Download Zisupton—A Novel Superfamily of DNA Transposable Elements

Zisupton—A Novel Superfamily of DNA Transposable
Elements Recently Active in Fish
Astrid Böhne,1 Qingchun Zhou,2 Amandine Darras,1 Cornelia Schmidt,2 Manfred Schartl,2
Delphine Galiana-Arnoux,1 and Jean-Nicolas Volff*,1
1
Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon 1, Centre National de la Recherche Scientifique,
Institut National de la Recherche Agronomique, Ecole Normale Supérieure de Lyon, Lyon, France
2
Department of Physiological Chemistry I, Biocenter, University of Würzburg, Würzburg, Germany
*Corresponding author: E-mail: [email protected].
Associate editor: Norihiro Okada
Abstract
Key words: transposon, platyfish, sex chromosomes, SUMO, Ulp1 protease, HMGXB3, testis.
Introduction
Transposable elements (TEs) are mobile genetic entities
found in the genome of almost all organisms. They are extremely abundant in many genomes, reaching over 90% of
the DNA content in some plants (Bennetzen 2000; Jurka
et al. 2007). TEs are traditionally classified into two large
classes according to their transposition mechanism
(Wicker et al. 2007; Kapitonov and Jurka 2008). Both TE
classes include autonomous and nonautonomous sequences. Autonomous elements encode proteins necessary for
their own transposition. In contrast, nonautonomous elements do not carry the complete set of genes required for
transposition and use proteins encoded by autonomous
elements for their mobilization.
Class I elements, also called retroelements, spread
throughout genomes via retrotransposition. This process is
based on the reverse transcription of a TE-encoded RNA into
cDNA and the integration of the new copy into another site
of the genome. This ‘‘copy and paste’’ mechanism leads to an
increase in copy number of the retroelement.
The second class of mobile sequences is constituted by
DNA transposons (class II elements). In contrast to retroelements, most class II elements transpose via a ‘‘cut and
paste’’ mechanism. Transposition is catalyzed by an enzyme called transposase, which is responsible for the excision of the element from one site and its integration into
another site. Other class II elements, rolling-circle DNA
transposons called Helitrons, have been proposed to transpose by single-strand transfer followed by replication
through the cellular machinery to create the second strand
(Kapitonov and Jurka 2001, 2007). Finally, TEs known as
Polintons or Mavericks are thought to transpose via selfsynthesis catalyzed by a DNA polymerase encoded by
the element itself (Feschotte and Pritham 2005; Kapitonov
and Jurka 2006; Pritham et al. 2007).
Many different genomics effects have been attributed to
TEs. Beside insertions, they can generate various types of
rearrangements through transposition and recombination,
such as deletions, duplications, or inversions (Gray 2000).
TEs can also modify the epigenetic context and therefore
© The Author 2011. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please
e-mail: [email protected]
Mol. Biol. Evol. 29(2):631–645. 2012 doi:10.1093/molbev/msr208
Advance Access publication August 27, 2011
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Research article
Transposable elements are widespread mobile DNA sequences able to integrate into new locations within genomes.
Through transposition and recombination, they significantly contribute to genome plasticity and evolution. They can also
regulate gene expression and provide regulatory and coding sequences (CDSs) for the evolution of novel gene functions.
We have identified a new superfamily of DNA transposon on the Y chromosome of the platyfish Xiphophorus maculatus.
This element is 11 kb in length and carries a single CDS of 24 exons. The N-terminal part of the putative protein, which is
expressed in all adult tissues tested, contains several nucleic acid– and protein-binding domains and might correspond to
a novel type of transposase/integrase not described so far in any transposon. In addition, a testis-specific splice isoform
encodes a C-terminal Ulp1 SUMO protease domain, suggesting a function in posttranslational protein modification
mediated by SUMO and/or ubiquitin small peptides. Accordingly, this element was called Zisupton, for Zinc finger SUMO
protease transposon. Beside the Y-chromosomal sequence, five other very similar copies were identified in the platyfish
genome. All copies are delimited by 99-bp conserved subterminal inverted repeats and flanked by copy-specific 8-nt target
site duplications reflecting their integration at different positions in the genome. Zisupton elements are inserted at
different genomic locations in different poeciliid species but also in different populations of X. maculatus. Such insertion
polymorphisms between related species and populations indicate relatively recent transposition activity, with a high
degree of nucleotide identity between species suggesting possible implication of horizontal gene transfer. Zisupton
sequences were detected in other fish species, in urochordates, cephalochordates, and hemichordates as well as in more
distant organisms, such as basidiomycete fungi, filamentous brown algae, and green algae. Possible examples of nuclear
genes derived from Zisupton have been identified. To conclude, our analysis has uncovered a new superfamily of DNA
transposons with potential roles in genome diversity and evolutionary innovation in fish and other organisms.
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Böhne et al. · doi:10.1093/molbev/msr208
the expression pattern of host genes (Lippman et al. 2004;
Haun et al. 2009). Finally, they can be considered as a kind
of evolutionary toolbox for the host, which can be used as
a source of new regulatory and coding sequences (CDSs)
(molecular domestication, Volff 2006; Bourque 2009). Importantly, genomes can considerably differ in their TE content at both the quantitative and qualitative levels, and
a same type of TE can have very different invasive success
in different species (Böhne et al. 2008). Hence, TEs have the
potential to generate biodiversity and evolutionary transitions through lineage-specific mutations and molecular domestication. Occasionally, TEs can be transferred between
reproductively isolated species through horizontal transfer.
This process, which occurs independently from classical
parent-to-offspring inheritance, also plays a role in genomic
diversity between species (Schaack et al. 2010). Examples of
horizontal transfer of TEs in vertebrates remain rare.
The numerous families of TEs identified in fish have been
proposed to be involved in biodiversity and speciation in
this group of animals (Volff et al. 2003; Volff 2005). With
almost 28,000 described species, fish make up roughly 50%
of all extant vertebrate species (Nelson 2006). They show
an exceptional diversity in body shape and plan, coloration,
ecology, behavior, social systems, and reproduction, among
others. Sex determination is particularly variable in fish. Fish
sex chromosomes are generally young and rarely heteromorphic, in contrast to differentiated and degenerated
sex chromosomes in mammals and birds (Ezaz et al.
2006). TEs frequently accumulate on sex chromosomes,
particularly in regions of suppressed recombination. TEs
and other repeats might play a role in the differentiation
process leading to the formation of heteromorphic sex
chromosomes during evolution.
Several new families of TEs in fish have been identified for the
first time on the sex chromosomes of the platyfish Xiphophorus
maculatus. This poeciliid freshwater fish belongs to a genus currently comprising 26 species inhabiting Atlantic drainages of
Mexico, Guatemala, and Honduras (Kallman and Kazianis
2006). Xiphophorus has been used as a model to study melanoma formation (Gordon 1927; Kosswig 1928; Meierjohann
and Schartl 2006), sexual selection (Offen et al. 2009), reproduction (Cummings and Gelineau-Kattner 2009), behavior
(Fernandez et al. 2008), and sex determination and sexual development (Schultheis et al. 2006; Böhne et al. 2009; Lampert
et al. 2010). Xiphophorus maculatus is particularly used to
study sex chromosome structure and evolution in poeciliids,
with the aim of identifying the master sex-determining gene
through positional cloning. For this purpose, bacterial artificial
chromosome (BAC) contigs covering the sex-determining
region of the X and Y chromosomes have been constructed
and partially analyzed (Froschauer et al. 2002). This region
was found to be infested by TEs (Volff et al. 1999; Volff, Körting,
Altschmied, et al. 2001; Volff, Körting, Froschauer, et al. 2001;
Froschauer et al. 2002; Zhou et al. 2006, 2010).
