Activity and Silencing of Transposable Elements in C. elegans

1. Introduction

Transposable elements (TEs), also called “jumping genes”, are a widespread class of mobile genetic elements that can move and replicate within a genome. Almost all eukaryotic genomes contain TEs. TEs can make up large proportions of eukaryotic genomes: around half the human genome and about 15% of the C. elegans genome are transposon sequence. The proportion of TEs in the genome can differ vastly in organisms of similar evolutionary complexity, which largely explains the vast differences in genome size between such organisms.

In addition to having profound effects on genome size, TEs are mutagenic, cause genome instability and are targeted by silencing mechanisms. Because TEs insert into new sites in the genome, they may disrupt genes. When TEs excise, they can leave footprints or trigger deletion of flanking sequences as a result of double-stranded break repair. TEs can also promote recombination and chromosomal rearrangements because of their repetitive nature. All these types of sequence changes may cause disease. On the other hand, transposons drive genome evolution and may be beneficial in the adaptation to changed conditions. Regulation of transposon silencing mechanisms by environmental stresses would allow for transposon activity under abrupt environmental changes and therefore potentially mediate adaptation [4].

TEs also affect genomes through the chromatin-based host silencing mechanisms directed at transposons. TE silencing mechanisms include chromatin modification, repressive complexes, RNA-directed DNA methylation, RNA interference and sequence-specific transcription factors. Constitutive heterochromatin in the centromeric and pericentromeric regions of human chromosomes is highly enriched in repeat elements such as TEs [5]. Similarly, the distal arms of the autosomal chromosomes of the holocentric animal C. elegans are enriched for heterochromatic proteins and marks, and TEs [6,7,8]. In addition to silencing of (TE) transcription in heterochromatin, homologous recombination is also suppressed in heterochromatin. This lack of homologous recombination is thought to contribute to the vast numbers of repetitive sequences accumulating in heterochromatin as these elements cannot be eliminated by homologous recombination[9].

In addition to generating sequence variation that may disrupt genes, TEs also contribute to the creation of new genes. As a result of transposition into genes, host protein domains can fuse with transposon-encoded protein domains, which can lead to novel host functions. This process is called co-option or domestication. Transposons can also provide new regulatory sequences to host genes since TEs contain promoters, polyadenylation signals and TF binding sites.

New TEs can enter host genomes through horizontal gene transfer, the non-sexual movement of genetic information between genomes. Horizontal gene transfer of TEs leads to phylogenetic incongruence, a strong conflict between the phylogenies of the transposon and the organism. Horizontal gene transfer of TEs has been well documented in flies [10], and has also recently been demonstrated in large scale studies of vertebrate genomes [11].

The nematode C. elegans was chosen as an animal model system to study development and behavior by Sydney Brenner in 1965 [12]. C. elegans was the first multicellular organism for which the genome was completely sequenced and published, in 1998 [13]. Genetic studies in the 1980s identified natural isolates of C. elegans, including one from Bergerac (France) that in comparison to the “wild type” or reference isolate from Bristol (England), showed a repeated sequence that was polymorphic and displayed high rates of spontaneous mutations [14,15,16]. These observations had several implications for C. elegans research in general and for the understanding of transposon silencing in particular. Tc1 was identified as an active DNA transposon in C. elegans. Genetic analysis showed that mutator loci in the genome were responsible for the mutator phenotype in Bergerac, indicating the possible existence of a silencing mechanism the Bristol strain. A practical benefit of these discoveries was the development of transposon tagging using Tc1 to rapidly identify genes mutated in forward genetic screens, and the development of transposon insertion libraries that were used to find knock-out mutations in genes of interest.

