Drosophila relics hobo and hobo-MITEs transposons as raw material for new regulatory networks.

Hypermutable strains of Drosophila simulans have been studied for 20 years. Several mutants were isolated and characterized, some of which had phenotypes associated with alteration in development; for example, showing ectopic legs with eyes being expressed in place of antennae. The causal agent of this hypermutability is a non-autonomous hobo-related sequence (hoboVA). Around 100 mobilizable copies of this element are present in the D. simulans genome, and these are likely mobilized by the autonomous and canonical hobo element. We have shown that hoboVA has transcription factor binding sites for the developmental genes, hunchback and even-skipped, and that this transposon is expressed in embryos, following the patterns of these genes. We suggest that hobo and hobo-related elements can be material for the emergence of new regulatory networks.

Introduction

The eukaryotic genomes sequenced thus far have shown that substantial portions of them are formed by transposable elements (TEs). These elements are extremely variable, and usually, the genomes are composed by dozens of different TE families, which are often represented by degenerated and inactive copies (review in Wicker ). TEs have parasitic characteristics, harboring mechanisms that enable them to self-multiply faster than the “host genome”. Furthermore, TEs are an important source of genetic variability to drive evolution. There are many ways that TEs can generate variability; for example, promoting mutations in coding or regulatory regions of genes, chromosome rearrangements, epigenetic alterations and others (reviews in Biémont and Vieira, 2006; Hua-Van ).

Recently, a growing body of evidence indicates that TEs are involved in rewiring gene regulatory networks (Feschotte, 2008). TEs typically carry a collection of regulatory elements, such as promotors, cis-regulatory sequences, enhancers, insulators, splice and poly(A) sites, usually used for their own expression. Also important in gene regulation involving TEs are those using miRNAs in the pre-translation process or in heterochromatin formation (Feschotte and Gilbert, 2012; Rebollo ; Chuong ).

This review will focus on cis-regulatory sequences, in particular on the potential of hobo relics elements to provide sequences for producing mutations in developmentally regulated genes or sequences in which developmental genes can act.

TEs can harbor many transcription factor binding sites (TFBSs) and, the mobile nature of TEs, which allows them to occupy almost any site of a genome, makes them a powerful route for the spread of “ready-to-use” cis-regulatory sequences. The addition of new TFBSs in regulatory regions can create novel patterns of gene expression. There are examples in diverse organisms of genes that have exapted TE-TFBSs (review in Chuong ). In mammals, Polavarapu found that 7-10% of experimentally characterized TFBSs in the human genome are derived from TEs. Sundaram studied 26 pairs of orthologous transcription factors (TFs) in two pairs of human and mouse cell lines and showed that 20% of binding sites were embedded within TEs. The expression of the human tumor suppressor protein, p53, is regulated by the p53 TFBS found in LTR (long terminal repeats) of ERV elements (Wang ). In insects, the domestication of the silkworm (Bombyx mori) involved the insertion of a partial TE (Taguchi) in cis-regulatory region of the ecdysone oxidase (EO) gene, enhancing the expression of this gene. It promotes a developmental uniformity of silkworm individuals, which is a desirable trait for domestication (Sun . The addition of new cis-elements from TEs on Cyps genes has been associated with the upregulation of these genes and consequent development of insecticide resistance. Different TEs or TEs regulatory sequences have been linked to this phenomenon as, for example, the retrotransposons Accord and HMS-Beagle, the transposons P and BARI, and the helitron DNAREP1 (Chung ; Schmidt ; Carareto ). In plants, many published examples describe the exaptation of TEs cis-regulatory regions. For instance, as the C4 photosynthesis system evolved, many genes involved in it acquired regulatory cis-elements from TEs (Cao ); and the hAT element Moshan, from Prunus, has cis-acting elements, recognized by MYB and WRKY transcription factors (TFs) (Wang ). Some transcription factors are products of the so-called “master regulatory genes”, originally defined by Susumu Ohno as “genes that occupy the very top of a regulatory hierarchy” acting over multiple downstream genes directly or through a cascade of gene expression changes (Ohno, 1979). Transposable elements that have TFBSs sensible to master genes are promising for producing evolutionary novelty. As stated by Britten and Davidson (1971), “major events in evolution require significant changes in patterns of gene regulation. These changes most likely consist of additions of novel patterns of regulation or reorganization or pre-existing patterns”. The hobo element of Drosophila has TFBSs for some master developmental genes and is potentially able to produce remarkable mutations. This can be an interesting example, as some evolutionary novelty can arise.