Here, we describe a new type of DNA transposon called
Zisupton, which was identified on the Y chromosome of the
platyfish. The Zisupton single coding region encodes a protein
with nucleic acid–binding and protein interaction domains
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as well as Ulp1 SUMO protease and F-box motifs specifically
expressed in testis. We provide evidence for the presence of
Zisupton in divergent organisms and for its recent activity
and possible horizontal transfer in Xiphophorus.
Materials and Methods
Fish
Platyfish (X. maculatus, population Rio Jamapa WLC1274,
closed colony stock derived from Jp163A) were kept under
standard conditions at the PRECI fish facility of the IFR128
Biosciences Gerland-Lyon Sud (Lyon, France). Samples from
X. couchianus, X. xiphidium, X. variatus, X. milleri, X. clemenciae, X. hellerii, X. andersi, and X. nezahualcoyotl were obtained from the fish facility of the Biozentrum at the
University of Würzburg (Germany).
In Silico Sequence Analysis
Zisupton-related protein and nucleotide sequences were
identified using BLAST/BLAT and PSI-BLAST searches
against GenBank databases (www.ncbi.nlm.nih.gov/blast/
Blast.cgi). BLAST analysis was also conducted using the current Ensembl genome assemblies of zebrafish (Danio rerio),
medaka (Oryzias latipes), green spotted puffer fish (Tetraodon nigroviridis), fugu (Takifugu rubripes), three-spined
stickleback (Gasterosteus aculeatus), and sea squirt (Ciona
intestinalis) (www.ensembl.org; version 57, March 2010).
Genome draft from acorn worm (Saccoglossus kowalevskii)
(http://www.hgsc.bcm.tmc.edu/project-species-o-Acorn%
20worm.hgsc) and amphioxus (Branchiostoma floridae)
(http://genome.jgi-psf.org/cgi-bin/runAlignment?db5Brafl1
&advanced51) was also analyzed. Hits were generally considered as significant below an E value cutoff of 1 1005.
Automatic sequence annotation was refined manually using expressed sequence tag data and related protein sequences with the help of FGENESH on the softberry web server
(http://linux1.softberry.com). Nucleotide and protein sequences were loaded into BioEdit (Hall 1999) and aligned using MUSCLE (Edgar 2004a, 2004b), T-Coffee (Notredame et al.
2000), and COBALT (Papadopoulos and Agarwala 2007).
Alignments were checked manually and ambiguously aligned
regions were removed before phylogenetic analysis.
The phylogenetic tree was constructed on a 166 amino
acid alignment generated through concatenation of domains
conserved in divergent Zisupton proteins. Maximum likelihood reconstruction was done using PhyML 3.0 (Guindon
and Gascuel 2003; Guindon et al. 2010) with 100 bootstraps
under the LG model. Similar results were obtained using the
neighbor joining method as implemented in MEGA4
(Tamura et al. 2007). MEGA4 was also used to visualize phylogenetic trees. Syntenic relationships between genomes
were established using Genomicus (Muffato et al. 2010).
Conserved protein regions in divergent Zisupton proteins were identified using MEME (http://meme.nbcr.net/
meme4_6_1/cgi-bin/meme.cgi). Annotation of conserved
protein motifs was performed with MotifScan (http://
myhits.isb-sib.ch/cgi-bin/motif_scan) and NCBI Conserved Domain Search (http://www.ncbi.nlm.nih.gov/
Structure/cdd/wrpsb.cgi).
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Ka/Ks calculation for puffer fish Zisupton-related sequences was performed using Ka/Ks Calculator under
the Modified version of Yang-Nielsen method model
(Zhang et al. 2006) on a pairwise nucleotide alignment
done with MUSCLE (Edgar 2004a, 2004b). Fisher’s exact
test and chi square with Yates’ correction were performed
on http://www.graphpad.com/quickcalcs/contingency1.cfm.
Xiphophorus maculatus Zisupton sequence has been deposited in GenBank under accession number JN255571.
Molecular Cloning
DNA fragments were cloned into pBluescript II KSþ (Stratagene) or using the TOPO TA Cloning Kit for Sequencing
(Invitrogen). BAC digestions for shotgun subcloning were
performed on 500 ng DNA under conditions recommended
by the manufacturer (Fermentas), with EcoRI (unique restriction site in exon 3 of Zisupton) and MscI (unique restriction
site in the 3# noncoding region of Zisupton). Cloning into
pBluescript II KSþ was done using Klenow fragment, T4
polynucleotide kinase, shrimp alkaline phosphatase, and
T4 DNA ligase (all from Fermentas). Ligations were done
overnight at 4 °C and subsequently used to transform chemical competent DH5a bacteria according to Ausubel et al.
(2010). Miniplasmid preparations were done from 5-ml overnight cultures using either standard protocols or the QIAprep Spin Miniprep Kit (Qiagen). DNA sequencing was carried
out by GATC Biotech (Constance, Germany).
Polymerase Chain Reaction Amplification
Polymerase chain reactions (PCRs) were performed using
the Taq DNA Polymerase Recombinant (Invitrogen), with
primer-dependent annealing temperatures ranging from
55 to 65 °C. Single-primer PCRs to characterize Zisupton
insertions were performed using one specific internal
Zisupton primer (5#-CCTTTGCTCATACAGTGCGG-3#, final concentration 6 ng/ll) on 100 ng of genomic DNA
(adapted from Parks et al. 1991). After an initial incubation
step at 94 °C for 5 min, the thermal cycling program was as
follows: 1) 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min
during 20 cycles; 2) 94 °C for 30 s, 40 °C for 30 s, and 72 °C
for 2 min during 30 cycles; and 3) 94 °C for 30 s, 55 °C for 30
s, and 72 °C for 2 min during 30 cycles. Amplification of the
X. hellerii-specific insertion was done using the QIAGEN
Long Range PCR kit with primers (5#-TCACATTTCAGCCACTATTTG-3#) and (5#-CTGTCGGAATCAGGCAAGT3#). Primer sequences for amplification and sequencing
are available upon request. Primers were ordered from
Invitrogen.
Expression Analysis
Total RNA was extracted from male and female tissues
(brain, eye, gill, liver, muscle, ovary, skin, spleen, and testis)
and embryos using the TRI Reagent (Molecular Research
Center, Inc). After DNase treatment (Fermentas), reverse
transcription was performed on 1 lg of total RNA using
the RevertAid First Strand Synthesis kit (Fermentas) and
random hexamer primers.
Classical real-time (RT) PCR experiments for Zisupton
(fig. 1) were performed with primers (5#-CTATCAATGGCACTGGCTT-3#) and (5#-TGGCATTCAGGGCAGTGT
CC-3#) for the testis-specific region and (5#-GCAGTGGG
ACAGACACAACTT-3#) and (5#-CAGCAAGTGATCGTTGAAG-3#) for the ubiquitously expressed region. PCR was
done with an annealing temperature of 58 °C (35 cycles).
The amount of cDNA template was adjusted between reactions using a beta-actin gene control.
Quantitative RT-PCRs were done on 2 ll of serial dilutions of cDNA starting from a dilution of factor 20 using
IQTM Custom SYBR Green Supermix (Bio-Rad). PCR amplification was monitored with a CFX96 Real-time System
(Bio-Rad). After one incubation step at 95 °C for 3 min, the
thermal cycling protocol was as follows: 95 °C for 10 s, 55–
62 °C (primer-dependent annealing temperature) for 20 s,
and 72 °C for 15 s during 40 cycles.
Primers were designed manually and ordered from Invitrogen. For each primer set, the efficiency of the PCR reaction was
measured in duplicate on serial dilutions (factor 10) of the
cDNA sample. RT-PCR efficiencies (E) were calculated for each
reaction from the slope of the standard curve using the equation E 5 10(1/slope) 1, as implemented in the software BioRad CFX manager V1.5, with 100% efficiency as an indicator of
a robust assay. All results reported are average of at least two
independent reverse transcription reactions and two RT-PCR
experiments for each reaction. For quantification, data were
analyzed using the 2(-Delta Delta C(T)) method (Livak and
Schmittgen 2001), with normalization to the housekeeping
gene ef1a (elongation factor 1 alpha). Expression was normalizedto eyetissue.Primer efficiencyandspecificitywas testedon
genomic DNA from each species before RT-PCR experiments.