2. Transposable Elements in the C. elegans Genome

Eukaryotic transposable elements are classified into two classes: Class I – retrotransposons and Class II – DNA transposons [17]. Retrotransposons transpose via an RNA intermediate that is reverse transcribed into a dsDNA intermediate which is integrated into the genome (Figure 1). The original copy remains at its genomic location, and therefore these TEs move through a “copy-and-paste” mechanism. This in contrast to DNA transposons that typically move through a “cut-and-paste” mechanism (Figure 1), although this does not apply to all types of DNA transposons. Typically, a DNA transposon is cut out by a transposase protein and integrates into another site in the genome. Within the two classes of TEs, transposons are further subdivided into subclasses and superfamilies based on mechanisms of replication and integration and phylogenetic relationships [18]. Finally, families of transposons are defined by DNA sequence similarity of 80% in at least 80% of the aligned sequence (either coding region or terminal repeats) with a minimum of 80 bp. Multiple transposon copies of one family can be aligned to generate a consensus sequence that represents the active ancestral TE. Many of the transposable elements present in the C. elegans genome are inactive because of mutations in the open reading frames of the transposon-encoded proteins required for mobilization [19], or because of mutations in the terminal inverted repeats (TIRs) or long terminal repeats (LTRs). However, since the transposon-encoded proteins can act in trans, a protein encoded by another element of the same family may be able to mobilize a transposon that no longer encodes the protein(s) required for transposon mobility. The most extreme example of this phenomenon is represented by miniature inverted-repeat elements (MITEs) that only consist of the inverted repeats of the parental transposon, but that can be mobilized in trans by a transposase protein encoded by a full length element [20]. These non-autonomous transposons are subfamilies of the full-length transposon family that harbors the transposon-mobility genes with which they share inverted repeat sequence that is recognized by the transposase.

The known active transposons in C. elegans are members of Class II DNA transposons of the Tc1/mariner superfamily, named after the C. elegans Tc1 transposon (Table I). The Tc1/mariner superfamily is the most widespread TE family among eukaryotes. Tc1 was identified in the 1980s as a repeated sequence that was polymorphic between two natural isolates of C. elegans [14,15,16]. This polymorphic sequence was shown to be mobile in studies of spontaneous mutations of two muscle genes that are reversible [21], and thus represented a transposon. Research into the underlying genetic loci that allow for mobilization of Tc1 in the germline [22], led to the discovery that RNA interference (RNAi) silences transposable elements [23,24]. We now know that RNAi is an evolutionarily deeply conserved mechanism to silence viruses and TEs.

Table I. Active transposons in the C. elegans genome.

Table I. Active transposons in the C. elegans genome.

Element	Class	Order	Superfamily	Family	Copy Num-ber	Length (bp)	Catal-ytic motif	IR / TIR length (bp)	Target site (duplication)
Cer1	Class I	LTR	Gypsy	Gypsy	1	8865	DDE	492
Tc1	Class II	TIR	Tc1/mariner	Tc1	32	1611	DD34E	54	TA (TA-TA)
Tc2	Class II	TIR	Tc1/mariner-Tc2/pogo group	Pogo	4	2074	DD35D	24	TA (TA-TA)
Tc3	Class II	TIR	Tc1/mariner	Tc1	22	2335	DD34E	462	TA (TA-TA)
Tc4/Tc4v	Class II	TIR	Tc1/mariner- Tc4 group	Tc4	10	1605/ 3483	DD37D	774	CTNAG (TNA-TNA)
Tc5	Class II	TIR	Tc1/mariner- Tc4 group	Tc4	4	3171	DD37D	491	CTNAG (TNA-TNA)
Tc7 (Tc1 MITE)	Class II	TIR	Tc1/mariner	Tc1	11	921	n/a	345	TA (TA-TA)

Shown are the transposons in the C. elegans genome that have been shown to be active, with the exception of the Cer1 retrotransposon, that generates VLPs but for which no new insertions have been found. Indicated are the classification of the transposons and their characteristics such as full length copy number, length of the transposon, the catalytic motif, length of the terminal repeats and the sequence of the target site and of the target site duplication after insertion.

