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24.2: DNA Transposable Elements

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    15190
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    Eukaryotic genomes contain an abundance of repeated DNA, and some repeated sequences are mobile. Transposable elements (TEs) are defined as DNA sequences that are able to 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 several fish species, 12 % of the C. elegans genome, 37% of the mouse genome, 45% of the human genome, and up to >80% of the genome of some plants like 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 less than enthusiastic reception by the genetic community. Her presentation at the 1951 Cold Spring Harbor Symposium was not understood and at least 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 some other discovery has been made. In this case it was the discovery of insertion sequences (IS) in bacteria by 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. It is possible that 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”.

    McClintock.png
    Figure 24.2.1: Barbara McClintock (1902-1992). Photo was taken in her lab within 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 for mobilization. There are several ways in which the activity of TEs 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 the expression of genes by insertion within introns, exons or regulatory regions. In addition, TEs can participate in the reorganization of a genome by the mobilization of non-transposon DNA or by acting as recombination substrates. This recombination would occur by homology between two sequences of a transposon located in the same or different chromosomes, which could be the origin for several types of chromosome alterations. Indeed, TEs can participate in the loss of genomic DNA by internal deletions or other mechanisms.

    The reduction in fitness suffered by the host due to transposition ultimately affects the transposon, since host survival is critical to perpetuation of the transposon. Therefore, strategies have been developed by host and transposable elements to minimize the deleterious impact of transposition, and to reach equilibrium. For example, some transposons tend to insert in nonessential regions in the genome, such as heterochromatic regions, where insertions will likely 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 to generate an inactive transposon, or can undergo self-regulation by overproduction-inhibition. On the other hand, host organisms have developed different mechanisms of defense against high rates of transposon activity, including DNA-methylation to reduce TE expression, several RNA interference mediated mechanisms, mainly in the germ line, or through the inactivation of transposon activity by the action of 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 are RAG proteins, which participate in V(D)J recombination during antibody class switching, and exhibit a high similarity to DNA transposons, from which these proteins appear 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 consists of the histone H3 methylase gene 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 view, it has been estimated that ~25% of human promoter regions and ~4% of human exons contain sequences derived from 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 and distinguished two classes of TEs characterized by their transposition intermediate: RNA (class I or retrotransposons) or DNA (class II or 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 hierarchical classification based on TEs structural characteristics and mode of replication (Fig. 24.2.2).

    TE.png
    Figure 24.2.2 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 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 reverse transcriptase phylogeny of these retroelements. Accidentally, the retrotranscriptase coded by an autonomous TE can reverse-transcribe another RNA present in the cell, e.g., mRNA, and produce a retrocopy of it, which 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 a viral particle coat and a pol gene that encodes a reverse transcriptase, ribonuclease H, and an 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 multistep process. Unlike LTR retrotransposons, exogenous retroviruses contain an env gene, which encodes an envelope that facilitates their migration to other cells. Some LTR retrotransposons may contain remnants of an env gene, but their insertion capabilities are limited to the originating genome. This would rather suggest that they originated in exogenous retroviruses by losing the env gene. However, there is evidence that suggests the contrary, given that LTR retrotransposons can acquire the env gene and become infectious entities. Presently, most of the LTR sequences (85%) in the human genome are found only as isolated LTRs, with the internal sequence being lost most likely 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 make up about 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 group 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 slime mold (Dictyostelium discoideum) genome in the early 1980s, and they are resent in all major phylogenetic lineages including vertebrates. It has been showed that they are also common in hydrothermal vent organisms.

    Another order, termed Penelope-like elements (PLE), has wide, though patchy distribution from amoebae and fungi to vertebrates with copy number up to thousands per genome. Interestingly, no PLE sequences have been found in mammalian genomes, and apparently they were lost from the genome of C. elegans. Although PLEs with an intact ORF have been found 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 simultaneous mobilization of several unrelated TE families in the progeny of dysgenic crosses. It seems that Penelope invaded D. virilis quite recently, and its invasive potential was 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, which can be in a direct or an inverse orientation, and have a functional intron.