Results
Zisupton Is Present on the Y But Not on the X
Chromosome in the Sex-Determining Region of the
Platyfish X. maculatus
In order to identify DNA sequences differentiating the Y
from the X chromosome of X. maculatus in the sexdetermining region, BAC contigs from each type of sex
chromosome were assembled and compared (Froschauer
et al. 2002). Sequence comparison revealed an approximately 11 kb sequence present in two overlapping Y-linked
BAC clones (F1 and O4) but absent from the homologous
X-linked clone K26. This sequence, which was called Zisupton, was found to contain one unique CDS (see below)
(fig. 1). Through exon walking on cDNA and rapid amplification of cDNA ends-PCR on testis RNA/cDNA, the
exon–intron structure of Zisupton was characterized.
The putative full-length CDS (from ATG to stop codon)
was 4,182 bp in length and consisted of 24 exons.
Zisupton Encodes a Putative Nucleic Acid–Binding
Protein Involved in Posttranslational Protein
Modification
The predicted full-length protein (1,393 amino acids) of the
Zisupton element contained putative nucleic acid–binding
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FIG. 1. Gene structure, predicted protein domains and expression of Zisupton in the platyfish. (A) The Y-specific copy of Zisupton identified in
BAC clones F1/O4 (24 exons). Symbols in the schematic protein above the gene structure of Zisupton indicate the conserved motifs. Numbered
boxes are exons, black boxes are introns, and untranslated regions are in gray. (B) RT-PCR analysis of the expression of Zisupton. Black arrows
indicate primer positions. The larger fragment in the RT-PCR experiment for the ubiquitous transcript corresponds to an unspliced form, as
demonstrated by DNA sequencing of RT-PCR products.
and protein interaction domains in its N-terminal part: two
C2H2 zinc fingers, a C3H zinc finger called the SWIM
(SWI2/SNF2 and MuDR) domain, and a SAP (SAF-A/B, Acinus, and PIAS) domain (fig. 1). C2H2 zinc fingers are often
a characteristic attribute of transcription factors with
sequence-specific DNA binding (for review, see Rosenfeld
and Margalit 1993; Iuchi 2001). C2H2 zinc fingers can also
confer binding to RNA (Brown 2005) or proteins (Brayer
and Segal 2008). The function of SWIM domains, characterized as cysteine–histidine zinc-chelating domains, remains unclear. However, due to structural similarities,
properties related to those of C2H2 zinc fingers have been
proposed (Makarova et al. 2002). Interactions of SWIM domains with either DNA or RNA have been reported for
some proteins (Banerjee et al. 2004; Cai et al. 2008; Lin
et al. 2008). The SAP motif is a DNA-binding domain found
in nuclear proteins (Aravind and Koonin 2000; Suzuki et al.
2009). This motif may bind to specific DNA sequences or
structures and possibly acts on chromosomal organization
(Aravind and Koonin 2000). Hence, the Zisupton protein
might be able to bind to DNA or RNA. A repetitive motif
of 14 amino acids (PAATDIQATQAPVR) of unknown function was located between both C2H2 fingers.
The C-terminal part of the Zisupton protein contained
an Ulp1 (ubiquitin-like protease 1) domain. This domain is
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a SUMO (Small Ubiquitin-like MOdifier)-specific protease
belonging to the subfamily of C48 cysteine peptidases
(Barrett and Rawlings 2001). SUMO proteases are involved
in the cleavage of SUMO from SUMO-conjugated substrates or poly-SUMO chains and the maturation of SUMO
itself (for review, see Yeh 2009). The Ulp1 protease domain
of Zisupton was flanked on its C-terminal side by a putative
F-box motif. This domain implicated in protein–protein interactions is present in F-box proteins that act as adapter
components of SCF (Skp, Cullin, F-box containing) complexes. SCF complexes are a family of huge modular E3
ubiquitin ligases (Craig and Tyers 1999; Jackson and
Eldridge 2002). Taken together, these observations suggested that the full-length Zisupton protein might be involved in posttranslational protein regulation mediated
by SUMO and/or ubiquitin small peptides. Interestingly
enough, the SWIM domain of the MEX E3 ubiquitin ligase
is involved in ubiquitination (Nishito et al. 2006), suggesting that this domain of Zisupton might also play a role in
posttranslational protein modification.
Zisupton Full-Length Isoform Is Testis Specific
Through exon walking on testis cDNA and expression analysis on adult organs and tissues, we could identify two
splice variants of Zisupton. One of them, encoding a shorter
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isoform, was lacking exon 15 and contained a premature stop
codon at the beginning of exon 16 (fig. 1B). At the protein
level, only the N-terminal part with both C2H2 zinc fingers,
the SWIM zinc finger and the SAP domain were present. By
RT-PCR, expression was detected in all adult organs and tissues tested (brain, eye, gill, liver, muscle, ovary, skin, spleen,
and testis). The additional upper band in figure 1B corresponded to a nonspliced product as confirmed by sequencing of the PCR products. In contrast, the longer splice variant,
in which inclusion of exon 15 restored the complete open
reading frame (ORF), was found to be expressed only in testis.
Hence, the Zisupton isoform with Ulp-1 and F-box domains
was apparently testis specific in adults.
Zisupton Is Multicopy in Platyfish Genome
In order to test for the presence of additional copies of Zisupton in the genome of X. maculatus, genomic DNA of females and males was cut with EcoRI (one unique restriction
site in Zisupton), and Southern blot DNA hybridization was
performed with a Zisupton-specific probe derived from exon
3. In males, several signals were obtained in addition to the
Y-specific copy, indicating that Zisupton was multicopy
(data not shown). All these additional signals were also
found in females, showing that Zisupton is not Y specific.
A BAC genomic library of male platyfish DNA (Froschauer
et al. 2002) was screened by Southern blot DNA hybridization
with the same Zisupton probe. Sixty-four Zisupton-containing
clones were isolated and analyzed through restriction fragment length polymorphism (RFLP) analysis. Besides BAC
clones containing the Y-specific copy already characterized,
five further RFLP groups were identified (fig. 2). None of them
belonged to the BAC contigs covering the sex-determining
region of the X and Y chromosomes of the platyfish. The
Zisupton-positive fragments from these groups were found
in both males and females (data not shown). Hence, these
copies were located either on autosomes or in the pseudoautosomal regions of the sex chromosomes. To conclude, at
least five copies were present in platyfish females and at least
six copies in males.
Zisupton Is a DNA Transposon
For each RFLP group, Zisupton and its flanking regions were
sequenced directly from representative BAC clones (fig. 2). All
six copies showed a high level of nucleotide identity (.99%)
in exons and introns. Strikingly, sequence identity between
copies always stopped at the same precise base pair position,
giving sharp 5# and 3# boundaries. No sequence similarity was
found between flanking sequences from different groups, indicating that they indeed corresponded to different copies of
Zisupton. Comparison of 5# and 3# extremities revealed the
presence of 99 bp terminal inverted repeats (TIRs) conserved
in all Zisupton copies. These repeats were subterminal, the
ends of the element being constituted by 8-nt segments with
no sequence similarity between both ends. Target site duplications (TSDs) of 8 bp flanked each copy of Zisupton.
TSDs from different copies were different and did not
display any obvious common motif. TIRs and TSDs are structural hallmarks of TE insertions. Therefore, Zisupton might
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correspond to a DNA TE (10,784 bp in length). The absence
of similarity between TSDs from different insertions indicated
that Zisupton integration is not site specific.