The C. elegans reference strain widely used in the laboratory, Bristol, shows very little transposition activity in the germline, although transposition does occur in somatic cells [25]. However, in other isolates of C. elegans, such as Bergerac, transposons do jump in germline cells. These isolates can have highly increased copy numbers of transposons with up to 748 Tc1 elements in Bergerac [26]. The most active transposons identified in such strains as well as in strains with mutator mutations isolated in screens for transposon activation, are the related transposons Tc1 and Tc3 present in 32 and 22 copies respectively in the Bristol genome (Table I). Tc1/mariner transposons move via a cut-and-paste mechanism described below. Whereas germline transposition is suppressed in Bristol, Tc1 and Tc3 are active in the germlines of mutator mutants such as mut-2/rde-3 and mut-7 (described below) [15], and Tc1 but not Tc3 is specifically mobilized in the germline of Bergerac and other uncloned mutator mutants [27,28]. Tc7 is a non-autonomous element related to Tc1, that is active in somatic cells of mut-6 and mut-7 mutants [29]. Also active in mutator mutants mut-2/rde-3 and mut-7 are the related Tc1/mariner transposons Tc4/Tc4v and Tc5 [30,31,32]. Tc2 is a pogo transposon that is related to Tc1/mariner transposons but may form a separate superfamily [34,35]. Tc2 is not active in mutator strains but is active in strains derived from Bergerac and Bristol crosses.

Only for these six DNA transposons is there evidence of active transposition in C. elegans: new insertions have been found in Bristol mutants or in C. elegans natural isolates. Other than these six elements, the C. elegans genome contains 752 TEs from 224 families [2]. These TEs include numerous additional Class II elements including of the Tc1/mariner, helitron, mutator, CACTA and maverick superfamilies. Whereas the human genome contains a large number of Class I elements covering over 40% of the genome [36], the C. elegans genome contains relatively few Class I TEs. There are 20 full-length copies of LTR retrotransposons of the gypsy and bel-pao superfamilies [38,39,40], as well as two SINE elements and LINE elements (non-LTR retrotransposons) of the RTE [41] and R2 superfamilies. LTR retrotransposons structurally and mechanistically resemble retroviruses but lack the envelope proteins required for the extracellular of the retroviral life cycle. Some C. elegans LTR retrotransposons do encode for envelope proteins and could therefore be endogenous retroviruses (ERVs). Whereas LTR retrotransposons typically only encode the gag, pol and possibly env polyproteins, several C. elegans LTR retrotransposons encode for additional proteins [43,44].

Some of the 752 TEs may be active under conditions of environmental stress or in mutant backgrounds that have not been studied. However, only a limited number of transposon-encoded genes in the genome appear to have the potential to be mobile [19]. (Recent) mobility of some of these elements in other genetic backgrounds was deduced from studies of naturally occurring polymorphisms and genome sequencing of C. elegans natural isolates [2,45]. A well-studied example of this is the Gypsy superfamily LTR retrotransposon Cer1 (Table I). The Bristol strain has a single insertion of Cer1 in its genome, inactivating a mucin gene [46]. This insertion is absent in two thirds of the natural isolates. Half of these do have Cer1 elsewhere in their genome. Cer1 produces viral-like particles (VLPs) in the Bristol strain but no new insertions of Cer1 have been observed in this strain. In a large-scale genome sequencing study of transposons in C. elegans natural isolates, over 60% of TEs shared sites in all natural isolates, indicating that these are inactive. Some of the 241 TE sites that were not present in all natural isolates, may represent transposons that are active under specific conditions or genetic backgrounds.

3. Mechanisms of Transposition in C. elegans

Biochemical studies and sequence analysis elucidated the mechanism of transposition of Tc1 and Tc3 transposition [48], (Figure 1). Tc1 contains one gene, encoding the Tc1 transposase, flanked by terminal inverted repeats (TIRs). Transposase is only protein required for movement, and can even catalyze transposition in cell free assays, which likely accounts for the successful spread of Tc1/mariner elements between widely diverged taxa. The transposase protein contains a bipartite N-terminal DNA binding domain [50,51], and a C-terminal endonuclease domain that contains a catalytic triad of acidic residues DDE/D found in transposases and retroviral integrases (Table I).

