNA and RNA:DNA hybrids trigger innate immune responses (illustrated by Aicardi-Goutières syndrome); and how endogenous retroviral Env protein overexpression has been linked to neurodegenerative diseases, including multiple sclerosis and ALS.

TE Domestication, Exaptation, and Contributions to Genome Function

• Describe three major examples of TE domestication in which transposon-derived sequences have been co-opted for essential host functions: the derivation of RAG1/RAG2 recombinases (mediating V(D)J recombination in adaptive immunity) from an ancestral DNA transposon ~500 million years ago; the repeated independent capture of ERV env genes as syncytins that mediate syncytiotrophoblast formation in placental mammals; and the domestication of a retroviral gag gene as the neuronal Arc protein involved in synaptic plasticity and memory, which retains its ancestral RNA-packaging activity for trans-synaptic RNA delivery.
• Explain how TEs contribute to cis-regulatory network evolution by dispersing promoters, enhancers, transcription factor binding sites, and insulator sequences throughout the genome—illustrating with examples such as the agouti coat color gene (regulated by methylation of an upstream TE) and TE sequences that have "wired" batteries of co-regulated genes in networks controlling pregnancy, stem cell pluripotency, innate immunity, and stress responses—and distinguish between biochemical activity (such as transcription factor binding) and functional significance requiring evidence of purifying selection or loss-of-function phenotypes.

Introduction

Eukaryotic genomes contain a substantial amount of repeated DNA, and some of these repeated sequences are capable of moving. Transposable elements (TEs) are defined as DNA sequences that can move from one location to another in the genome. TEs have been identified in all organisms, prokaryotic and eukaryotic, and can occupy a high proportion of a species’ genome. For example, transposable elements comprise approximately 10% of the genomes of several fish species, 12% of the C. elegans genome, 37% of the mouse genome, 45% of the human genome, and up to more than 80% of the genomes of some plants, such as maize. From bacteria to humans, transposable elements have accumulated over time and continue to shape genomes through their mobilization.

TEs were discovered by Barbara McClintock during experiments conducted in 1944 on maize. Since they appeared to influence phenotypic traits, she named them controlling elements. However, her discovery was met with a less-than-enthusiastic reception by the genetic community. Her presentation at the 1951 Cold Spring Harbor Symposium was not well understood and was not very well received. She had no better luck with her follow-up publications, and after several years of frustration, decided not to publish on the subject for the next two decades. Not for the first time in the history of science, an unappreciated discovery was brought back to life after another discovery had been made. In this case, it was the discovery of insertion sequences (IS) in bacteria by the Szybalski group in the early 1970s. In the original paper, they wrote: “Genetic elements were found in higher organisms which appear to be readily transposed from one to another site in the genome. Such elements, identifiable by their controlling functions, were described by McClintock in maize. They might be somehow analogous to the presently studied IS insertions”. The importance of McClintock’s original work was eventually appreciated by the genetic community with numerous awards, including 14 honorary doctoral degrees and a Nobel Prize in 1983 “for her discovery of mobile genetic elements. Her picture is shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): Barbara McClintock (1902-1992). The photo was taken in her lab at the Department of Genetics, Carnegie Institution at Cold Spring Harbor, New York. This photo was taken when McClintock received the American Association of University Women Achievement Award in 1947 for her work on cytogenetics. She received the Nobel Prize in Physiology or Medicine in 1983 for her discovery of mobile genetic elements. Image from: Smithsonian Institution

The mobilization of TEs is termed transposition or retrotransposition, depending on the nature of the intermediate used. TE activity can positively and negatively impact a genome; for example, TE mobilization can promote gene inactivation, modulate gene expression, or induce illegitimate recombination. Thus, TEs have played a significant role in genome evolution. For example, DNA transposons can inactivate or alter gene expression by insertion into introns, exons, or regulatory regions. In addition, TEs can participate in genome reorganization by mobilizing non-transposon DNA or by acting as substrates for recombination. This recombination occurs through homology between two transposon sequences located on the same or different chromosomes, which may be the origin of various types of chromosomal alterations. Indeed, TEs can contribute to the loss of genomic DNA through internal deletions or other mechanisms.

The reduction in host fitness due to transposition ultimately affects the transposon, since host survival is critical to its perpetuation. Therefore, strategies have evolved in host and transposable elements to minimize the deleterious impact of transposition and to reach equilibrium. For example, some transposons tend to insert in non-essential regions of the genome, such as heterochromatic regions, where insertions are likely to have a minimal deleterious impact. In addition, they might be active in the germ line or embryonic stage, where most deleterious mutations can be selected against during fecundation or development, allowing only non-deleterious or mildly deleterious insertions to pass to successive generations. New insertions may also occur within an existing genomic insertion, generating an inactive transposon, or may undergo self-regulation via overproduction-inhibition. On the other hand, host organisms have developed various mechanisms of defense against high rates of transposon activity, including DNA methylation to reduce TE expression, several RNA interference-mediated mechanisms, primarily in the germ line, and the inactivation of transposon activity by specific proteins.