Strikingly, three copies (copies III, IV, and V), clearly corresponding to different insertions, were 100% identical over
their complete length (almost 11 kb) (fig. 2). They differed
from the Y-specific copy at only two nucleotide positions:
an A-G transition in exon 8, leading to a histidine–arginine
nonconservative change (CAT-CGT) outside of the conserved
domains identified in Zisupton, and a T missing in the 3# untranslated region of the Y copy. Neither frameshift nor nonsense mutations were found in these four copies. Taken
together, the presence of (almost) identical noncorrupted
copies suggested that Zisupton was recently—and might
be still—active in the genome of the platyfish. The two remaining insertions have been inactivated through mutations.
Sequencing of regions surrounding the different Zisupton insertions revealed that at least two copies were inserted in repeat-containing regions (copies I and IV).
Copy III was inserted directly 3# behind the pol ORF of
a Bel/Pao long terminal repeat retrotransposon. Copy IV
insertion was located in an intergenic region 360 nt 5# from
an apparently intact glucosidase gene (1.3 kb).
The Y-chromosomal copy was found to be inserted in a
nonautonomous Zisupton-like element called little-Zisupton
(liZi, 3,372 bp). LiZi was located at the same position on the X
and on the Y chromosome. LiZi was delimited by TIRs (21 nt)
and flanked by TSDs (10 nt), indicating that it also corresponded to a TE. Detailed examination of the insertion
on the Y chromosome indicated that Zisupton TSD sequence corresponded to the first 5# nucleotides of the 3#
TIR of liZi. Hence, we assume that Zisupton was integrated
into the 3# TIR of liZi on the Y chromosome.
Recent Transposition Activity and Putative
Horizontal Transfer of Zisupton in the Poeciliidae
Fish Family
Using Southern blot hybridization and PCR amplification
with X. maculatus primers, we could detect Zisupton elements in other fish species from the family Poeciliidae (interspecific divergence 3–20 My; Hrbek et al. 2007). We
amplified by PCR, sequenced and assembled large overlapping fragments of Zisupton from other Xiphophorus species
including X. clemenciae, X. andersi, X. milleri, X. nezahualcoyotl, X. xiphidium, and X. hellerii (table 1).
Strikingly, all Zisupton sequences from these species
were extremely similar, with at least 99.9% of nucleotide
identity. Inclusion of different PCR controls and detection
of rare species-specific single nucleotide polymorphisms indicated that these results were not due to contaminations.
No rearrangements such as deletions, insertions, or inversions were detected in the amplified fragments.
In order to test if the low interspecific variability observed
for Xiphophorus was in the range of variation observed for
other nuclear genes, we amplified and sequenced parts of
the b-actin genes from different species. Published data for
exon 3 of rag1 were also included (Meyer et al. 2006). Only
presumed neutral changes (substitutions at synonymous sites
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FIG. 2. Structural comparison of different Zisupton insertions in Xiphophorus maculatus. Boxes at the boundaries of the sequences: target site
duplications (TSDs), white arrowheads: terminal inverted repeats (TIRs), white boxes: Zisupton exons, black boxes: introns, little white boxes
above exon 3: repetitive sequence region showing numbers of repetitions, black letters: synonymous/silent nucleotide substitutions, and gray
letters in italics: nonsynonymous nucleotide changes. Neighbor sequences were identified using BLAST and are marked as boxes; gene names
are given according to most significant BLAST hits. Dashed line indicates incomplete sequence data for copy V.
in exons and substitutions in intronic sequences) were considered. No intronic data were available for rag1.
In most cases, Zisupton transposons from two Xiphophorus species were significantly more similar than nuclear
genes (table 1). This was particularly true in comparisons
with X. nezahualcoyotl, which was the most divergent species included compared with X. maculatus. As expected,
differences were less or not significant for species closely
related to X. maculatus, such as X. milleri and X. xiphidium.
Even if these data must be completed by other nuclear sequences for extensive comparison, they are consistent with
horizontal transfer of Zisupton in Xiphophorus.
In order to investigate the transposition dynamics of Zisupton, we tested X. maculatus insertion sites by PCR on
genomic DNA of different poeciliid species as well as on
different populations of X. maculatus. Primers were designed for 5# and 3# flanking genomic regions and used
each in combination with a Zisupton-specific internal
primer to test for the presence of the insertion. Primers
for 5# and 3# flanking genomic regions were combined
to test for the presence of ‘‘empty’’ sites without transposon insertion. We performed this test for the six Zisupton
insertions identified in X. maculatus. For all 10 Xiphophorus
species tested, the flanking regions were conserved enough
to be amplified by PCR. For copy V, only the 3# insertion
test could be done since no sequence information was
available for the 5# flanking genomic region.
Results demonstrated that all six genomic sites carrying
a Zisupton insertion in X. maculatus were devoid of insertions in other Xiphophorus species. Similarly, no orthologous insertion was detected in non-Xiphophorus species
(Gambusia affinis, several Poecilia species). We also tested
two X. maculatus populations (Rio Usumacinta and South
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Rio Grijalva) different from the population used to construct the genomic BAC library (Rio Jamapa). Both Rio Usumacinta and Rio Grijalva populations contained Zisupton,
as shown by PCR and Southern blot hybridization (data not
shown). Strikingly, all the above characterized insertions
were Rio Jamapa specific; only empty sites were detected
in the Rio Usumacinta and Rio Grijalva population. None of
the empty sites contained any ‘‘footprint,’’ that is, any small
rearrangement left after transposon excision. These findings indicated recent activity of the transposon after separation of the different populations.
We were able to identify a further Zisupton insertion in
the swordtail X. hellerii. Using only one PCR primer matching the second exon of Zisupton, the 5# flanking region of
an X. hellerii insertion was amplified by single-primer PCR.
The fragment obtained was cloned and sequenced. The
flanking region, showing similarity with the hAT transposon Trilian from the puffer fish T. rubripes (Smit 2002), did
not correspond to any insertion site identified in X. maculatus. PCR assays showed that this insertion was not present in other Xiphophorus species. Importantly, the
sequence of Zisupton in this X. hellerii-specific insertion
is identical over 4 kb with the 5# part of the Y copy of Zisupton in X. maculatus, excluding PCR contamination and
suggesting again interspecific horizontal transfer.
We also tested for the presence of intrapopulational insertion polymorphism in the Rio Jamapa strain of X. maculatus using 24 males and 17 females. The Y-specific
insertion was present in all males and absent from all females tested. No insertion polymorphism was detected for
copies I to IV. In contrast, copy V was apparently absent
from 10 males and 6 females, as tested by ‘‘3# insertion’’
PCR. However, absence of sequence data for the 5# region
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Zisupton Transposon in Xiphophorus · doi:10.1093/molbev/msr208
C2H2
repetitive
motif
SWIM
C2H2
Ulp1
protease
SAP
STOP
ATG
1
2
3
4
5
6
7
8
9
10
11 12
13 14 15
16
17 18 19 20 21 22
23
24
Maculatus
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
11.00
10.00
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
B
E
G
L
O/T
S
M
Embryos
head
tail
15.00
10.00
3.00
2.00
5.00
1.00
0.00
0.00
O/T
S
M
B
E
G
L
O/T
S
M
L
5.00
4.00
3.00
2.00
1.00
0.00
S
B
M
S
M
O6days
E
Clemenciae
G
L
OandE6days
O/T
S
head
tail
500.00
400.00
300.00
200.00
100.00
0.00
B
E
G
L
O/T
S
B
M
E
G
L
O/T
S
M
Couchianus
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
B
M
Embryos
Millerii
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
6.00
O/T
O/T
Variatus
7.00
L
0.00
G
Couchianus
Variatus
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
G
0.50
E
160.00
140.00
120.00
100.00
80.00
60.00
40.00
20.00
0.00
4.00
E
1.00
Xiphidium
5.00
B
1.50
B
6.00
L
2.00
0.00
OandE6days
7.00
G
2.50
300.00
Millerii
20.00
E
400.00
100.00
Xiph idium
B
3.00
200.00
O6days
25.00
Maculatus Embryonic
500.00
E
G
L
O/T
S
M
B
E
G
B
E
G
L
O/T
S
M
Nezahualcoyotl
7.00
12.00
6.00
8.00
4.00
3.00
6.00
2.00
4.00
1.00
2.00
0.00
0.00
B
E
G
L
O/T
S
M
B
E
G
Andersi
L
T/O
S
M
100.00
0.00
B
E
G
S
M
O/T
S
M
0.00
0.00
B
E
G
L
T
S
M
S
T
S
M
8.00
6.00
4.00
400.00
200.00
O/T
10.00
600.00
1.00
L
Hellerii
800.00
2.00
O/T
L
1000.00
3.00
L
200.00
1200.00
4.00
G
300.00
Andersi
5.00
E
400.00
Hellerii
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
B
500.00
400.00
350.00
300.00
250.00
200.00
150.00
100.00
50.00
0.00
10.00
5.00
2.00
0.00
B
E
G
L
O/T
S
M
B
E
G
L
M
FIG. 3. Quantitative RT-PCR expression analysis of Zisupton in the genus Xiphophorus. The expression of Zisupton was tested using two primer
combinations, one reaching from exon 3 to exon 4 (left side, gray box) and the other one from exon 15 to exon 19 (right side, black box).