The N-terminal domain of Tc1 transposase binds to specific sequences within the TIRs. The transposase hydrolyzes the phosphodiester bonds at the termini TIRs to cut the transposon out of its genomic location and form a synaptic complex. At the target site, which is always a TA dinucleotide, the transposase catalyzes a transesterification reaction to insert the transposon into the target site. The staggered cuts result in duplication of the TA target sequence. Host DNA double-stranded break repair mechanisms repair the transposon excision site via non-homologous end-joining (NHEJ), via homology-directed repair (HDR)(homologous recombination), or via single strand annealing (SSA) (Figure 1). Depending on the specific repair pathway, a short footprint is left at the excision site (NHEJ), a larger deletion around the excision site is created (SSA), or the site is repaired via homologous recombination using sister chromatid or other homologous copies, retaining the transposon at original location. The latter mechanism results in an increase in transposon copy number. Homologous recombination can also contribute to the rapid evolution of TEs by the creation of internally deleted transposon copies via strand slippage or template switching [53]. Which repair pathway is used is in part dependent on the tissue: repair by homologous recombination is most prevalent germ line of C. elegans, while NHEJ and SSA more active in somatic cells. However, all three pathways are used for repair after excision of Tc1 in the germline of C. elegans: sequence exchange between Tc1 elements demonstrated the use of HDR [54], whereas footprints found after Tc1 excisions are a consequence of NHEJ[55]. Finally, deletions of sequences flanking the Tc1 excision site are a consequence of SSA repair [56].

Although Tc1 and Tc3 insert into TA dinucleotides, the sequences immediately flanking the TA affect target choice [57,58,59]. On the level of the chromosomes, the Tc1 elements jump from and into all chromosomes, but reside in regions of repressed chromatin and are underrepresented in domains with highly active chromatin marks [26]. In general, DNA transposons are found on the autosomal chromosome arms of C. elegans [2,60]

LTR retrotransposons like Cer1 transpose via an RNA intermediate (Figure 1). After transcription, some of the retrotransposon transcripts are used for translation of the LTR retrotransposon gag and pol genes. The Gag proteins form virus-like particles (VLPs) that contain the pol proteins (protease, integrase and reverse transcriptase) and dimeric retrotransposon genomic RNA (gRNA) transcripts that interact with the Gag protein via CCHC Zn fingers in the nucleocapsid domain. In the VLPs, the RNA is reverse transcribed to form a double-stranded complementary DNA (cDNA) that forms a complex with the integrase. This pre-integration complex moves to a new genomic location and the integrase cleaves the genomic DNA via a mechanism similar to the related protein Tc1 transposase, and integrates the LTR retrotransposon DNA. Like DNA transposons, LTR retrotransposons are found on chromosome arms [40]. Although Cer1 produces VLPs, new insertions of Cer1 have not been identified in the reference strain, suggesting that Cer1 may not be active.

4. Silencing of Transposable Elements

The observation that Tc1 elements are active in the germline of the Bergerac isolate but not in the reference strain indicated that a silencing mechanism exists in the germline of the reference strain. Although the loci from the Bergerac strain that allow for Tc1 hopping have not been identified, genetic screens for mutator mutants in which Tc1 and/or Tc3 are desilenced in the reference strain have uncovered core factors in transposon silencing pathways. At the same time, similar screens for RNAi-defective mutants identified an overlapping set of genes indicative of the role of RNAi in silencing transposable elements.

In most animals, Piwi proteins together with Piwi-interacting RNAs (piRNAs) silence TEs in the germline. In C. elegans PIWI and piRNAs also act in transposon silencing and are required for silencing of Tc3 in the germ line (Figure 2) [61]. Most of the ~15,000 C. elegans piRNAs are transcribed from piRNA genes located in two large clusters on chromosome IV [61]. These clusters contain TEs that have inserted behind piRNA promoters. After processing of a piRNA precursor into a mature 21 nt RNA, the piRNA associates with the C. elegans Piwi protein PRG-1 (reviewed in [62]). The PRG-1-piRNA complex sequence-specifically recognizes its target (transposon) mRNA through base pairing in perinuclear P granules. Base pairing between piRNA and target mRNA accommodates multiple mismatches [63]. The large number of piRNAs in combination with such relaxed targeting allows for silencing of newly acquired transposons or rapidly diverging elements without a need to produce new small RNAs specific to the transposon [64]. Conversely, many non-transposon genes are targeted by piRNAs. To counteract silencing of self genes and to promote their expression in the germline, the CSR-1 Argonaute in association with endogenous siRNAs protects these self genes against piRNA-mediated silencing.