Hobo, its relics and MITEs

The hobo transposon is a class II transposable element and a member of the hAT superfamily (Wicker ). The main characteristics of this superfamily are: i) presence of short terminal inverted repeats (TIRs), 10-25 bp in length; ii) target site duplications (TSDs) of 8 bp as a consequence of the transposition process; iii) when complete, elements encode for a transposase of 500-800 amino acids; and iv) different elements of this superfamily share between 20 and 60% of amino acid transposase sequence similarity. This enzyme has an amino acid triad (DDE or DDD) in its catalytic domain (Ladevèze ). The hAT superfamily is also characterized as being widely present in eukaryotes (Calvi ).

Currently, it is proposed that the hAT superfamily is formed by three families; Ac, Buster and Tip (Rossato ). In Drosophila, the Ac family is more representative: in 12 analyzed Drosophila genomes, members of this family were found in 11, corresponding to 39 different hAT elements, of which 29 were potentially autonomous. However, as the elements are found as multiple copies, most (92.9%) are non-autonomous (Ortiz and Loreto, 2009). The Buster family is represented in the Drosophila genus only by the Mar element, present in species of the willistoni group, mainly as MITEs (Deprá ). The only element of the Tip family described in Drosophila so far is But2, occurring in some species of groups melanogaster, repleta, and willistoni (Rossato ).

A remarkable characteristic of many hAT elements is the formation of short, but mobilizable elements, the “Miniature Inverted repeat TEs” (MITEs). They normally have less than 800 bp, with no coding capacity, but with conserved TIRs, and often reach high copy numbers in the genomes (Feschotte and Pritham 2007). In Drosophila, 68% of the described elements of the Ac family have potentially mobilizable elements with less than 600 bp (Ortiz ). Also, the MITEs copies are the most abundant in Buster and Tip family (Deprá ; Rossato ).

The hobo element belongs to Ac family and was discovered in D. melanogaster by McGinnis , as a 1.3 kbp sequence inserted in the Sgs-4 gene. Soon after, a complete and active element was described and shown as able to produce hybrid dysgenesis (Blackman ), and was used as a vector for genetic transformation (Blackman ). This 2,959 bp active hobo, called a canonical element, presents an ORF encoding a TPase, short TIRs of 12 bp, and produces a target site duplication (TSD) of 8 bp (Figure 1A). Complete canonical elements have two sites for the restriction enzyme XhoI, producing a 2.6 kbp diagnostic band in Southern blot analyses. Population studies showed that some populations had a 2.6-kbp band of complete elements, called H (hobo), and other populations had no band, called E (empty). Short bands resulting from internally deleted elements can be present; the most frequent being elements that produce a 1.1 kbp band in Southern blot analyses (Daniels ; Periquet , 1994) (Figure 1A). A second form of the hobo element is called “relics” (Figure 1B). Even E populations show, in Southern blots, bands with high molecular size, which had lost XhoI sites and were characterized as degenerate sequences, diverging in 10-20% of the canonical elements (Simmons, 1992). A third form is the miniature inverted-repeat transposable element, MITE (Figure 1C) (Ortiz and Loreto, 2008). MITEs are characteristically 80-500 bp in size (but they can sometimes reach lengths of up to 1.6 kbp).

Figure 1

Hobo, relics and MITEs. A) Two forms of canonical hobo; the complete and deleted elements. Open triangle = TIR (terminal inverted repeats); complete elements have a transposase gene (ORF); deleted elements normally lack the central part of the sequence (del 1.4 kbp); X= XhoI restriction site, which produces a 2.6 kbp fragment in complete elements and, generally, a fragment of 1.1 kbp in deleted elements. These fragments are used to identify complete and deleted elements in Southern Blots studies; B) Relics hobo elements are present in two forms: mobilizable, those that have TIRs and conserved subterminal sequences; and immobile elements are defective in one TIR. The inner parts of elements are degenerated (striped) and can be AT rich. The XhoI site may or may not be present (indicated by “?”). In D. simulans, when the XhoI site is present, the more abundant relic element generates a 0.6 kbp fragment. The lengths of fragments generated by immobile copies are variable (?) C) hobo elements can be found as MITEs (80-700 bp).