Amplified fragments are marked with gray and black boxes, respectively. B, brain; E, eye; G, gill; L, liver; O, ovary; T, testis; S, skin; M, muscle;
O6d, ovary 6 days after fertilization; OþEmb, Ovary with embryos 10 days after fertilization; Em, embryos 10 days after fertilization; H, head of
1-day-old fry; and Bo, body of 1-day-old fry. Male tissues are in black, female tissues in white, and embryonic samples in gray.
flanking this insertion did not allow to confirm these results
through ‘‘5# insertion’’ and ‘‘empty site’’ PCR tests.
Taken together, insertion polymorphism analysis in Xiphophorus, added to the presence of noncorrupted (almost) identical copies of Zisupton, suggests that this
transposon was recently active in poeciliids and could have
been subject to horizontal transfer.
Expression Analysis of Zisupton in the Genus
Xiphophorus
First classical RT-PCR experiments (fig. 1) showed that exon
15 of Zisupton is only expressed in testis in X. maculatus.
Without this exon, the Zisupton-transcript contains
a premature stop codon in exon 16. In testis, inclusion
of exon 15 restores the ORF and leads to the production
of a longer protein. In order to know if this splicing and
expression pattern was conserved among Xiphophorus species, we tested several species in quantitative RT-PCR experiments (fig. 3): X. maculatus Rio Jamapa as a reference,
X. xiphidium, X. milleri, X. variatus, X. couchianus, X. andersi
as other platyfishes and the swordtails X. clemenciae, X. nezahualcoyotl, and X. hellerii. The amplified fragments and
primer-binding sites were 100% identical between these
species, allowing direct interspecific comparison of expression levels. We tested two primer combinations for Zisupton: the first combination was located in the 5# part of
637
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Böhne et al. · doi:10.1093/molbev/msr208
Table 1. Comparison of interspecific sequence divergence for Zisupton and two nuclear genes, b-actin and rag1.
Sequence
Zisupton
b-actin
rag1
X.
X.
X.
X.
X.
X.
Species
clemenciae
andersi
milleri
nezahualcoyotl
xiphidium
hellerii
Sequence
length in nt
2785
2785
2785
2785
2785
2785
Synonymous changes in exons/
introns compared with
X. maculatus
0/0
0/0
0/0
0/1
0/3
0/3
X. clemenciae
584
2/6
X. andersi
821
0/2
X. milleri
821
1/2
X. nezahualcoyotl
821
2/7
X. xiphidium
821
0/2
X. hellerii
821
0/4
X. clemenciae
1,573
9/—
X. andersi
1,573
4/—
X. milleri
1,573
1/—
X. nezahualcoyotl
1,573
7/—
X. xiphidium
1,573
4/—
X. hellerii
1,573
8/—
P-value
<0.0001**
<0.0001**
0.052
0.078
0.012*
0.012*
<0.0001**
<0.0001**
0.312
0.699
0.051
0.085
0.0001**
0.0003**
0.017*
0.003*
0.36
0.77
0.004**
0.008**
0.261
0.443
0.022*
0.026*
NOTE.—Sequences were compared with X. maculatus (for Zisupton, the Y-specific sequence was used as a reference). Nucleotide divergence was calculated using only substitutions
at synonymous sites in exons (for all three sequences, first value) and substitutions in intronic sequences (for Zisupton and b-actin, second value). Two-tailed P values (first values:
Fisher’s exact test; second values: chi square with Yates’ correction) are provided to assess if the interspecific divergence observed between nuclear genes is statistically higher than
the divergence observed between Zisupton elements (calculated using the sum of synonymous changes; **extremely/very statistically significant, *statistically significant). Zisupton
and b-actin data have been obtained by PCR; data for exon 3 of rag1 are from Meyer et al. (2006). No intronic sequence was available for rag1.
Zisupton (exon 3 to exon 4, gray box, fig. 3) and the second
one started from the exon specifically expressed in X. maculatus testis (exon 15) until exon 19 (black box, fig. 3).
The observed expression pattern with the first primer
combination located in the 5# part confirmed a broad expression pattern in adult tissue. However, expression was
stronger in testis and male/female brain in all tested species. This part was also expressed in head of 1-day-old fry of
X. maculatus.
With the primer combination amplifying exons 15–19,
exclusive testis-specific expression reaching much higher
expression levels was observed in X. maculatus, X. xiphidium,
X. milleri, X. clemenciae, and X. nezahualcoyotl. Xiphophorus
andersi showed additionally high expression in male brain.
Xiphophorus hellerii displayed lower expression than the
other species tested, with an expression pattern more
similar to that obtained with the first primer combination.
Xiphophorus variatus and X. couchianus, two platyfishes, did
not show any preferential testis expression with the second
primer combination, with overall lower gene expression. All
together, the 3# part is, if strongly expressed, restricted to
testis, with preferential expression in brain in some species.
638
Distribution of Zisupton-Like Transposons
To test if Zisupton is related to any known TE in fish and other
organisms, we used the platyfish Zisupton sequence to search
against available TE databases (Jurka et al. 2005; Kohany et al.
2006 and http://www.repeatmasker.org). Zisupton did not
match any entry and was not recognized as a TE.
To gain insights into the distribution and evolution of Zisupton transposons, available sequence databases were
screened in silico with Zisupton protein sequences as queries.
Zisupton-related sequences were identified in all five sequenced fish genomes (zebrafish, medaka, stickleback,
and the two puffer fishes T. nigroviridis and T. rubripes)
(figs. 4–6). The largest number of Zisupton-related sequences
(about 30 copies) was found in zebrafish. In all these species,
sequences with TIRs and TSDs were identified, suggesting that
Zisupton is also a transposon in other fish species. As observed
in the platyfish, Zisupton inverted repeats are subterminal in
other fish species.
No Zisupton transposon was detected in tetrapods, suggesting loss from this lineage during evolution. Sequences
were found in Ciona (urochordate) as well as in the amphioxus B. floridae (cephalochordate) and the acorn worm
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Zisupton Transposon in Xiphophorus · doi:10.1093/molbev/msr208
S. kowalevskii (hemichordate). In Ciona savignyi, two very related Zisupton sequences (8.3–8.4 kb in length) were found to
be integrated at different positions in the genome (accessions
AACT01001128 and AACT01065526). Presence of empty
sites without insertion in the genome database allowed identifying 8-nt TSDs for both insertions. Imperfect subterminal
inverted repeats were identified for both sequences. Hence,
Zisupton probably behaves as a TE in Ciona too.