    LINEs do not have LTRs; however, they have a poly-A tail at the 3′ end 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 retroposon 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 noticed in the malaria mosquito Anopheles gambiae, where around 100 divergent LINE families compose only 3% of its genome. LINEs in plants, e.g., Cin4 in maize and Ta11 in Arabidopsis thaliana, seem rare as compared with 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. The ORF1p functions as 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 fly, mosquito, zebrafish, pufferfish, turtle, and chicken. 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 pair long. They do not encode their own retrotranscription machinery and are considered as nonautonomous elements and in most cases are mobilized by the L1 machinery. The outstanding member of this class from the human genome is the Alu repeat, which contains a cleavage site for the AluI restriction enzyme that gave 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 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. Usually, they contain the hallmarks of the retroposition, i.e., they are flanked by TSDs and terminated by a poly(A) tail. It seems that SVA elements are nonautonomous retrotransposons mobilized by L1 machinery, and they are thought 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 they form the youngest retrotransposon family with about 3000 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, as sometimes retropseudogenes are called, have been generated in vitro at a low frequency in the human HeLa cells via mRNA from a reporter gene. The source of the reverse transcription machinery in humans and other vertebrates seems to be active 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 retroposed more often than others. For instance, in the human genome there are over 2000 retropseudogenes of ribosomal proteins. A genome-wide study showed that the human genome harbors about 20,000 pseudogenes, 72% of which most likely arose through retroposition. Interestingly, the vast majority (92%) of them are quite recent transpositions that occurred after primate/rodent divergence. Some of the retroposed genes may undergo quite complicated evolutionary paths. An example could be the RNF13B retrogene, which replaced its own parental gene in the 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 intron gain via changing a part of coding sequence into 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 the subclass I, and they are classified as the TIR order. They are characterized by terminal inverted repeats (TIR) and encode a transposase that binds near the inverted repeats and mediates mobility. This process is not usually a replicative one, unless the gap caused by excision is repaired using the sister chromatid. When inserted at a new location, the transposon is flanked by small gaps, which, when filled by host enzymes, cause duplication of the sequence at the target site. The length of these TSDs is characteristic for 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, now it is clear that they have a wide distribution, including animals and heterokonts. A heterogeneous, small, nonautonomous group of elements MITEs also belong to the TIR order, which in some genomes 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 the target site duplication. 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 that have been found in different eukaryotic lineages excluding plants. They encode various numbers of proteins that include 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 to a (sub)family, superfamily, subclass, and class. This is summarized in Figure 24.2.3. However, much like the taxonomy of species, the classification of TEs is in constant flux, perpetually subject to revision due to the discovery of completely novel TE types, the introduction of new levels of granularity in the classification, and ongoing development of methods and criteria to detect and classify TEs.

    Figure 3.png
    Figure 24.2.3 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 may be viewed as an ecosystem inhabited by diverse communities of TEs, which seek to propagate and multiply through sophisticated interactions with each other and with other components of the cell. 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 (Figure 24.2.4). TEs exhibit various levels of preference for insertion within certain features or compartments of the genome. These are often guided by opposite selective forces, a balancing act of facilitating future propagation while mitigating deleterious effects on host cell function. At the most extreme 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 and budding and fission yeast have evolved independently, but convergently, the capacity 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 4.pngFigure 24.2.4 Ten things you should know about transposable elements (TEs). Examples of how TEs can impact genomes in direct and indirect ways. Blue boxes represent TEs, gray boxes represent canonical exons, and the black box represents a sequencing read. Right-angled arrows represent gene 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, but 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), and 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 ongoing activity of several families of LTR elements are 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 reaching fixation in the population do so largely by genetic drift and are subsequently eroded by point mutations that accumulate neutrally. Over time, these mutations result in TEs that can no longer encode transposition enzymes and produce new integration events. For instance, our (haploid) genome contains ~ 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 a set of ~ 100 active L1 elements, and most of these are young insertions still segregating within the human population. Thus, as for any other organism, the ‘reference’ human genome sequence does not represent a comprehensive inventory of TEs in humans. Thousands of ‘non-reference’, unfixed TE insertions have been catalogued through whole genome sequencing and other targeted approaches. On average, any two human haploid genomes differ by approximately a thousand 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 large body of evidence supporting the idea that horizontal transposon transfer is a common phenomenon that affects virtually every major type of 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.