In some cases, transposable elements have been “domesticated” by the host to perform a specific function in the cell. A well-known example is the RAG protein family, which participates in V(D)J recombination during antibody class switching and is highly similar to DNA transposons, from which these proteins appear to be derived. Another example is the centromeric protein CENP-B, which seems to have originated from the pogo-like transposon. The analogous human mariner Himar1 element has been incorporated into the SETMAR gene, which encodes the histone H3 methylase and the Himar1 transposase domain. This gene is involved in the non-homologous end-joining pathway of DNA repair and has been shown to confer resistance to ionizing radiation. From a genome-wide perspective, approximately 25% of human promoter regions and 4% of human exons contain sequences derived from transposable elements (TEs). Thus, we are likely underestimating the rate of domestication events in mammalian genomes.

The first TE classification system was proposed by Finnegan in 1989, distinguishing two classes of TEs based on their transposition intermediates: RNA (class I, retrotransposons) or DNA (class II, DNA transposons). The transposition mechanism of class I is commonly called “copy and paste,” and that of class II, “cut and paste.” In 2007, Wicker et al. proposed a hierarchical classification based on the structural characteristics and mode of replication of TEs, as shown in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): Structures of eukaryotic mobile elements. See text for detailed discussion Image from Makalowski, W, et. al. (2019)

Class I: Mobile Elements

As mentioned above, class I transposable elements (TEs) transpose through an RNA intermediary. The RNA intermediate is transcribed from genomic DNA and then reverse-transcribed into DNA by a TE-encoded reverse transcriptase (RT), followed by reintegration into a genome. Each replication cycle produces one new copy, and as a result, class I elements are the major contributors to the repetitive fraction in large genomes. Retrotransposons are divided into five orders: LTR retrotransposons, DIRS-like elements, Penelope-like elements (PLEs), LINEs (long interspersed elements), and SINEs (short interspersed elements). This scheme is based on the mechanistic features, organization, and phylogeny of the reverse transcriptase of these retroelements. Accidentally, the retrotranscriptase encoded by an autonomous transposable element (TE) can reverse-transcribe another RNA present in the cell, such as mRNA, producing a retrocopy that, in most cases, results in a pseudogene.

The LTR retrotransposons are characterized by the presence of long terminal repeats (LTRs) ranging from several hundred to several thousand base pairs. Both exogenous retroviruses and LTR retrotransposons contain a gag gene that encodes the viral particle coat, and a pol gene that encodes reverse transcriptase, ribonuclease H, and integrase, which provide the enzymatic machinery for reverse transcription and integration into the host genome. Reverse transcription occurs within the viral or viral-like particle (GAG) in the cytoplasm, and it is a multi-step process. Unlike LTR retrotransposons, exogenous retroviruses contain an env gene that encodes an envelope protein that facilitates their entry into other cells. Some LTR retrotransposons may contain remnants of an env gene, but their insertion capabilities are limited to the genome from which they originated. This would suggest that they originated from exogenous retroviruses that lost the env gene. However, evidence suggests the contrary, as LTR retrotransposons can acquire the env gene and become infectious entities. Currently, most long terminal repeat (LTR) sequences (85%) in the human genome are found only as isolated LTRs, with the internal sequence likely lost due to homologous recombination between flanking LTRs. Interestingly, LTR retrotransposons target their reinsertion to specific genomic sites, often around genes, with putative important functional implications for a host gene. It is estimated that 450,000 LTR copies comprise approximately 8% of our genome. LTR retrotransposons inhabiting large genomes, such as maize, wheat, or barley, can contain thousands of families. However, despite the diversity, very few families comprise most of the repetitive fraction in these large genomes. Notable examples are Angela (wheat), BARE1 (barley), Opie (maize), and Retrosor6 (sorghum).

The DIRS order clusters structurally diverged groups of transposons that possess a tyrosine recombinase (YR) gene instead of an integrase (INT) and do not form target site duplications (TSDs). Their termini resemble either split direct repeats (SDR) or inverted repeats. Such features indicate a different integration mechanism than that of other class I mobile elements. DIRS were discovered in the genome of the slime mold (Dictyostelium discoideum) in the early 1980s and are present in all major phylogenetic lineages, including vertebrates. They have also been shown to be common in hydrothermal vent organisms.

Another order, termed Penelope-like elements (PLE), has a wide, though patchy, distribution, ranging from amoebae and fungi to vertebrates, with copy numbers of up to thousands per genome. Interestingly, no PLE sequences have been found in mammalian genomes, and it appears that they were lost from the C. elegans genome. Although PLEs with an intact open reading frame (ORF) have been identified in several genomes, including Ciona and Danio, the only transcriptionally active representative, Penelope, is known from Drosophila virilis. It causes the hybrid dysgenesis syndrome, characterized by the simultaneous mobilization of several unrelated transposable element families in the progeny of dysgenic crosses. Penelope appears to have invaded D. virilis relatively recently, and its invasive potential has been demonstrated in D. melanogaster. PLEs harbor a single ORF that codes for a protein containing reverse transcriptase (RT) and endonuclease (EN) domains. The PLE RT domain more closely resembles telomerase than the RT from LTRs or LINEs. The EN domain is related to GIY-YIG intron-encoded endonucleases. Some PLE members also have LTR-like sequences that can be in either direct or inverted orientation and contain a functional intron.