In the Zisupton proteins, C2H2 and SWIM zinc fingers
were detected in fish, urochordates, cephalochordates, and
hemichordates (fig. 5). The SAP motif was predicted in fish
and acorn worm, the Ulp1 protease, and F-box domains only
in fish. Interestingly, the amphioxus and acorn worm sequences contained protease domains like Zisupton, but these domains were different. The protease domain in amphioxus was
a C19 protease, also known as ubiquitin carboxyl-terminal hydrolase. The protease in the acorn worm sequence was an
OTU domain, also a cysteine protease (Makarova et al. 2000).
Through PSI-BLAST analysis, divergent Zisupton-related sequences were identified in basidiomycete fungi (Lacaria, Coprinopsis, Serpula, Postia, Moniliophthora, and Cryptococcus),
filamentous brown algae (Ectocarpus), and green algae (Volvox
and Chlamydomonas) (figs. 4–6). Most of these sequences were
multicopy and showed structures typical of TEs. In some cases,
transposon ends and TSDs were precisely characterized
through comparison with empty sites present in the databases.
For instance, in the fungus Lacaria bicolor, two insertions of 12.8
and 13.3 kb displayed imperfect TIRs and 8-nt TSDs (accessions
ABFE01001864 and ABFE01001823).In Postia placenta,another
fungus, a 6-kb sequence with 277-nt TIRs and 8-nt TSDs has
been identified (accession ABWF01004332). In the filamentous
brown alga Ectocarpus siliculosus, a 14.7 kb Zisupton-like transposon with subterminal TIRs and 8-nt TSDs was found in the
genome (accession CABU01009984). Importantly, none of the
sequences recovered through PSI-BLAST did correspond to any
known TE. Only the SWIM domain was detected in Zisupton
proteins from fungi and green algae but not from Ectocarpus
(fig. 4 and 5).
Sequence alignment of predicted Zisupton proteins
from divergent organisms allowed identifying four domains
of higher sequence conservation containing amino acid residues potentially critical for transposition activity. Domain I
contained the SWIM domain. Domains II and III (between
SWIM and SAP domains in fish proteins) to IV (between
SAP and Ulp1 protease domains) did not carry any amino
acid signatures found in other transposon proteins. From
the phylogenetic point of view, sequences from chordates/
hemichordates, fungi, green algae, and brown algae were
well differentiated (fig. 6). The presence of one fish sequence in the Ciona group might be explained by in silico
contamination, presence of several ancient Zisupton lineages in chordates or horizontal transfer.
Domesticated Zisupton Transposons in Vertebrate
Genomes
In puffer fishes, only one Zisupton-related sequence was found
in T. nigroviridis and two in the Fugu T. rubripes. Interestingly,
synteny analysis revealed that the unique Tetraodon sequence
and one Fugu sequence were orthologous and inserted at the
same position in their respective genome. This indicated conservation of an insertion event that must have taken place
before the split of these two species approximately 18–30
Ma (Van de Peer 2004). Neither TIRs nor TSDs were detected,
questioning the functionality of both sequences as transposons. Both orthologous sequences contained an intact coding
region that encoded a putative protein with similarities only
to the N-terminal part of the Zisupton protein (fig. 5). When
we compared the rate of nonsynonymous (Ka) with the rate
of synonymous substitutions (Ks) between both puffer fish
sequences, we obtained a Ka/Ks value of 0.28. This suggested
that they evolved under purifying selection and probably did
not correspond to pseudogenes. Hence, these sequences
might correspond to a ‘‘domesticated’’ form of Zisupton performing functions useful to the host, as reported for other TEs
(for review, see Volff 2006).
A family of vertebrate proteins called HMGXB3 (high
mobility group [HMG] box domain containing 3, also
known as SMF) was found to be related to Zisupton proteins (E value 5 2 1021 for BLAST comparison between
platyfish Zisupton and mouse HMGXB3; figs. 4–6).
HMGXB3 proteins possess an HMG DNA-binding motif
found in a variety of transcription factors that preferentially
bind to distorted DNA (Thomas 2001). Significant similarity with Zisupton proteins was found in conserved domains
II, II, and IV. HMGXB3 proteins lack the SWIM, SAP, ULP1
protease, and F-box domains. Presence of HMGXB3 in fish,
amphibians, birds, and mammals suggested an ancient
event of molecular domestication that occurred at least
450 Ma in a common ancestor of fish and tetrapods.
Discussion
We have described here Zisupton, a fish TE encoding a protein with DNA-binding domains and SUMO protease/
F-box motifs. Related elements were identified in urochordates, cephalochordates, hemichordates, basidiomycete
fungi, filamentous brown algae, and green algae. Zisupton
generally contains introns and does not encode any reverse
transcriptase or other proteins typical of retroelements.
Zisupton does not share any homology with already described TEs at both the nucleotide and protein levels.
We therefore propose that Zisupton represents a new superfamily of DNA transposons. A region containing four
conserved domains, including the SWIM motif, might correspond to a new type of transposase, which remains to be
analyzed at the functional level. No aspartate/aspartate/
glutamate catalytic triad classically found in transposases
and integrases could be unambiguously identified through
comparison of divergent Zisupton proteins.
Some of the predicted functional domains detected in
Zisupton proteins have been found in other transposons.
Ulp1 protease domains are encoded by GIN/Ginger transposons in animals (Bao et al. 2010; Marı́n 2010) and a large
family of Mutator-like TEs in plants (Hoen et al. 2006; van
Leeuwen et al. 2007). A SWIM domain has been identified
in plant MuDr transposases (Makarova et al. 2002). At the
structural level, subterminal inverted repeats found in fish
639
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Böhne et al. · doi:10.1093/molbev/msr208
I
65-556aa
76-198aa
SWIM
II
36-76aa
Platyfish
Ciona
Amphioxus
Saccoglossus
Lacaria
Coprinopsis
Serpula
Postia
Moniliophthora
Cryptococcus
Chlamydomonas
Volvox
Ectocarpus
Hmgxb3_Mouse
:
:
:
:
:
:
:
:
:
:
:
:
:
:
-----NTWHCACTEG-------KRSCTHKATAK-----VTLTGVVKGISTYRKTCPNCS----MVYRYQDWEDGIHNFNDHLLLSIHFC-----YLSFEG
-----GTWSCQCPRASG-----TSTCLHESIAK-----YGINKIWKGTTMITKLCRSCK----CITRFQNYKSGFHNYNNQIILSICLC-----FYHFCA
-----NILNCTCCKI-------KRGCIHKGISK-----VGLQHISTDIEVFVRKCPQCH----HVFRYQEYEDGLLNYNNKTFLTLQLC-----YLLFEV
-----NTWHCRCCK--------KRGCGHKALAR-----VAMNGLIKGVETYCRKCSLCD----LVYRYQEHCDQLHNYDDKWIFSVRLL-----YEHFEA
-----LIVSCMCPASTSRSTGLSASCFHIKFLL-----IYGLNCAWESMIEVQPCGTCA---KRNIGPDCQDLGLFNYDNDRLVTHALM-----WFAYVQ
-----IHTVCTCPHGMED----EVLCIHRRFIE-----VYTLTTVYVHQIQLQRCPQCPPSRRRFIGPDPRHEGLFNYNNSVLVSHELL-----WFAYVS
-----IVVVCFCLR--------GSTCFHVQFFQ-----IYTLTNAWTSVIEVQSCTSCPPHKCCFIGPDLQEHGLFNLNNWLLFTHDLL-----LFAYVQ
-----LGVACTCPLFKA-----QRNCTHRRVLL-----LYGLTMRNQVSIEVYPCPSCHH-HQRFIGPDLGCEGIFNWNNLFLFTHELL-----WFAFVR
-----LTVTCLCPDGDS-----FRRCVHEQFYL-----VYTLTGCISRRIELQQCSQCHQRRHCYVGPEPRDIGLFNYNNTVLFTHELM-----WFAFAT
-----GEKSCDCPAYRS-----NQDCVHVLLYI-----VLHTTGVEKVRLETVRCPRPGC--GRDIGPDLASHGLVNFNNEIGFSRNVF-----FFAFLK
-----PRMTCTCSSFRVVGAA-VSRCLHTAYVS-----VGPVRAHLFELYSGCSCLACVMAYDGVAD------GLFRYSRSSFFCLRHM-----WQQFEA
-----GSPCCSCEIYGTT----GVSCLHLEYVH-----MGPRHAAEYMLRSRCGRSACNIVYDGFGD------GLFRHSSRSYFSLRHM-----WYKFIT
-----GGTGCTSSKKGS-----SGKCVHTHIVG-----LVPGPDLVEAVLKIKGCVDCGV--EAVTGDDWQEHGYFVFGETLLVHLSHM-----YLAFEA
-----KDRTEKTPKAI------EASSPHPDVPS-----LLTANRLQVVTAQVKMCLNPHC--LALHSFTDIYTGLFNVGNKLLVSLDLL-----YWAFEC
Platyfish
Ciona
Amphioxus
Saccoglossus
Lacaria
Coprinopsis
Serpula
Postia
Moniliophthora
Cryptococcus
Chlamydomonas
Volvox
Ectocarpus
Hmgxb3_Mouse
:
:
:
:
:
:
:
:
:
:
:
:
:
:
LSDHDY-QYSCDSCGYHPAIVVMDL-----SGGWAVITCPCGIIYSVKFNLRAESPRD-FADLLLSWKFFPNITVYDYPRGLVSHLKKRCA----LKDFSY-DFNCLKCGHSPKILIADG-----GGGVLHFRCPHGICYYFKTLLRQESARD-FVDGLLSLKQQPIVFISDVASQVAHHGENRKT----LSGHQY-EYYCIRCGHHPPVLIMDL-----SGGLLVAACPHRVVYAAKFLLRGESPRD-FVDILLSMQWQPTVTIADMPQIIAAHGNGRCP----LTDHDY-TFNCVICGSNPPVIITDL-----SGGWLSASCPHRIVYAVKFLIRAESPRD-HVDLIRSFKVQPSVVIVDMPHMVARHGNIRHP----LQCFDG-DMICGVCGPTPEDTIWDG-----TGGLMCVWCPHSVCYGFHNIPKGEGRNDVFSAIVTRWEKAPKRIIYDFACALGPYCMTREP----LQGWEN-DMACKRCGPSPETVIWDG-----TGGIMVAWCTHSICYGFHCIAESEGRDDVFSALVTRWPIAPKRVVYDFACALGPYCMLREP----LQQFNN-DMCCPRCGPLPQETIWDG-----TGGIMCAWCTHSICYGFHCIPNGEGRNDVFSAMITRWPKAPKVVVYDFACALGPYCLTCEP----LQSLDS-KMQCPTCGPCPKVVIADG-----TGGILILXCSHSICLGFHSIPVAEGRNDVFSAIYTHFPQAPDIIIYDFACQLAQYCLVREA----VQEMRD-DMVCPDCGDAPENLIWDG-----TGGIMCAWCTHSICYGFHCIPTGEGRNDVFSAMVTRWPIAPRRVIYDFACALEPYCMTREP----LIGWGT-VMTCPECGPNPEVVIMDG-----TGGIAGIWCPHGICLAYQVMPTSEGRDDFFSLLKCFFPVPPKVIVYDFACSLATYCMLRDP----RLDRFH-TFKCPICDWRPRVLICDA-----TGGIFTVFCEHGVCYAFFVLPRAEGRNEMYSWMVSFLPRAPEVVVYDFACSLHEYCLNRAP----HLDLPY-KFTCPVCGTSPRRLICDG-----TGGVFTLFCEHRICYAFFILPKAEGRNEVYSWMVSYLEKAPDLVVYDFACNLHEYCMNRAP----LTDHPY-GFFSFIDGFFPLLSSFDG-----EGGYRPD----MFMLRAMFMLRAESPTD-LAVLLLHHVHMPILVITDIMCRVGPIVHDRCQ----LTVRDYNDMICGICGVAPKVEMAQR-----TGGKIYKVCPHQVVCGSKYLVRGESARD-HVDLLASSRHWPPVYVVDMATPVALCADLCYP-----
III
149-650aa
IV
92-733aa
FIG. 4. Identification of conserved regions in divergent Zisupton-related proteins. The four regions of higher conservation (I to IV) and the
SWIM domain are shown. Amino acid size variations in nonconserved regions are indicated (size might be affected by models used to predict
gene structure from genomic DNA). Identical residues are white in black boxes, similar residues in black on gray boxes. Accession numbers:
Ciona intestinalis EAAA01002232, Amphioxus Branchiostoma floridae (Florida lancelet) XP_002610779, Saccoglossus kowalevskii
ACQM01108926, Laccaria bicolor ABFE01001864, Coprinopsis cinerea AACS02000001, Serpula lacrymans AEQC01001111, Postia placenta
ABWF01004332, Moniliophthora perniciosa ABRE01017974, Cryptococcus neoformans AAEY01000005, Ectocarpus siliculosus CABU01013191,
Chlamydomonas reinhardtii ABCN01003575, Volvox carteri ACJH01007750, and Hmgxb3 mouse EDL09783.
Zisupton sequences are reminiscent of the structures
found in some transposons from the Mutator superfamily
(Marquez and Pritham 2010 and references therein).
Hence, a distant relationship between Mutator and
Zisupton superfamilies appears to be possible.
The expression pattern of Zisupton is conserved in the
majority of Xiphophorus species tested, with a strong expression in the testis. In addition, initial unpublished data
obtained for Zisupton in zebrafish and medaka also point to
a preferential expression in gonads, and the acorn worm
database sequence was obtained from testis. This is of
course not unexpected for a TE, which must be active
in the germ line to ensure transmission of new insertions
to the next generation. Indeed, preferential activity in male
or female germ line has been reported for many elements
(Weiner et al. 1986; Jurka et al. 2004). In contrast, TE expression is often downregulated in somatic tissues, probably to restrain damage caused by transposition. The
expression of Zisupton in testis is extremely high, especially
in X. maculatus. This might be linked to the presence of one
copy on the Y chromosome, where sequences preferentially expressed in testis are frequently located (Skaletsky
et al. 2003). Depending on alternative splicing, the Zisupton
protein can contain (in testis) or not (in all tissues tested)
the Ulp1 SUMO protease and the F-box domains. Alternative splicing of transposon mRNA generating different
640
transcripts and eliminating premature stop codons in a tissue-specific manner has already been described for other
TEs (Rio 1990; Fujino et al. 2009).
In the genus Xiphophorus, Zisupton is extremely conserved between species having diverged 3–20 Ma (Hrbek
et al. 2007). In addition, all transposon insertions tested
so far were species specific and even population specific
within a same species. Taken together, these observations
indicate that Zisupton has recently been active in Xiphophorus and has possibly been subject to horizontal transfer.
Horizontal transfer has been reported for many elements,
especially DNA transposons. This mode of transmission is
considered as a way to escape vertical extinction in their
original host lineage and to infect nonrelated organisms
(Schaack et al. 2010). However, much more sequence information must be obtained for other cellular genes from
different Xiphophorus species to assess if interspecific sequence divergence for Zisupton is significantly lower than
the divergence between their host genomes.
Within vertebrates, Zisupton elements have been
widely detected in fish. In contrast, they could not be identified through in silico analysis in any tetrapod lineage.