The canonical hobo is also found in D. simulans and D. mauritiana (Boussy and Daniels, 1991). The high similarity observed between the sequences of this element in these species led Simmons (1992) to suggest that horizontal transfer could have occurred for this TE between these species. The “relics” hobo has a wide distribution. Although it is mainly restricted to the melanogaster subgroup, these sequences are present in D. melanogaster, D. simulans, D. sechellia, D. mauritiana, D. santomea, D. yakuba, D. teissieri and D. erecta (Ortiz and Loreto, 2008).

A hypermutable strain and the occurrence of developmental mutants

We have characterized a hypermutable strain of Drosophila simulans (Dshs), originated from a single spontaneous mutant male, collected in nature, showing the lozenge phenotype. The genetic characterization of this mutant revealed that the females are sterile due absence of spermathecae. Therefore, to maintain the mutants in the laboratory, the males were crossed with a wild strain (D. simulans Eldorado). During this process, new mutations were observed. The strain was followed for roughly 100 generations, and during the mutation screening, several of the isolated mutants corresponded to developmental genes (Loreto ). One interesting mutant, which can represent the potential of transposons to create “evolutionary novelties” is the one showing an antennapedia phenotype, where legs grow in place of antennae. In addition, in this particular mutant, ectopic eyes grow on homeotic legs. This allele is dominant, and flies show a phenotype with variable expressivity, ranging from normal antennae to homeotic legs, with approximately 6% of flies expressing ectopic eyes on the homeotic legs (Figure 2). This gene was mapped to the 3L chromosome in the region corresponding to the eyegone locus, although no molecular evidence has confirmed the mutation’s presence in this gene (unpublished result).

Figure 2

Variable expression of the Zp (Zoinho-na-pata) mutant. This mutant shows ectopic expression of legs in the antennae and, sometimes the expression of ectopic eyes. The mutation is dominant, but some individuals show normal antennae (A), a weak transformation of antennae to leg (B-C), a complete leg in place of antenna (E), or eye structures in the ectopic leg (D, F, G).

Other mutations were characterized and mapped to, for example, the decapentaplegic gene (dpp); lozenge (lz); blistered (bs); and white (w) (Figure 3) (Loreto ; Torres ). The blistered (bl) mutant (Figure 3A) is dominant, showing incomplete penetrance, which is sensitive to temperature, with stronger expression at higher temperatures. The same increase in phenotypic expression was observed in the Zp mutant (Loreto ELS, 1997, Doctoral thesis, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS). The mutant decapentaplegic (dpp) is recessive, and the homozygous flies have wings, which are held out laterally (Figure 3B).

Figure 3

Mutant phenotypes A) Phenotypic appearance of blistered mutant (bl); B) dpp mutant; C) somatic mutation in which half the thorax and one wing was not formed; D) mosaic fly in which one eye has a wild type phenotype and the other has a lozenge appearance.

In the hypermutable strain, the occurrence of a high rate of somatic mutations is suggested, as it has been observed in many flies with severe phenotypic alterations that are not inherited (Figure 3C) (Loreto ).

The putative causal agent of this hypermutability: an old, hectic, energetic and degenerated hobo element

The molecular characterization of a “de novo” white mutation isolated in the hypermutable strain showed that it was caused by an insertion of a “relic” non-autonomous hobo element. This is a 1.2 kbp element, with conserved regions of 12 bp TIRs, 8 bp TSD, and subterminal sequences (Figure 4A). The 5’ region was 381 bp in length and showed 93% similarity with canonical hobo. The 3’ region was 341 bp in length and 85% similar to canonical hobo. The inner region is AT rich and has low similarity with canonical hobo (Torres ). This non-autonomous element is mobilizable in the hypermutable strain, and it is involved in “de novo” mutations and contains sufficient sequences for transposition (a minimum of 141 bp on the 5’ end and 65 bp on the 3’ end) (Kim ). The source of transposase to induce mobilization is postulated as the canonical hobo, which is present in this strain (Torres ; Deprá ).

Figure 4

Canonical hobo and hoboVA. A) Schematic representation of canonical hobo and hoboVA. TIR are represented as a triangle. The red line shows the probe region used in in situ hybridization (C). The hoboVA element has the best conserved extremities and a more divergent inner sequence. The similarity for each region is indicated in %, and the sizes of the regions are indicated in base pairs (bp); B) in situ hybridization of polytene chromosomes using the complete hoboVA as probe; C) in situ hybridization of polytene chromosomes using the inner portion of canonical hobo as probe. Arrows point to the hybridization sites.