After target recognition by the piRNA, secondary siRNA generation is triggered in perinuclear phase-separated mutator foci (Figure 2). Loss of components of mutator foci such as the exonuclease MUT-7 and MUT-16, results in a loss of transposon silencing and the activation of transposons in the germline [66,67]. Secondary siRNA generation may be initiated by slicing or cleavage of the mRNA by the endonuclease RDE-8. The poly(UG)-polymerase RDE-3 (also known as MUT-2) adds a poly(UG) tail to the 3’end of the target mRNA fragment [68]. The pUG tail recruits an RNA-dependent polymerase (RdRP) that generates secondary siRNAs using the transposon RNA as a template [69]. Twelve distinct Argonaute (AGO) proteins can be loaded with these siRNAs: WAGO proteins that act in post-transcriptional gene silencing (PTGS) and promote continued siRNA generation by RDE-3 and the RdRP, and the nuclear Argonaute proteins HRDE-1 and NRDE-3 that in complex with siRNAs and other nuclear RNAi factors, direct transcriptional gene silencing (TGS) of the transposon. Transcriptional gene silencing is mediated by a direct inhibition of RNA polymerase II transcribing the target transposon, and by the deposition of repressive chromatin marks directed by the nuclear RNAi machinery. HRDE-1 induces heterochromatin formation and chromatin compaction [72,73]. Transcription within heterochromatin generates additional small RNAs that are loaded onto HRDE-1 to promote further small RNA amplification in mutator foci [72].

TEs and other repeats in C. elegans are enriched in regions with heterochromatin marks such as the presence of HPL-2, one of two C. elegans heterochromatin protein 1 orthologs, and histone H3K9me2 and H3K9me3 [8,60,74]. In addition to HPL-2, heterochromatin proteins MET-2/SETDB1, LIN-61, LIN-13 and LET-418/Mi-2 are enriched at repetitive elements and are required for transcriptional silencing of a subset of TEs [7]. Many of the TEs suppressed by the heterochromatin proteins are silenced by piRNAs [8].

The nuclear RNAi machinery including HRDE-1 mediates trans-generational silencing of transposons, that once established, does not require the piRNA trigger [75]. Recent reviews describe trans-generational RNAi in detail [76,77]. The heritable nature of RNAi-mediated transposon silencing allows for an immediate silencing response to invading TEs that have sequence similarity to previously encountered TEs, reminiscent of the adaptive immune system in human.

Despite the large number of piRNAs that are expressed and the promiscuity with which they target mRNAs, some transposons that are not silenced by piRNAs. For example, a set of non-conserved genes that includes several retrotransposons, is silenced through primary siRNAs that are loaded onto the Argonaute ERGO-1 triggering secondary siRNA generation and nuclear RNAi [78]. It is likely that other mechanisms of transposon recognition upstream of secondary siRNA generation exist.

One feature of transposon RNA that may be recognized by silencing pathways, is the inefficiency or absence of splicing of transposon RNA, because of poor splice sites, lack of introns or other suboptimal features of transposon pre-mRNAs [79]. The Tc1 transposase gene contains an intron but is only spliced in a small fraction of transcripts [80]. The retention of Tc1 transcripts on spliceosomes suggests that defects in splicing could trigger silencing [79]. Indeed, the presence of bona fide introns protects C. elegans genes against silencing [81]. EMB-4, a homolog of the intron-binding helicase Aquarius, interacts with HRDE-1 and initiates silencing of an overlapping set of TEs, but is only required to silence intron-containing target RNAs [82]. Thus, EMB-4 is important to overcome the protection against silencing of piRNA targets that do contain introns.

Another feature of most DNA transposons is the presence of TIRs that can form dsRNA structures. These structures are not only targets of the RNAi enzyme Dicer but can also be targeted by other dsRNA-binding and dsRNA-modifying proteins such as the dsRNA receptor RIG-I and the ADAR enzymes that edit dsRNA. There are no reports of a role of ADARs in the silencing of Tc1 and Tc3 transposons, but ADARs do act on small palindromic repeat elements such as Tc7 [43]. ADAR editing of these repeats competes with silencing of these dsRNAs by the RNAi pathway. It is unknown if and how such RNA editing would affect mobilization of these palindromic repeat elements.