LINEs do not have LTRs; however, they have a poly-A tail at the 3′ ends and are flanked by the TSDs. They comprise about 21% of the human genome, and among them, L1, with about 850,000 copies, is the most abundant and best-described LINE family. L1 is the only LINE retrotransposon still active in the human genome. In the human genome, there are two other LINE-like repeats, L2 and L3, distantly related to L1. A contrasting situation has been observed in the malaria mosquito Anopheles gambiae, where approximately 100 divergent LINE families comprise only 3% of its genome. LINEs in plants, such as Cin4 in maize and Ta11 in Arabidopsis thaliana, appear to be rare compared to LTR retrotransposons. A full copy of mammalian L1 is about 6 kb long and contains a PolII promoter and two ORFs. The ORF1 codes for a non-sequence-specific RNA-binding protein that contains zinc finger, leucine zipper, and coiled-coil motifs. ORF1p functions as a chaperone for the L1 mRNA. The second ORF encodes an endonuclease, which makes a single-stranded nick in the genomic DNA, and a reverse transcriptase, which uses the nicked DNA to prime reverse transcription of LINE RNA from the 3′ end. Reverse transcription is often unfinished, leaving behind fragmented copies of LINE elements; hence, most of the L1-derived repeats are short, with an average size of 900 bp. LINEs are part of the CR1 clade, which has members in various metazoan species, including fruit flies, mosquitoes, zebrafish, pufferfish, turtles, and chickens. Because they encode their own retrotransposition machinery, LINE elements are regarded as autonomous retrotransposons.

SINEs evolved from RNA genes, such as 7SL and tRNA genes. By definition, they are short (up to 1000 base pairs). They do not encode their own retrotranscription machinery and are considered nonautonomous elements; in most cases, they are mobilized by the L1 machinery. The outstanding member of this class in the human genome is the Alu repeat, which contains a cleavage site for the AluI restriction enzyme, hence its name. With over a million copies in the human genome, Alu is probably the most successful transposon in the history of life. Primate-specific Alu and its rodent relative, B1, have a limited phylogenetic distribution, suggesting their relatively recent origins. The mammalian-wide interspersed repeats (MIRs), by contrast, spread before eutherian radiation, and their copies can be found in different mammalian groups, including marsupials and monotremes. SVA elements are unique primate elements due to their composite structure. They are named after their main components: SINE, VNTR (a variable number of tandem repeats), and Alu. Typically, they exhibit the hallmarks of retroposition, i.e., they are flanked by TSDs and terminated by a poly(A) tail. SVA elements appear to be non-autonomous retrotransposons mobilized by the L1 machinery and are believed to be transcribed by RNA polymerase II. SVAs are transpositionally active and are responsible for some human diseases. They originated less than 25 million years ago and form the youngest retrotransposon family, with approximately 3,000 copies in the human genome.

Retro(pseudo)genes are a special group of retroposed sequences, which are products of reverse transcription of a spliced (mature) mRNA. Hence, their characteristic features are an absence of promoter sequence and introns, the presence of flanking direct repeats, and a 3′-end polyadenosine tract. Processed pseudogenes, also known as retropseudogenes, have been generated in vitro at a low frequency in human HeLa cells using mRNA from a reporter gene. The reverse transcription machinery in humans and other vertebrates appears to be active in L1 elements. However, not all retroposed messages have to end up as pseudogenes. About 20% of mammalian protein-encoding genes lack introns in their ORFs. It is conceivable that many genes lacking introns arose by retroposition. Some genes are known to be more frequently retroposed than others. For instance, in the human genome, there are over 2000 retropseudogenes of ribosomal proteins. A genome-wide study revealed that the human genome contains approximately 20,000 pseudogenes, 72% of which are likely to have arisen through retroposition. Interestingly, the vast majority (92%) of these are quite recent transpositions that occurred after the divergence of primates and rodents. Some retroposed genes may follow quite complex evolutionary paths. An example is the RNF13B retrogene, which replaced its parental gene in mammalian genomes. This retrocopy was duplicated in primates, and the evolution of this primate-specific copy was accompanied by the exaptation of two TEs, Alu and L1, and by intron gain via the reclassification of part of the coding sequence as an intron, leading to the origin of a functional, primate-specific retrogene with two splicing variants.

Class II: Mobile Elements

Class II elements move by a conservative cut-and-paste mechanism; the excision of the donor element is followed by its reinsertion elsewhere in the genome. DNA transposons are abundant in bacteria, where they are called insertion sequences, but are also present in all phyla. Two subclasses of DNA transposons have been distinguished, based on the number of DNA strands that are cut during transposition.

Classical “cut-and-paste” transposons belong to subclass I, and they are classified as the TIR order. They are characterized by terminal inverted repeats (TIRs) and encode a transposase that binds near the inverted repeats, mediating mobility. This process is not typically replicative, unless the gap caused by excision is repaired using the sister chromatid. When inserted at a new location, the transposon is flanked by short gaps that, when filled by host enzymes, cause duplication of the sequence at the target site. The length of these TSDs is characteristic of particular transposons. Nine superfamilies belong to the TIR order, including Tc1-Mariner, Merlin, Mutator, and PiggyBac. The second-order Crypton consists of a single superfamily of the same name. Originally thought to be limited to fungi, it is now clear that they are widespread, including in animals and heterokonts. A heterogeneous, small, nonautonomous group of elements, MITEs, also belongs to the TIR order, which in some genomes is amplified to thousands of copies, e.g., Stowaway in the rice genome, Tourist in most bamboo genomes, or Galluhop in the chicken genome.