Although the only mutation for which the causal agent was fully characterized as being the hoboVA element was the white mutant, two other facts lead us to suggest that the causal agent of the hypermutability in this strain is the hoboVA. First, an insertion of the same size of that element, 1.2 kbp, was also observed in the lozenge mutant generated in this hypermutable strain (Loreto ). Second, it has long been known that the cis-regulatory heldout region of the decapentaplegic (dpp) gene is a preferential site for hobo insertions (Newfeld and Takaesu, 1999). One of the mutants we have isolated is with the heldout phenotype of dpp.

Aiming to verify the abundance of sequence similar to hoboVA in the D. simulans genome, we performed an in silico analysis on the genome available after the publication of the 12 Drosophila genomes by Clark . In that study, the genome of D. simulans was assembled using a mix of seven strains. The analysis showed that these 1.2 kbp sequences, similar to hoboVA, are abundant, with 147 copies scattered across all chromosomes. These comprise 92 putatively mobilizable sequences and 72 with TSDs, indicative of recent mobilization. However, the sequenced strains only had two copies of the putative autonomous hobo element (Ortiz and Loreto, 2008). Also, we have performed a quantification of hoboVA sequences in our hypermutable D. simulans strain, showing that this element is also abundant in the strain. Figure 4B shows the fluorescent in situ hybridization (FISH) of polytene chromosomes with the hoboVA element, where at least 90 hybridization sites can be identified. In contrast, when the polytene chromosomes were hybridized with the inner portion of the hobo element, found exclusively in the complete elements, only six hybridization sites were observed (Figure 4C).

Another characteristic of these hobo-related elements, hoboVA, is that they have apparently been maintained for an evolutionary time that is prior to the D. sechellia and D. simulans speciation event, estimated at 0.4 MYA. Sequences similar to hoboVA are found in both species, suggesting that this element has been maintained as a non-autonomous element in the genomes of these species for all this time (Torres ; Ortiz and Loreto, 2008). The presence of short, non-autonomous but mobilizable elements in a higher number, contrasting with low copy numbers of autonomous elements, appears to be a pattern for hAT elements (Ortiz ).

The data described above suggest that the hypermutable strain could have an autonomous hobo element, free of silencing mechanisms, and in this way, able to mobilize hoboVA elements. Because these “relics” elements are maintained for a long time, and are very active, we call it hobo “Velho Assanhado” (VA), which in Portuguese means “a very animated elder”.

hoboVA and his cis-regulatory developmental sites

The transcription factor binding sites in the hoboVA element were predicted using the “motility” toolkit, which allowed us to search for sequence motifs using position weight matrices. For this analysis, we searched high scoring binding sites for six homeotic genes (bicoid, even-skipped, fushi-tarazu, hunchback, knirps and krüppel) using matrices described in Ho et al. (2009). Max-scoring matches were found for even-skipped and hunchback (Figure 5A).

Figure 5

hobo transcriptional regulation. A) Transcription factor binding sites (TFBSs) in the hoboVA element predicted by “motility” toolkit. For this analysis, we searched for high scoring binding sites of six homeotic genes (bicoid, even-skipped, fushi-tarazu, hunchback, knirps and kruppel) using matrices described in Ho . Possible CAAT and TATA boxes were found using the description of hobo elements by Streck , for reference in the alignments. TFBSs are represented by colored boxes. A red star indicates a perfect match of TFBS and the hoboVA sequence; B) in situ hybridization whole-mount embryos of the Drosophila simulans hypermutable strain, using hoboVA as probe (RNA). hoboVA can be seen expressing as hunchback and even-skipped in two different developmental stages.

Experimental evidence that the cis-regulatory sites for hunchback and even-skipped are functional in hoboVA were shown by Deprá . In situ hybridization in embryos of flies belonging to hypermutable and other strains, using hoboVA as a probe showed expression comparable to that observed for hunchback and even-skipped (Figure 5B).