An alternative way to silence the effects of transposable elements is to ensure that even when a transposon is inserted into a gene, excision of the transposable element does not inactivate the gene. A high proportion of Tc1 insertions into genes is silent. The silent nature of Tc1 insertions can be the result of frequent excision while leaving minimal footprints that retain the reading frame, or what has been observed, the frequent use of these footprints as alternative splice sites to restore the open reading frame [83]. Even if Tc1 is not excised from the DNA, Tc1 is spliced out in a high fraction of mRNAs [84].

5. Other Mechanisms of Transposon Regulation: Adaptation and Domestication

A proposed benefit for the host of the presence of TEs in the genome is the potential to adapt to unfavorable or changed conditions by desilencing of TEs to generate random mutations. In relation to transposon silencing in C. elegans, the most-studied stresses are growth at elevated temperatures and acute heat stress. The latter stress induces DNA damage and Tc1 mobilization in the male germline but not the female germ line. Tc3 is not mobilized by heat stress, again indicating that these transposons are differentially regulated [85]. Longer term exposure to elevated temperatures that are not lethal to wild type animals, results in sterility and elevated transposition rates in mutator mutants [66]. These reports show that heat stress does result in an increase in transposon mobilization. Many small RNA pathway mutants, including mutator mutants display a progressive decline in fertility, a mortal germline, over generations that for some mutants is dependent on elevated temperature. This sterility is not caused by accumulation of DNA damage due to an increase in TE mobilization but is more likely a consequence of a more broad mis-regulation of gene expression in the germline [86].

Evidence of the potentially beneficial nature of TEs to the host is found in the domestication or co-option of TEs. In C. elegans, the Cer1 Gag protein and VLPs act in the horizontal transfer of memory of pathogen avoidance to naïve animals [87]. Co-option of transposons as regulatory sequences in C. elegans is widespread: a third of the germ-line specific promoters have been co-opted from two miniature inverted-repeat transposable elements (MITEs) [88]. A protein with DNA-binding domains related to a transposase protein regulates these promoters. Whereas the transposons have become part of host genes in the case of the above-mentioned MITEs, transposons can also regulate host genes in their vicinity. Over half of the of Heat Shock Elements (HSEs) are found within Helitron DNA transposons in C. elegans [89]. HSEs are bound by Heat Shock Factor 1 (HSF-1) in response to elevated temperatures to drive transcription of genes required for reduce the proteotoxic effects of heat stress. HSF-1 binding to the Helitron HSEs drives transcription of neighboring genes upon heat stress. Mobility of Helitron transposons thus mediates the incorporation of new genes into the HSR pathway, driving evolution through changes in gene regulation.

6. Conclusions and Future Directions

This breadth of this review underscores the wide-ranging effects of TEs on the C. elegans genome, from the mutagenic properties of TEs to the complex and intricate silencing mechanisms that the host mounts to silence transposable elements.

Transposon research in C. elegans started with a natural isolate in which the Tc1 transposon hops. While C. elegans research into RNAi and transposon silencing has contributed a wealth of knowledge and understanding of these processes, it is still unknown why Tc1 is desilenced in this Bergerac natural isolate. Using CRISPR-Cas, transposon-specific silencing pathways could be uncovered that may be independent of known RNAi factors. It will be of particular interest to determine if there are mechanisms of transposon recognition other than piRNA complementarity, especially given that not all transposons appear to be silenced through Piwi and piRNAs. It is also unclear if TE silencing is affected by environmental stresses other than heat stress and how TEs contribute to adaption of the host to environmental stresses.

The large number of C. elegans natural isolates and related nematodes are a valuable resource to study additional potentially active transposons and silencing mechanisms, especially given the rapid evolution of transposons and silencing pathways. Caenorhabditis inopinata is a nematode related to C. elegans that has undergone a massive expansion in TEs, including Tc1/mariner transposons and LTR retrotransposons. It will be of interest to explore the genetic differences that could underlie this burst in transposon activity. Given that C. inopinata has lost the ERGO-1 pathway, this pathway may play a major role in curtailing transposon proliferation. Finally, whereas most research has focused on transposons that are active, transposons that do not appear active may have been co-opted and fulfill new functions in the host, such as the retrotransposon Cer1 and the Helitron transposons, and therefore should not be discarded as junk.