Zisupton-like sequences have also been found in Ciona, amphioxus, and acorn worm. Hence, Zisupton might have
been present in ancestral chordates and has been possibly
lost during early evolution of the tetrapod lineage. This
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Zisupton Transposon in Xiphophorus · doi:10.1093/molbev/msr208
I
II III
IV
C2H2
SWIM
SAP
Ulp1
protease
C2H2
SWIM
SAP
Ulp1
protease
SWIM
SAP
Ulp1
protease
C2H2
SWIM
SAP
?
C2H2
C2H2
SWIM
SAP
Tetraodon
Takifugu
C2H2
C2H2
SWIM
Amphioxus
C2H2
C2H2
SWIM
C2H2
SWIM
C2H2
SWIM
Platyfish
C2H2
Zebrafish
C2H2
Medaka
C2H2
Stickleback
C2H2
Takifugu
Ciona
repetitive
motif
C2H2
C19
protease
SAP
Fungi
SWIM
SAP
Chlamydomonas
Volvox
SWIM
SAP
OTU
domain
Ectocarpus
HMGXB3
HMGB
FIG. 5. Schematic representation of conserved domains in Zisupton-related proteins. Predicted functional motifs within proteins are shown, as
well as the four conserved regions, which are represented by vertical gray bars (I to IV, fig. 4).
observation confirms that fish genomes contain many
more families of TEs than tetrapod genomes and that
the evolution of the lineage leading to mammals has undergone a drastic reduction in TE diversity (Volff et al. 2003;
Fischer et al. 2005; Böhne et al. 2008). In fish, Zisupton
seems to have experienced differential success in different
lineages. This element is present with several highly conserved and probably intact copies in Xiphophorus genomes
and is represented by 20–30 copies in medaka and zebrafish. In contrast, Zisupton is rare in stickleback and has apparently lost its ability to transpose in puffer fishes.
The unique sequence of Tetraodon and one sequence of
Fugu are located at the same position in syntenic regions of
their respective genomes. These sequences are much shorter than other fish Zisuptons and do not have any TIRs and
TSDs. Such a conservation between a transposition-
deficient TE in species having diverged approximately
18–30 Ma (Van de Peer 2004) would be quite unusual
for simply dead elements. Rather, we suggest that they correspond to bona fide cellular genes derived from a Zisupton
transposon. In vertebrates and many other lineages, numerous host genes with important cellular functions have
been derived from TEs. This phenomenon is called molecular domestication (Volff 2006 and references therein;
Sinzelle et al. 2009). Accordingly, Ka/Ks analysis indicated
an evolution under purifying selection, suggesting that
puffer fish Zisupton-like orthologous sequences do not
correspond to pseudogenes.
Interestingly, Zisupton shows phylogenetic relationship
with bona fide cellular genes from the vertebrate HMGXB3
family. One unique orthologous HMGXB3 gene is present
within syntenic genomic regions in all major vertebrate
641
MBE
Böhne et al. · doi:10.1093/molbev/msr208
Ectocarpus
Hmgxb3-Tetraodon
Hmgxb3-Fugu Hmgxb3-Medaka
Hmgxb3-Zebrafish
Hmgxb3_Stickleback
Ciona2
Ciona1
Ciona3
Hmgxb3_Chicken
Hmgxb3_Xenopus
Hmgxb3_Mouse
Hmgxb3_Human
Ciona4
Stickleback
Amphioxus
90/83
100/99
56/-
Saccoglossus
70/73
40/90
Medaka2
Medaka1
79/73
Fugu
Platyfish
Zebrafish3
Zebrafish4
Zebrafish2
Zebrafish5
Zebrafish6
Zebrafish1
Tetraodon-dom.
Fugu-dom.
100/100
77/56
Cryptococcus
77/99
70/-
Postia
Serpula2
80/Serpula1
Chlamydomonas3
Serpula4
Chlamydomonas4
Serpula3
Chlamydomonas1
Lacaria2
Moniliophthora2
Chlamydomonas2 Volvox Lacaria1
Moniliophthora1
Lacaria3
Coprinopsis4 Coprinopsis1
Coprinopsis3
Coprinopsis2
FIG. 6. Molecular phylogeny of Zisupton-related proteins. The tree was constructed using maximum likelihood analysis with 100 bootstrap
replicates (first values) on concatenated conserved regions shown in figure 4. Bootstrap values in italics (second values) have been obtained
using neighbor joining reconstruction (1,000 bootstrap replicates).
lineages including fish, amphibians, birds, and mammals.
The evolutionary relationship between HMGXB3 and Zisupton remains to be elucidated. Because of the presence of
Zisupton sequences in nonvertebrate chordates, hemichordates, fungi, and algae, we propose that HMGXB3 has been
derived from a Zisupton TE via an event of molecular domestication in a common ancestor of fish and tetrapods at
least 450 Ma. Alternatively, the Zisupton transposon might
have been formed from an HMGXB3-like gene and transmitted to more divergent organisms through horizontal
transfer.
Consistent with a fish-specific acquisition of the Cterminal region, the Ulp1 SUMO protease/F-box region
was detected only in fish Zisupton sequences. The amphioxus
sequence contains a C19 protease, also known as ubiquitin
carboxyl-terminal hydrolase. This protease is involved in the
processing of poly-ubiquitin precursors and ubiquitinated
proteins, a function similar to that of Ulp1 in the SUMO
pathway (Jentsch et al. 1991; Barrett and Rawlings 2001).
In the acorn worm, the protease domain is an OTU protease
(cysteine protease) (Makarova et al. 2000). The evolution of
TEs is often modular; different regions in one element can
have different evolutionary origins (Lerat et al. 1999; Capy
and Maisonhaute 2002). Accordingly, Zisupton might have
captured an Ulp1 SUMO protease gene during evolution,
possibly early in the fish lineage. The closest cellular proteases
642
related to Zisupton are SENP proteins, sentrin-specific proteases (sentrin is another name for SUMO). Sentrin proteases
catalyze the removal of SUMO from its substrates as well as
its activating cleavage from a precursor form (Drag and
Salvesen 2008; Yeh 2009). Hence, Zisupton transposons might
have recruited a sentrin-specific protease, together with
a protein–protein interaction domain called F-box, which
is present in the C-terminal region too. F-box proteins are
implicated in the ubiquitin pathway in the ligation step of
ubiquitin (Skowyra et al. 1997). The C-terminal part of
the Zisupton protein might interfere with ubiquitin/SUMO
posttranslational protein modification and enhance the
transposition efficiency of the element, for example, through
the inhibition of protein degradation in the proteasome. Accordingly, ubiquitin/SUMO pathways have been shown to
modulate the activity of several TEs (Kang et al. 1992; Menees
and Sandmeyer 1996).
To conclude, Zisupton represents a new type of DNA
transposon active in fish and detected in various divergent
organisms. This element does not share any obvious similarity with already described TEs. Enzymes catalyzing its transposition must be now characterized at the functional level.
Zisupton distribution, evolutionary dynamics, and effects on
genomes outside of poeciliid fish must be further analyzed.
Of particular interest are its potential ability to be transferred
horizontally between species and to serve as a source for the
Zisupton Transposon in Xiphophorus · doi:10.1093/molbev/msr208
formation of novel genes, the function of which is still to be
elucidated. From all these features, Zisupton has the potential of a powerful agent of genomic diversity and evolutionary novelties in fish and other organisms.
Acknowledgments
This work was supported by a European Mobility PhD fellowship from the French Ministry of National Education,
Research and Technology to A.B. and by grants from the
BioFuture program of the Bundesministerium für Bildung
und Forschung (BMBF), the Agence Nationale de la
Recherche (ANR), the Institut National de la Recherche
Agronomique (INRA, département PHASE), the Centre
National de la Recherche Scientifique (CNRS), and the Fondation pour la Recherche Médicale (FRM) to J.-N.V.
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