Subclass II includes two orders of TEs that, just as those from subclass I, do not form RNA intermediates. However, unlike “classical” DNA transposons, they replicate without double-strand cleavage. Helitrons replicate using a rolling-circle mechanism, and their insertion does not result in duplication of the target site. They encode tyrosine recombinase along with some other proteins. Helitrons were first described in plants, but they are also present in other phyla, including fungi and mammals. Mavericks are large transposons found across different eukaryotic lineages, excluding plants. They encode various proteins, including DNA polymerase B and an integrase. Kapitonov and Jurka suggested that their life cycle includes a single-strand excision, followed by extrachromosomal replication and reintegration to a new location.

TEs are not randomly distributed in the genome

As seen in the previous section, TEs are highly diverse and, in principle, every TE sequence in a genome can be affiliated with a (sub)family, superfamily, subclass, and class. This is summarized in Figure \(\PageIndex{3}\). However, much like the taxonomy of species, the classification of TEs is in constant flux, perpetually subject to revision due to the discovery of novel TE types, the introduction of new levels of granularity in classification, and the ongoing development of methods and criteria for detecting and classifying TEs.

Figure \(\PageIndex{3}\): Classification of Eukaryotic Transposable Elements. This schematic shows the key features and relationships between TE classes, subclasses, superfamilies, and families. Blue circles represent TE-encoded enzymes. circDNA circular DNA intermediate, DIRS Dictyostelium repetitive sequence, dsDNA linear double-stranded DNA intermediate, EN endonuclease, IN integrase, PLEs Penelope-like elements, HUH, Rep/Helicase protein with HUH endonuclease activity, RT reverse transcriptase, TP transposase, TPRT target primed reverse transcription, YR tyrosine recombinase. Image from: Bourque, G., et. al. (2018) Genetic Biology 19:199

The genome can be viewed as an ecosystem inhabited by diverse communities of transposable elements (TEs), which seek to propagate and multiply through sophisticated interactions among themselves and with other cellular components. These interactions encompass processes familiar to ecologists, such as parasitism, cooperation, and competition. Thus, it is perhaps not surprising that TEs are rarely, if ever, randomly distributed in the genome, as shown in Figure \(\PageIndex{4}\). TEs exhibit varying degrees of preference for insertion into specific features or compartments of the genome. These are often guided by opposing selective forces, a balancing act that facilitates future propagation while mitigating deleterious effects on host cell function. At the end of the site-selection spectrum, many elements have evolved mechanisms to target specific loci where their insertions are less detrimental to the host but favorable for their propagation. For instance, several retrotransposons in species as diverse as slime mold, budding yeast, and fission yeast have evolved independently but convergently to target the upstream regions of genes transcribed by RNA polymerase III, where they do not appear to affect host gene expression but retain the ability to be transcribed themselves.

Figure \(\PageIndex{4}\): Ten things you should know about transposable elements (TEs). Examples of how TEs can affect genomes directly and indirectly. Blue boxes represent TEs, gray boxes represent canonical exons, and the black box represents a sequencing read. Right-angled arrows represent genes or TE promoters. Image from: Bourque, G., et. al. (2018) Genetic Biology 19:199

TEs are an extensive source of mutations and genetic polymorphisms

TEs occupy a substantial portion of the genome of a species, including a large fraction of the DNA unique to that species. In maize, where Barbara McClintock did her seminal work, an astonishing 60 to 70% of the genome is comprised of LTR retrotransposons, many of which are unique to this species or its close wild relatives. Still, the less prevalent DNA transposons are currently the most active and mutagenic (Fig. 24.2.4). Similarly, the vast majority of TE insertions in Drosophila melanogaster are absent at the orthologous site in its closest relative D. simulans (and vice versa). Most are not fixed in the population. Many TE families are still actively transposing, and the process is highly mutagenic; more than half of all known phenotypic mutants of D. melanogaster isolated in the laboratory are caused by spontaneous insertions of a wide variety of TEs. Transposition events are also common and mutagenic in laboratory mice, where the ongoing activity of several families of LTR elements is responsible for 10–15% of all inherited mutant phenotypes. This contribution of TEs to genetic diversity may be underestimated, as TEs can be more active when organisms are under stress, such as in their natural environment.

Because TE insertions rarely provide an immediate fitness advantage to their host, those that reach fixation in the population do so largely through genetic drift and are subsequently eroded by point mutations that accumulate neutrally. Over time, these mutations result in transposable elements (TEs) that can no longer encode transposition enzymes and therefore produce new integration events. For instance, our haploid genome contains approximately 500,000 L1 copies, but more than 99.9% of these L1 copies are fixed and no longer mobile due to various forms of mutations and truncations. It is estimated that each person carries approximately 100 active L1 elements, most of which are young insertions still segregating within the human population. Thus, like any other organism, the ‘reference’ human genome sequence does not represent a comprehensive inventory of transposable elements (TEs) in humans. Thousands of ‘non-reference’, unfixed TE insertions have been cataloged through whole-genome sequencing and other targeted approaches. On average, any two human haploid genomes differ by approximately 1,000 transposable element (TE) insertions, primarily from the L1 or Alu families. The number of TE insertion polymorphisms in a species with much higher TE activity, such as maize, dwarfs the number in humans.

If TEs bring no immediate benefit to their host and are largely decaying neutrally once inserted, how do they persist in evolution? One key to this conundrum is the ability of TEs not only to propagate vertically but also horizontally between individuals and species. There is now a substantial body of evidence supporting the idea that horizontal transposon transfer is a common phenomenon that affects virtually every major type of transposable element (TE) and all branches of the tree of life. While the cellular mechanisms underlying horizontal transposon transfer remain murky, it is increasingly apparent that the intrinsic mobility of TEs and ecological interactions between their host species, including those with pathogens and parasites, facilitate the transmission of elements between widely diverged taxa.

TEs are ass ociated with genome rearrangements and unique chromosome features

Transposition is a potent mechanism of genome expansion that, over time, is counteracted by DNA removal via deletion. The balance between the two processes is a major driver in the evolution of genome size in eukaryotes. Several studies have demonstrated the impact and range of this shuffling and cycling of genomic content on the evolution of plant and animal genomes. Because the insertion and removal of TEs are often imprecise, these processes can indirectly affect surrounding host sequences. Some of these events occur at a high enough frequency to result in extensive duplication and reshuffling of host sequences, including genes and regulatory elements. For example, a single group of DNA transposons, known as MULEs, has been responsible for capturing and reshuffling approximately 1,000 gene fragments in the rice genome. Such studies have led to the conclusion that the rate at which transposable elements (TEs) transpose, which is in part under host control, is a significant driver of genome evolution.

In addition to rearrangements induced as a byproduct of transposition, TEs can promote genomic structural variation long after they have lost the capacity to mobilize. In particular, recombination events can occur between the highly homologous regions dispersed by related TEs at distant genomic positions and result in large-scale deletions, duplications, and inversions (Fig. 24.2.4). TEs also provide regions of microhomology that predispose to template switching during repair of replication errors, leading to another source of structural variants. These non-transposition-based mechanisms for TE-induced or TE-enabled structural variation have contributed substantially to genome evolution. These processes can also complicate the identification of actively transposing elements in population studies that infer their existence by detecting non-reference insertions.

TEs also contribute to the specialized features of chromosomes. An intriguing example is found in Drosophila, where LINE-like retrotransposons form and maintain telomeres in place of the telomerase enzyme, which was lost during dipteran evolution. This domestication event could be viewed as a replay of what may have occurred much earlier in eukaryotic evolution to solve the ‘end problem’ posed by the linearization of chromosomes. Indeed, the reverse transcriptase component of telomerase is thought to have originated from an ancient lineage of retroelements. TE sequences and domesticated transposase genes also play structural roles at centromeres.

There is an intricate balance between TE expression and repression

To persist in evolution, TEs must strike a delicate balance between expression and repression (Fig. 24.2.4). Expression should be sufficient to promote amplification, but not so vigorous as to lead to a fitness disadvantage for the host that would offset the benefit to the TE of increased copy numbers. This balancing act may explain why TE-encoded enzymes are naturally suboptimal for transposition and why some transposable elements (TEs) have evolved self-regulatory mechanisms that control their copy numbers. A variety of host factors are also employed to control TE expression, including small RNAs, chromatin and DNA modification pathways, and sequence-specific repressors such as the recently profiled KRAB zinc-finger proteins. However, many of these silencing mechanisms must be at least partially released to permit the developmental regulation of host gene expression programs, particularly during early embryonic development. For example, genome-wide loss of DNA methylation is necessary to reset imprinted genes in primordial germ cells. This affords TEs an opportunity, as reduced DNA methylation often promotes their expression. Robust expression of a transposable element (TE) in the germ lineage (but not necessarily in the gametes themselves) is often its own downfall. In one example of a clever trick employed by the host, TE repression is relieved in a companion cell derived from the same meiotic product as flowering plant sperm. However, this companion cell does not contribute genetic material to the next generation. Thus, although TEs transpose in a meiotic product, the events are not inherited. Instead, TE activity in the companion cell may further dampen TE activity in sperm via the import of TE-derived small RNAs.

Another important consequence of the intrinsic expression/repression balance is that the effects of TEs on a host can vary considerably among tissue types and stages of an organism’s life cycle. From the TE’s perspective, an ideal scenario is to be expressed and active in the germline, but not in the soma, where expression would gain the TE no advantage, only disadvantages. This phenomenon is indeed observed among many species, with ciliates representing an extreme example of this division—TEs are actively deleted from the somatic macronucleus but retained in the micronucleus, which serves as the germline. Another example is the P-elements in Drosophila, which are differentially spliced in the germline versus soma. Many organisms, including plants, do not differentiate germline cells early in development; rather, they are specified from somatic cells shortly before meiosis commences. Thus, TEs that transpose in somatic cells of plants have the potential to be inherited, suggesting that the interests of TEs and hosts conflict in many more cells and tissues than in animals with a segregated germline.

TEs are insertional mutagens in both the germline and soma

Like other species, humans contend with a contingent of currently active transposable elements (TEs), in which the intrinsic balance between expression and repression remains at play. For us, this includes L1 and other mobile elements that depend on L1-encoded proteins for retrotransposition. These elements are responsible for new germline insertions that can lead to genetic diseases. More than 120 independent TE insertions have been associated with human disease. The rate of de novo germline transposition in humans is approximately one in 21 births for Alu and one in 95 births for L1.

Historically, little attention has been given to transposition in somatic cells and its consequences, as somatic transposition may be viewed as an evolutionary dead end for the transposable element (TE) with no long-term consequences for the host species. Yet, there is abundant evidence that TEs are active in somatic cells in many organisms (Fig. 24.2.4). In humans, L1 expression and transposition have been detected in various somatic contexts, including early embryos and specific stem cells. There is also considerable interest in the expression and activity of mobile elements in the mammalian brain, where L1 transposition has been proposed to diversify neuronal cell populations. One challenge in assessing somatic activity has been the development of reliable single-cell insertion site mapping strategies.

Somatic activity has also been observed in human cancers, where tumors can acquire hundreds of new L1 insertions. As for human polymorphisms, somatic activity in human cancers is driven by a small number of so-called ‘hot’ L1 loci. The activities of these master copies vary depending on the individual, tumor type, and the tumor's clonal evolution timeframe. Some of these de novo L1 insertions disrupt critical tumor suppressors and oncogenes and thus drive cancer formation, although the vast majority appear to be ‘passenger’ mutations. Host cells have evolved several mechanisms to regulate transposable elements (TEs). However, as the force of natural selection diminishes with age and drops to zero in post-reproductive life, TEs may become more active.

TEs can be damaging in ways that do not involve transposition

TEs are best known for their mobility and their ability to transpose to new locations. While the breakage and insertion of DNA associated with transposition represent an obvious source of cell damage, this is not the only, nor perhaps even the most common, mechanism by which TEs can harm their host. Reactivated transposons harm the host in multiple ways. First, derepression of transposon loci, including their own transcription, may interfere with transcription or processing of host mRNAs through a myriad of mechanisms. Genome-wide transcriptional derepression of TEs has been documented during replicative senescence of human cells and several mouse tissues, including the liver, muscle, and brain. De-repression of LTR and L1 promoters can also cause oncogene activation in cancer. Second, TE-encoded proteins, such as the endonuclease activity of L1 ORF2p, can induce DNA breaks and genomic instability. Third, accumulation of RNA transcripts and extrachromosomal DNA copies derived from TEs may trigger an innate immune response, leading to autoimmune diseases and sterile inflammation (Fig. 24.2.4). Activation of the interferon response is now a well-documented property of transcripts derived from endogenous retroviruses and may give immunotherapies a boost in identifying and attacking cancer cells. The relative contributions of the above mechanisms to organismal pathologies remain to be determined.

Following transcription (and sometimes splicing) of TEs, the next step in the process involves the translation of the encoded proteins and, for retroelements, reverse transcription of the TEs into cDNA substrates suitable for transposition. Once engaged by a TE-encoded reverse transcriptase protein, the resulting cytosolic DNAs and RNA:DNA hybrids can activate inflammatory pathways. An example of this is seen in patients with Aicardi–Goutières syndrome, in whom the accumulation of TE-derived cytosolic DNA results from mutations in pathways that normally block TE processing or degrade TE-derived DNA. Although not all TEs encode functional proteins, some do, including a few endogenous retroviruses that can produce Gag, Pol, or envelope (Env) proteins. Overexpression of these Env proteins can be cytotoxic and has been linked to at least two neurodegenerative diseases, multiple sclerosis and amyotrophic lateral sclerosis. Small accessory proteins produced by the youngest human endogenous retrovirus (HERV) group, HERV-K (HML-2), may play a role in some cancers, but the evidence remains circumstantial.

Key coding and non-coding RNAs are derived from TEs

Although usually detrimental, there is growing evidence that TE insertions can provide the raw material for the emergence of protein-coding genes and non-coding RNAs, which can take on important and, in some cases, essential cellular function (Fig. 24.2.4). The process of TE gene ‘domestication’ or exaptation over evolutionary time contributes to both deeply conserved functions and more recent, species-specific traits. Most often, the ancestral or a somewhat modified role of a TE-encoded gene is harnessed by the host and conserved, while the rest of the TE sequence, and hence its ability to autonomously transpose, has been lost. Spectacular examples of deeply conserved TE-derived genes are Rag1 and Rag2, which catalyze V(D)J somatic recombination in the vertebrate immune system. Both genes, and probably the DNA signals they recognize, were derived from an ancestral DNA transposon around 500 million years ago. Indeed, DNA transposases have been co-opted multiple times to form new cellular genes.

The gag and env genes of LTR retrotransposons or endogenous retroviruses (ERVs) have been domesticated numerous times to perform various functions in placental development, contribute to host defense against exogenous retroviruses, influence brain development, and play other diverse roles. One of the most intriguing examples of TE domestication is the repeated, independent capture of ERV env genes, termed syncytins, which appear to function in placentation by facilitating cell–cell fusion and the formation of syncytiotrophoblasts. Notably, one or more syncytin genes have been found in virtually every placental mammalian lineage where they have been sought, strongly suggesting that ERVs have played essential roles in the evolution and extreme phenotypic variability of the mammalian placenta. Another example of viral-like activity repurposed for host-cell function is the neuronal Arc gene, which arose from the gag gene of an LTR retrotransposon domesticated in the common ancestor of tetrapod vertebrates. Genetic and biochemical studies of murine Arc show that it is involved in memory and synaptic plasticity and has preserved most of the ancestral activities of Gag, including the packaging and intercellular trafficking of its own RNA. Remarkably, flies appear to have independently evolved a similar system of trans-synaptic RNA delivery involving a gag-like protein derived from a similar yet distinct lineage of LTR retrotransposons. Thus, the biochemical activities of TE-derived proteins have been repeatedly co-opted during evolution to foster the emergence of convergent cellular innovations in different organisms.

TEs can donate their own genes to the host, but they can also add exons, rearrange, and duplicate existing host genes. In humans, intronic Alu elements are particularly prone to being captured as alternative exons through cryptic splice sites residing within their sequences. L1 and SVA (SINE/VNTR/Alu) elements also contribute to exon shuffling through transduction events of adjacent host sequences during their mobilization. The reverse transcriptase activity of retroelements is also responsible for the trans-duplication of cellular mRNAs to create ‘processed’ retrogenes in a wide range of organisms. The L1 enzymatic machinery is thought to generate tens of thousands of retrogene copies in mammalian genomes, many of which remain transcribed and some of which have acquired new cellular functions. This process is still actively shaping our genomes; it has been estimated that one in every 6,000 humans carries a novel retrogene insertion.

TEs also make substantial contributions to the cell's non-protein-coding functions. They are major components of thousands of long non-coding RNAs in human and mouse genomes, often transcriptionally driven by retroviral long terminal repeats (LTRs). Some of these TE-driven lncRNAs appear to play crucial roles in maintaining stem cell pluripotency and other developmental processes. Numerous studies have shown that TE sequences embedded within lncRNAs and mRNAs can directly modulate RNA stability, processing, or localization, with significant regulatory consequences. Furthermore, TE-derived microRNAs and other small RNAs processed from transposable elements (TEs) can also adopt regulatory roles, serving host cell functions. The myriad mechanisms by which TEs contribute to the generation of coding and non-coding RNAs illustrate the multifaceted interactions between these elements and their hosts.

TEs contribute cis-regulatory DNA elements and modify transcriptional networks

Cis-regulatory networks coordinate the transcription of multiple genes that function in concert to orchestrate entire pathways and complex biological processes. In line with Barbara McClintock’s insightful predictions, there is now mounting evidence that TEs have been a rich source of material for the modulation of eukaryotic gene expression (Fig. 24.2.4). Indeed, TEs can disperse vast amounts of promoters and enhancers, transcription factor binding sites, insulator sequences, and repressive elements. The varying coat colors of agouti mice provide a striking example of a host gene controlling coat color whose expression can be altered by the methylation levels of a transposable element (TE) upstream of its promoter. In oil palm, the methylation level of a transposable element (TE) located within a gene important for flowering ultimately controls whether the plants bear oil-rich fruit.

As TE families typically populate a genome with multiple related copies, it has long been postulated that they can donate the same cis-regulatory module to ‘wire’ batteries of genes dispersed throughout the genome. An increasing number of studies support this model, suggesting that TEs have provided the building blocks for the assembly and remodeling of cis-regulatory networks during evolution. These networks underlie processes as diverse as pregnancy, stem cell pluripotency, neocortex development, innate immunity in mammals, and the response to abiotic stress in maize. Indeed, TE sequences harbor all the necessary features of a ‘classical’ gene regulatory network. They are bound by diverse sets of transcription factors that integrate multiple inputs (activation/repression), respond to signals in both cis and trans, and can coordinately regulate gene expression. In this context, TEs are highly suitable agents to modify biological processes by creating novel cis-regulatory circuits and fine-tuning pre-existing networks.

Outlook

As potent insertional mutagens, TEs can have both positive and negative effects on host fitness. Still, it is likely that the majority of TE copies in any given species—and especially those in species such as humans with small effective population sizes—have reached fixation through genetic drift alone and are now largely neutral to their host. When can we say that TEs have been co-opted for cellular function? The publication of the initial ENCODE paper, which asserted ‘function for 80% of the genome’, was the subject of much debate and controversy. Technically speaking, ENCODE assigned only ‘biochemical’ activity to this large fraction of the genome. Yet critics objected to the grand proclamations in the popular press (The Washington Post Headline: “Junk DNA concept debunked by new analysis of the human genome”) and to the ENCODE consortium’s failure to prevent this misinterpretation. To these critics, ignoring evolutionary definitions of function was a major misstep.

This debate can be easily extended to include TEs. TEs make up the vast majority of what is often referred to as ‘junk DNA’. Today, the term is mainly used—and abused—by the media, but it has deep roots in evolutionary biology. Regardless of the semantics, what evidence is needed to assign a TE a function? Many TEs encode a wide range of biochemical activities that normally benefit their own propagation. For example, TEs often contain promoter or enhancer elements that hijack cellular RNA polymerases for transcription, and autonomous elements encode proteins with various biochemical and enzymatic activities, all of which are necessary for the transposon to replicate. Do these activities make them functional?

The vast differences in TEs between species make standard approaches to establishing their regulatory roles particularly challenging. For example, intriguing studies on the impact of HERVs, particularly HERV-H, in stem cells and pluripotency must be interpreted using novel paradigms that do not invoke deep evolutionary conservation to imply function, as these particular ERVs are absent outside of the great apes. Evolutionary constraints can be measured at shorter time scales, including the population level, but this remains a statistically challenging task, especially for non-coding sequences. Natural loss-of-function alleles may exist in the human population, and their effects on fitness can be studied when their impact is apparent; however, such alleles are quite rare and do not allow for systematic studies. It is possible to engineer genetic knockouts of a particular human transposable element (TE) locus to test its regulatory role; however, such approaches are typically restricted to in vitro systems, especially when the orthologous TE is absent from the model species. In this context, studying the impact of TEs in model species with powerful genome-engineering tools and vast collections of mutants and other genetic resources, such as plants, fungi, and insects, will continue to be extremely valuable.