    <h3sec4">TEs are associated with genome rearrangements and unique chromosome feature

    Transposition represents a potent mechanism of genome expansion that over time is counteracted by the removal of DNA 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 is often imprecise, these processes can indirectly affect surrounding host sequences. Some of these events occur at high enough frequency to result in vast amounts of duplication and reshuffling of host sequences, including genes and regulatory sequences. For example, a single group of DNA transposons (MULEs) has been responsible for the capture and reshuffling of ~ 1,000 gene fragments in the rice genome. Such studies have led to the conclusion that the rate at which TEs transpose, which is in part under host control, is an important 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 make the identification of actively transposing elements more difficult in population studies that infer the existence of active elements through the detection of non-reference insertions.

    TEs also contribute to specialized chromosome features. An intriguing example is in Drosophila, where LINE-like retrotransposons form and maintain the telomeres in replacement of the telomerase enzyme which has been lost during dipteran evolution. This domestication event could be viewed as a replay of what might have happened much earlier in eukaryotic evolution to solve the ‘end problem’ created 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.

    <h3sec5">There is an intrinsic 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 TEs have evolved self-regulatory mechanisms controlling their own copy numbers. A variety of host factors are also employed to control TE expression, which includes a variety of small RNA, chromatin, and DNA modification pathways, as well as 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 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 TE expression. Robust expression of a 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 disadvantage. This 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, or 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 germ lineage cells early in development; rather, they are specified from somatic cells shortly before meiosis commences. Thus, TEs that transpose in somatic cells in plants have the potential to be inherited, which suggests that the interest of TEs and host are in conflict across many more cells and tissues than in animals with a segregated germline.

    <h3sec6">TEs are insertional mutagens in both germline and soma

    Like other species, humans contend with a contingent of currently active TEs where the intrinsic balance between expression and repression is still 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 cause genetic disease. 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, because somatic transposition may be viewed as an evolutionary dead-end for the 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 a variety of somatic contexts, including early embryos and certain stem cells. There is also a great deal of interest in mobile element expression and activity in the mammalian brain, where L1 transposition has been proposed to diversify neuronal cell populations. One challenge for assessing somatic activity has rested with 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. Just like for human polymorphisms, somatic activity in human cancers is caused by small numbers of so-called ‘hot’ L1 loci. The activities of these master copies varies depending on the individual, tumor type, and timeframe in the clonal evolution of the tumor. 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 keep TEs in check. However, as the force of natural selection begins to diminish with age and completely drops in post-reproductive life, TEs may become more active.

    <h3sec7">TEs can be damaging in ways that do not involve transposition

    TEs are best known for their mobility, in other words their ability to transpose to new locations. While the breakage and insertion of DNA associated with transposition represents an obvious source of cell damage, this is not the only or perhaps even the most common mechanism by which TEs can be harmful to their host. Reactivated transposons harm the host in multiple ways. First, de-repression of transposon loci, including their own transcription, may interfere with transcription or processing of host mRNAs through a myriad of mechanisms. Genome-wide transcriptional de-repression of TEs has been documented during replicative senescence of human cells and several mouse tissues, including 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 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 contribution of all the above mechanisms in organismal pathologies remains to be determined.

    Following transcription (and sometimes splicing) of TEs, the next step in the process involves 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 alert inflammatory pathways. An example of this is seen in patients with Aicardi–Goutières syndrome, where accumulation of TE-derived cytosolic DNA is due to 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 capable of producing 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 amytrophic 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.