The presence of cis-regulatory sequences of developmental genes, mainly those expressed in the initial phase of embryonic development, have been described for many TEs. For example, several retrotransposons of Drosophila have these sequences (Ding and Lipshitz, 1994; Borie ), as do LINEs in mammals (Loh ; Gerdes ). Transcription factor binding sites related to genes involved in the initial phases of development can be selectively advantageous for TEs, which could maximize their chance of increasing their presence in the next generation. For organisms whose germlines and somatic cells are separated, it is important for TEs to be active in phases when transposons can increase their copy number in the germ line, but not in somatic cells. Transposition in germ cells can be selectively advantageous from TE perspectives, yet transposition is normally detrimental in somatic cells (Haig, 2016).

Creating new regulatory networks

From Figure 5B, it can be seen that hoboVA are scattered across all chromosomes, carrying its regulatory sequences. When some of these transposons are mobilized, they can be inserted in nearby genes, leading to a new position for their transcription factor binding sites, and this can modify the expression of genes in these new locations. Although we do not have a molecular characterization of the Zoinho-na-pata (Zp) mutation, we can hypothesize that the ectopic expression of Zoinho-na-pata mutants, as well as other mutants observed in the hypermutable strain, could be a product of hoboVA insertion. Master control genes, such as eyegone, which is involved in antennal and eye development and morphogenesis (Dominguez ; Yao and Sun, 2005; Wang ), can activate a new spatiotemporal pattern of gene expression when they receive insertions of new cis-regulatory sequences in their regulatory region. Therefore, transcription factor binding sites (TFBSs) for hunchback and even-skipped, present in hoboVA, can produce new phenotypes if inserted in such genes. These TFBSs are known as promoters of spatiotemporal gene control compatible with those observed in Zp mutant phenotype.

From an evolutionary point of view, the spread of cis-regulatory sequences can rewire gene regulatory networks. This can occur with the gradual addition of these sequences in the promotor regions of new genes, such as products of new TE insertions. As consequence of these insertions, genes can show new regulatory patterns by answering to transcription factors in which they were not respondent before. This rewiring can later undergo fine-tuning, resulting by natural selection of other mutations in the involved genes and the regulatory sequences that were added to the system by TEs. The involvement of TEs in rewiring gene networks is well supported in the literature (Feschotte, 2008; Feschotte and Gilbert, 2012; Rebollo ; Chuong ). The classical Darwinian view of evolution as a gradual process, in which no leaps are taken, fits well in this scenario of the rewiring of gene networks. Also, it has been shown in the literature that complex structures can evolve gradually as, for example, complex organs such as eyes found in vertebrates, insects or cephalopods have evolved from photoreceptor cells, in which many intermediary steps can be found throughout the animals phylogeny (reviewed in Gehring, 2002).

The idea of large mutations producing great leaps of adaptation, as originally proposed by Richard Goldschmidt, in his hopeful monster theory, was refuted for a long time. Now, some examples indicate these “monsters” could have a place in evolutionary theory, though not exactly as frequently credited to Goldschmidt’s original proposition, as mutations with dramatic alterations in phenotype, producing an organism perfectly adapted to the environment. However, Chouard (2010) has revised some examples where single-gene changes promoting large phenotype effect can confer large adaptive value. These examples are not in disagreement with the Darwinian theory, they only open space for mutations with large phenotypic consequences, which, when viable in natural situations, could be initial steps for evolutionary novelties.

Master control genes are at the top of networks to build structures, body parts, and metabolic routes. Many master control genes are themselves transcription factors. When transposons carrying transcription factor binding sites (TFBSs) insert into the regulatory region of a master control gene, they can, theoretically, imbricate phenotypic building cascades, leading to evolutionary novelties.

The appearance of antennae with eyes could constitute a large evolutionary leap. Unfortunately, after some year of maintenance in the laboratory, we lost the Zp strains, making it impossible to show if hoboVA was involved in this particular mutation. The difficulty in maintaining this strain in the laboratory is `per se’ indicative that such mutations normally are inviable in nature. However, we can imagine that insertions of TEs, such as hoboVA, carrying TFBSs for master control genes, can bring new regulatory patterns for other master control genes, producing new phenotype patterns. If so, maybe some “hopeful monsters” could be the products of TE insertions, as is suggested by this hypothetical example. Maybe, hopeful monsters need a “lucky spot”. Large phenotypic alteration, when occurring in particular environments, could be the initial point for evolutionary novelties, and TEs can be part of this process.