Finally, a growing consensus calls for greater rigor in assigning cellular functions to TEs, particularly with respect to the host's fitness benefit. Indeed, a TE displaying biochemical activity (such as those bound by transcription factors or lying within open chromatin regions) cannot be equated to a TE that shows evidence of purifying selection at the sequence level or, when genetically altered, results in a deleterious or dysfunctional phenotype. Recent advances in editing and manipulating the genome and the epigenome en masse, yet with precision, including repetitive elements, offer the promise of a systematic assessment of the functional significance of TEs.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic

Transposable elements (TEs) are mobile DNA sequences capable of moving from one genomic location to another. Discovered by Barbara McClintock in maize in 1944 and initially dismissed, their importance was recognized after the parallel discovery of bacterial insertion sequences in the 1970s; McClintock received the Nobel Prize in 1983. TEs have accumulated in all sequenced genomes, comprising ~12% of the C. elegans genome, ~37% of the mouse genome, ~45% of the human genome, and over 80% of some plant genomes such as maize, making them major architectural components of eukaryotic genomes.

TEs are classified into two major classes based on their transposition intermediates. Class I retrotransposons transpose via an RNA intermediate: genomic DNA is transcribed, reverse-transcribed into cDNA by a TE-encoded reverse transcriptase, and reintegrated—a "copy-and-paste" mechanism that increases copy number with each cycle. The major Class I orders include LTR retrotransposons (structurally similar to retroviruses, encoding gag and pol genes, comprising ~8% of the human genome with ~450,000 copies; exogenous retroviruses differ by additionally carrying an env gene enabling cell-to-cell transmission), LINEs (autonomous elements encoding their own reverse transcriptase and endonuclease; L1 is the only currently active LINE in humans, with ~500,000 total copies of which 99.9% are inactivated by accumulated mutations, leaving approximately 100 active copies per person), and SINEs (nonautonomous elements derived from tRNA and 7SL RNA genes, mobilized by L1 machinery; the Alu element with over one million copies in the human genome is the most abundant TE in evolutionary history). SVA elements, composite primate-specific retrotransposons containing SINE, VNTR, and Alu components, represent the youngest human retrotransposon family (~3,000 copies). Class II DNA transposons use a conservative "cut-and-paste" mechanism without an RNA intermediate, excising from one location and reinserting elsewhere. The TIR order—including the superfamilies Tc1-Mariner, PiggyBac, and MITEs—is characterized by terminal inverted repeats and transposase-mediated mobility, thereby generating target-site duplications. Helitrons replicate by a rolling-circle mechanism without TSD formation, while Mavericks undergo single-strand excision followed by extrachromosomal replication.

The genomic impact of TEs is profound and multifaceted. Active transposition is mutagenic: insertions within coding or regulatory sequences disrupt gene function, and more than 120 TE insertions are associated with human genetic disease. De novo germline transposition rates in humans are approximately 1 in 21 births for Alu and 1 in 95 for L1, and somatic L1 activity in human cancers can generate hundreds of new insertions per tumor. Beyond direct mutagenesis, TEs promote large-scale genome rearrangements through homologous recombination between dispersed copies of related TE sequences, generating deletions, duplications, and inversions long after the elements have lost the capacity to transpose. TE-derived proteins and nucleic acids also cause harm through non-transposition mechanisms: L1 ORF2p endonuclease activity induces DNA breaks; cytosolic TE-derived DNA and RNA:DNA hybrids trigger innate immune responses (as in Aicardi-Goutières syndrome); and endogenous retroviral Env proteins have been linked to multiple sclerosis and ALS.

Host genomes have evolved multiple defense mechanisms against excessive TE activity, including DNA methylation silencing of TE promoters, piRNA-mediated repression in the germline, and KRAB zinc-finger protein sequence-specific silencing. These mechanisms must be at least partially released during developmental reprogramming events such as genome-wide DNA methylation erasure in primordial germ cells, creating windows of TE vulnerability. TEs have evolved to respond by preferentially transposing in the germline, where new insertions can be inherited, and by targeting insertions to genomic regions where effects on host fitness are minimized.

Counterbalancing these costs, TEs have made extraordinary positive contributions to genome function through domestication and exaptation. The RAG1/RAG2 recombinases mediating V(D)J recombination in vertebrate adaptive immunity were derived from an ancestral DNA transposon ~500 million years ago. ERV env genes have been independently captured as syncytins in virtually every placental mammalian lineage, where they mediate syncytiotrophoblast formation and placental development. The neuronal Arc gene—derived from a retroviral gag gene domesticated in the tetrapod ancestor—retains its ancestral RNA-packaging activity for intercellular RNA trafficking underlying synaptic plasticity and memory. TEs have also seeded transcriptional regulatory networks by dispersing promoters, enhancers, transcription factor binding sites, and insulator elements throughout the genome, thereby contributing to the coordinated regulation of gene batteries that control pregnancy, stem cell pluripotency, innate immunity, and developmental programs. Approximately 25% of human promoter regions and 4% of human exons contain TE-derived sequences—likely an underestimate of the true extent of TE domestication. The critical challenge for the field is distinguishing between TEs displaying mere biochemical activity (such as transcription factor binding or open chromatin) and those genuinely under purifying selection with demonstrable functional roles, a distinction that will increasingly be addressed through precision genome- and epigenome-editing technologies.

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References

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