    <h3sec8">A number of key coding and non-coding RNAs are derived from TEs

    Although usually detrimental, there is growing evidence that TE insertions can provide 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, that 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 also been domesticated numerous times to perform functions in placental development, contribute to host defense against exogenous retroviruses, act in 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 syncytiotrophoblast formation. Notably, one or more such 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 a viral-like activity re-purposed for host cell function is provided by the neuronal Arc gene, which arose from the gag gene from a 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 and rearrange and duplicate existing host genes. In humans, intronic Alu elements are particularly prone to be 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 be involved in the generation of tens of thousands of retrogene copies in mammalian genomes, many of which remain transcribed and some of which have acquired new cellular functions. This is a process still actively shaping our genomes; it has been estimated that 1 in every 6000 humans carries a novel retrogene insertion.

    TEs also make substantial contributions to non-protein coding functions of the cell. They are major components of thousands of long non-coding RNAs in human and mouse genomes, often transcriptionally driven by retroviral LTRs. Some of these TE-driven lncRNAs appear to play important roles in the maintenance of stem cell pluripotency and other developmental processes. Many studies have demonstrated that TE sequences embedded within lncRNAs and mRNAs can directly modulate RNA stability, processing, or localization with important regulatory consequences. Furthermore, TE-derived microRNAs and other small RNAs processed from TEs can also adopt regulatory roles serving host cell functions. The myriad of mechanisms by which TEs contribute to coding and non-coding RNAs illustrate the multi-faceted interactions between these elements and their host.

    <h3sec9">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 provides a striking example of a host gene controlling coat color whose expression can be altered by the methylation levels of a TE upstream of its promoter. In the oil palm, the methylation level of a TE that sits within a gene important for flowering ultimately controls whether or not the plants bear oil-rich fruit.

    As TE families typically populate a genome as a multitude of related copies, it has long been postulated that they have the potential to donate the same cis-regulatory module to ‘wire’ batteries of genes dispersed throughout the genome. An increasing number of studies support this model and suggest that TEs have provided the building blocks for the assembly and remodeling of cis-regulatory networks during evolution, including pathways underlying processes as diverse as pregnancy, stem cell pluripotency, neocortex development, innate immunity in mammals, or 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 integrate multiple inputs (activation/repression), respond to signals in both cis and trans, and are capable of co-ordinately regulating 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.

    <h3sec11">Outlook

    As potent insertional mutagens, TEs can have both positive and negative effects on host fitness, but it is likely that the majority of TE copies in any given species—and especially those such as humans with small effective population size—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 mostly used (and abused) by the media, but it has in fact deep roots in evolutionary biology. Regardless of the semantics, what evidence is needed to assign a TE with 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 highjack 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 establish their regulatory roles particularly challenging. For example, intriguing studies on the impact of HERVs, in particular 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 great apes. Evolutionary constraint 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 effect on fitness can be studied if their impact is apparent, but these are quite rare and do not allow systematic studies. It is possible to engineer genetic knockouts of a particular human TE locus to test its regulatory role but those are restricted to in-vitro systems, especially when the orthologous TE does not exist in 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 also continue to be extremely valuable.

    Finally, a growing consensus is urging more rigor when assigning cellular function to TEs, particularly for the fitness benefit of the host. 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, result 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 for a systematic assessment of the functional significance of TEs.

    References

    1. Munoz-Lopez, M. and Garcia-Perez, J.L. (2010) DNA Transposons: Nature and Applications in Genomics. Curr Genomics 11(2):115-128. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2874221/

    2. Makalowski, W., Gotea, V., Pande, A. and Makalowska, I. (2019) Transposable Elements: Classification, Identification, and Their Use As a Tool For Comparative Genomics. In: Anisimova M. (eds) Evolutionary Genomics. Methods in Molecular Biology, vol 1910. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9074-0_6

    3. Bourque, G., Burns, K.H., Gehring, M., Borbunova, V., Seluanov, A., Hammell, M., Imbeault, M., Izvak, Z., Levin, H.L., Macfarlan, T.S., Mager, D.L., Feschotte, C. (2018) Ten things you should know about transposable elements. Genome Biology 19: 199. Available at: https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1577-z#Fig1


    24.2: DNA Transposable Elements is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski.