RESEARCH Open Access Crypton transposons: identification ...Crypton domestication, and one of them,...

RESEARCH Open Access

Crypton transposons: identification of new diversefamilies and ancient domestication eventsKenji K Kojima and Jerzy Jurka*

Abstract

Background: “Domestication” of transposable elements (TEs) led to evolutionary breakthroughs such as the originof telomerase and the vertebrate adaptive immune system. These breakthroughs were accomplished by theadaptation of molecular functions essential for TEs, such as reverse transcription, DNA cutting and ligation or DNAbinding. Cryptons represent a unique class of DNA transposons using tyrosine recombinase (YR) to cut and rejointhe recombining DNA molecules. Cryptons were originally identified in fungi and later in the sea anemone, seaurchin and insects.

Results: Herein we report new Cryptons from animals, fungi, oomycetes and diatom, as well as widely conservedgenes derived from ancient Crypton domestication events. Phylogenetic analysis based on the YR sequencessupports four deep divisions of Crypton elements. We found that the domain of unknown function 3504 (DUF3504)in eukaryotes is derived from Crypton YR. DUF3504 is similar to YR but lacks most of the residues of the catalytictetrad (R-H-R-Y). Genes containing the DUF3504 domain are potassium channel tetramerization domain containing1 (KCTD1), KIAA1958, zinc finger MYM type 2 (ZMYM2), ZMYM3, ZMYM4, glutamine-rich protein 1 (QRICH1) and“without children” (WOC). The DUF3504 genes are highly conserved and are found in almost all jawed vertebrates.The sequence, domain structure, intron positions and synteny blocks support the view that ZMYM2, ZMYM3,ZMYM4, and possibly QRICH1, were derived from WOC through two rounds of genome duplication in earlyvertebrate evolution. WOC is observed widely among bilaterians. There could be four independent events ofCrypton domestication, and one of them, generating WOC/ZMYM, predated the birth of bilaterian animals. This isthe third-oldest domestication event known to date, following the domestication generating telomerase reversetranscriptase (TERT) and Prp8. Many Crypton-derived genes are transcriptional regulators with additional DNA-binding domains, and the acquisition of the DUF3504 domain could have added new regulatory pathways viaprotein-DNA or protein-protein interactions.

Conclusions: Cryptons have contributed to animal evolution through domestication of their YR sequences. TheDUF3504 domains are domesticated YRs of animal Crypton elements.

Keywords: tyrosine recombinase, Crypton, domestication, transposon, DUF3504

BackgroundThe structural and mechanistic variety of transposable ele-ments (TEs) is well-documented [1]. They encode proteinsthat include diverse functional domains involved in cataly-sis or interaction with DNA, RNA and other proteins.Because of this diverse repertoire, TEs can supply func-tional modules to generate new genes. “Molecular domes-tication” of transposable elements [2] led to evolutionary

milestones such as the origin of telomerase and the verte-brate adaptive immune system. Telomerase reverse tran-scriptase (TERT) provides a solution for end replicationproblems accompanying linear chromosome replicationand was derived from a reverse transcriptase (RT) relatedto Penelope-like elements in the very early stage of eukar-yote evolution [3,4]. V(D)J recombination is a mechanismused in jawed vertebrates to generate a variety of immuno-globulins and T-cell receptors. It is catalyzed by therecombination activating gene 1 (RAG1) derived from atransposase encoded by the Transib family of DNA trans-posons [5]. Different kinds of transposon proteins were

* Correspondence: [email protected] Information Research Institute, 1925 Landings Drive, Mountain View,CA 94043, USA

Kojima and Jurka Mobile DNA 2011, 2:12http://www.mobilednajournal.com/content/2/1/12

© 2011 Kojima and Jurka; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

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domesticated, including transposase, integrase, RT, envel-ope and gag proteins [6]. Herein we report in-depth stu-dies of another type of transposon enzyme, tyrosinerecombinase (YR), which was repeatedly domesticated inthe history of animals.To date four types of enzymes are known to catalyze

DNA integration of eukaryotic transposons: DDE-trans-posase, YR, rolling-circle replication initiator and thecombination of RT and endonuclease (EN) [7]. DDE-transposase is the most abundant gene in nature [8] andis carried by many DNA transposon superfamilies, self-synthesizing transposons (Polinton), as well as long term-inal repeat (LTR) retrotransposons (Gypsy, Copia, BELand endogenous retroviruses) [1,9-11]. They share threeconserved amino acids (DDD or DDE) at their catalyticsites, which are separated by amino acid sequences ofvarying length. Some domesticated DDE-transposasesbecame DNA-binding proteins, such as CENP-B in mam-mals and Daysleeper in Arabidopsis thaliana [12,13].Non-LTR retrotransposons and Penelope-like elementsuse a combination of RT and EN in their transposition[14-17]. Helitron is the only group of eukaryotic transpo-sons encoding rolling-circle replication initiator [9].YR genes are ubiquitous in prokaryotes but rare in

eukaryotes [18,19]. All YRs found in eukaryotes areencoded by mobile elements: yeast 2-micron circle plas-mids [20], ciliate Euplotes crassus transposons (Tec1,Tec2 and Tec3) [21,22], three groups of retrotransposons(DIRS/Pat, Ngaro and VIPER) [23-25], and Cryptons [19].The YR encoded by the yeast 2-micron plasmid, knownas “flippase” (FLP), is widely used for site-specific recom-bination in the FLP-FRT system [26]. Tec1 and Tec2transposons encode a DDE-transposase in addition toYR, and therefore the YR domains in these transposonsare probably involved in resolving transposition inter-mediates. To date the only YR-encoding transposonsfound in the vertebrate genomes are DIRS and Ngaro ret-rotransposons. Cryptons were originally found in a basi-diomycete Cryptococcus neoformans and severalpathogenic fungi. Their boundaries are difficult to char-acterize because they have neither terminal invertedrepeats (TIRs) nor long direct repeats. Instead they haveshort direct repeats at both termini. These 4- or 6-bpdirect repeats are considered substrates for recombina-tion. By analogy to prokaryotic YR-encoding transposons,Goodwin et al. [19] proposed that Cryptons are excisedfrom the host genome as an extrachromosomal circularDNA and integrated at a different locus in the genome.YR typically recognizes recombination sites consisting oftwo inverted repeats that are 11 to 13 bp long and sepa-rated by a segment 6 to 8 bp long [27]. Recently, transpo-sons encoding only a YR have been found in sea urchin,insects and cnidarians and classified as Cryptons [28,29].YR contains four catalytically important residues (R-H-R-

Y), but their overall sequence identity is very low amongdifferent genes and transposons [18,19]. The conservedtyrosine residue directly binds to DNA in the recombina-tion reaction. In this paper, we report Cryptons from var-ious species, including medaka fish, and six human genesoriginated from ancient domestication events of CryptonYRs.

ResultsThe diversity of Crypton elements in terms of theirsequence and domain structureWe identified 94 Crypton elements from 24 speciesrepresenting animals, fungi and stramenopiles thatinclude oomycetes and diatom (Figure 1, Table 1 andAdditional file 1). Phylogenetic clustering of Cryptons onthe basis of their YR domain sequences revealed fourgroups reflecting the systematics of their hosts (Figure 2,open circles), but two of them were not strongly sup-ported phylogenetically because of the low bootstrapvalues. Herein we designate them as CryptonF, CryptonS,CryptonA and CryptonI to indicate their correspondinghosts: fungi, stramenopiles, animals and insects. Cryp-tonA and CryptonI are structurally similar; however,CryptonF, CryptonS and CryptonA/CryptonI have distinctprotein domain structures (see Figure 1 and detaileddescription in the next three sections). Because of thelow resolution of the phylogenetic tree, we could notdetermine whether there is any relationship betweenthese four Crypton groups and to other YR-encoding ele-ments, and we cannot rule out the possibility that theyhave originated independently.

CryptonF elements from fungi and oomycetes, andCryptonF-derived genesWe identified CryptonF elements in nine species of fungiand four species of oomycetes (Table 1 and Additional file1). These elements encode a protein that includes YR andGCR1_C DNA-binding domains (Figure 1). Most of thefungal Cryptons and the five oomycete Cryptons are asso-ciated with 6-bp terminal direct repeats, which are likelysubstrates for Crypton integration (Additional file 1). InFusarium oxysporum, Crypton is fused with a Mariner-type DNA transposon and this composite transposon ishearafter named MarCry-1_FO (Figure 1). The analysis offour MarCry-1_FO copies with more than 97% identity toeach other revealed the presence of 16-bp TIRs and targetsite duplications (TSDs) of the TA dinucleotide, indicatingthat their Mariner-type DDE-transposase is responsiblefor transposition. CryptonF-2_PS from Phytophthora sojaeand related elements encode a C48 peptidase (Ulp1 pro-tease) in addition to a YR (Figure 1). The oomycete Cryp-tonF elements are nested in fungal CryptonF elements inthe phylogenetic tree (Figure 2), indicating a horizontaltransfer between fungi and oomycetes.


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Four genes from Saccharomyces cerevisiae were derivedfrom CryptonF elements (Figure 3 and Additional files 2and 3). It was previously reported that the GCR1_C pro-tein domain encoded by Gcr1, Msn1 and Hot1 genes issimilar to the C-terminal part of fungal Cryptons [19]. Inaddition to these three genes, we found that Cbf2/Ndc10contains a C-terminal domain similar to CryptonF pro-teins. The central portions of Cbf2 and Gcr1 are similarto CryptonF YR domains, but the catalytic site is not pre-served (data not shown). Vanderwaltozyma polysporacarries two paralogous genes of Gcr1 and Msn1. Candidatropicalis and related species (Candida albicans, Pichiastipitis and Pichia guilliermondii) harbor another genederived from a CryptonF element, represented by

XP_002548716 in C. tropicalis. It is designated herein asCrypton-derived gene 1 (Cdg1) (Figure 3). The onlydomain shared by CryptonF elements and all Crypton-derived genes is the GCR1_C domain. The phylogeneticanalysis of GCR1_C domains (Figure 3C) indicates thatHot1 and Msn1 are paralogous and that the gene relatedto Hot1/Msn1 in C. tropicalis represents an outgroup ofboth genes. Therefore, it is likely that four domesticationevents (for Hot1/Msn1, Gcr1, Cbf2 and Cdg1) occurred inthis group.We could not find any Crypton insertions in the sub-

phylum Saccharomycotina (including S. cerevisiae,C. tropicalis and related species). The distribution ofCrypton-derived genes indicates that Crypton was active

GCR1_C Crypton-Cn1

YR

600 bp/200 aa

MarCry-1_FO GCR1_C DDE YR

CryptonF-2_PS GCR1_C C48 YR

CryptonS-1_PI YR

CryptonS-5_PR HTH C48 YR

CryptonA-1_OL YR

CryptonI-1_RPro YR

intron

Figure 1 Schematic structures of Cryptons. Crypton-Cn1 and MarCry-1_FO belong to the CryptonF group. YR = tyrosine recombinase; GCR1_C =DNA-binding domain; DDE = DDE-transposase; C48 = C48 peptidase; HTH = helix-turn-helix motif.

Table 1 Distribution of Crypton elements

Classification Phylum orClass

Speciesa

Fungi Basidiomycota Cryptococcus neoformans [19]

Ascomycota Coccidioides posadasii [19], Histoplasma capsulatum [19], Chaetomium globosum, Fusarium oxysporum, Ajellomycescapsulatus, Coccidioides immitis, Microsporan canis, Talaromyces stipitatus, Neosartorya fischeri

Zygomycota Rhizopus oryzae

Animals Chordata Oryzias latipes

Echinodermata Strongylocentrotus purpuratus [28]

Hemichordata Saccoglossus kowalevskii

Mollusca Lottia gigantea

Arthropoda Nasonia vitripennis [29], Tribolium castaneum [29], Rhodnius prolixus, Aedes aegypti, Culex quinquefasciatus

Cnidaria Nematostella vectensis [28]

Stramenopiles Oomycetes Phytophthora infestans, Phytophthora sojae, Phytophthora ramorum, Pythium ultimum, Saprolegnia parasitica,Hyaloperonospora arabidopsidis, Albugo laibachii

Diatoms Phaeodactylum tricornutumaCrypton elements from species without references are found in this study.


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CryptonI

CryptonS

CryptonF

CryptonA &DUF3504 genes

KCTD1

KIAA1958b/L

WOC/ZMYM/QRICH1

KIAA1958a

Figure 2 Phylogeny of Cryptons, DUF3504 genes and other eukaryotic tyrosine recombinases. The numbers at nodes are bootstrap valuesover 40. Open circles indicate the clusters of Cryptons, and filled circles show the clusters of DUF3504 genes. YR = tyrosine recombinase. Prefixes ofnames are as follows. Cry = Crypton; 1958 = KIAA1958. Accession numbers of DUF3504 genes are shown in Additional file 5. Sequences of thetransposable elements are deposited in Repbase http://www.girinst.org/repbase/. Other abbreviations and accession numbers are as follows. FLP =FLP recombinase of the 2-micron plasmid in Saccharomyces cerevisiae (NP_040488); FLP_Klac = FLP recombinase of the plasmid pKD1 inKluyveromyces lactis (YP_355327); CRE = Cre recombinase of the enterobacteria phage P1 (YP_006472); Vlf1_AcNPV = very late expression factor 1from the Autographa californica nucleopolyhedrovirus (NP_054107); Tn916 = Tn916 transposase from Enterococcus faecalis (NP_0687929); XerD =XerD from Escherichia coli (NP_417370); Lambda = lambda phage recombinase (NP_040609); At_Ti = recombinase from the Agrobacteriumtumefaciens Ti plasmid (NP_059767); SpPat1 from Strongylocentrotus purpuratus (obtained at http://biocadmin.otago.ac.nz/fmi/xsl/retrobase/home.xsl). Suffixes for species names are as follows. Animals: Hs = human, Homo sapiens; Oa = platypus, Ornithorhynchus anatinus; Gg = chicken, Gallusgallus; Tg = zebra finch, Taeniopygia guttata; Ac/ACa = lizard, Anolis carolinensis; Xt/XT = frog, Xenopus tropicalis; Dr/DR = zebrafish, Danio rerio; OL =medaka, Oryzias latipes; Cm = chimaera, Callorhinchus milii; SP = sea urchin, Strongylocentrotus purpuratus; SK = acorn worm, Saccoglossuskowalevskii; Dm = fruit fly, Drosophila melanogaster; Tc/TC/TCa = beetle, Tribolium castaneum; NVi = parasitic wasp, Nasonia vitripennis; CQ =southern house mosquito, Culex quinquefasciatus; AA = yellow fever mosquito, Aedes aegypti; DPu = water flea, Daphnia pulex; Acal = sea hare,Aplysia californica; Sm = bloodfluke, Schistosoma mansoni; NV = sea anemone, Nematostella vectensis. Fungi: RO = Rhizopus oryzae; CGlo =Chaetomium globosum; TS = Talaromyces stipitatus; CI = Coccidioides immitis; FO = Fusarium oxysporum. Stramenopiles: PI = Phytophthora infestans;PS = Phytophthora sojae; PU = Pythium ultimum; HAra = Hyaloperonospora arabidopsidis; ALai = Albugo laibachii; PTri = Phaeodactylum tricornutum.Plants: CR = Chlamydomonas reinhardtii.


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http://www.girinst.org/repbase/

http://biocadmin.otago.ac.nz/fmi/xsl/retrobase/home.xsl

http://biocadmin.otago.ac.nz/fmi/xsl/retrobase/home.xsl

GCR1_CCrypton-Cn1

YR

HOT1

MSN1

GCR1

CBF2/NDC10

CDG1

500 aa

Saccharomyces cerevisiae

Candida glabrata

Vanderwaltozyma polyspora

Zygosaccharomyces rouxii

Lachancea thermotolerans

Kluyveromyces lactis

Ashbya gossypii

Candida tropicalis

Gcr1 Msn1 Hot1 Cbf2 Cdg1

+ + + +

+ + + +

++ ++ +

+ + +

+ + + +

+ + + +

+ + + +

+ +

417 MYA

311 MYA

A

B

C

Msn1

Hot1

Cbf2

Cdg1

Gcr1

Figure 3 Distribution and schematic structures of Crypton-derived genes in Saccharomycetaceae fungi. (A) Schematic protein structuresencoded by Crypton-derived genes and Cryptons. (B) Distribution of Crypton-derived genes. Each gene identified in the haploid genome isrepresented by a plus symbol. (C) The phylogeny of Crypton-derived genes and Cryptons using the GCR1_C domain sequences. The numbers atnodes are bootstrap values over 50. Accession numbers of genes are shown in Additional file 2. “Cry” stands for Crypton. Suffixes for species namesare as follows. Sc = Saccharomyces cerevisiae; Cg = Candida glabrata; Vp = Vanderwaltozyma polyspora; Zr = Zygosaccharomyces rouxii; Lt =Lachancea thermotolerans; Kl = Kluyveromyces lactis; Ag = Ashbya gossypii; Ct = Candida tropicalis; Ca = Candida albicans; Ps = Pichia stipitis; Pg =Pichia guilliermondii.


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in the past and that the DNA-binding domain GCR1_Cwas most likely derived from Cryptons.

CryptonS, a new group of Cryptons from oomycetes anddiatomWe found CryptonS elements in seven oomycete and onediatom species (Figure 1, Table 1 and Additional file 1).CryptonS elements do not encode any GCR1_C domain,but the C-terminal region is conserved among CryptonSelements. CryptonS elements are associated with 5- or 6-bp terminal direct repeats. The majority of CryptonS ele-ments share TATGG termini. Some CryptonS elementsencode an additional protein containing a C48 peptidasedomain. The peptidases encoded by CryptonS and Cryp-tonF elements in oomycetes belong to the same family andare related to the Ulp1 protease family. Domain shufflingbetween two groups of Crypton elements could explainthe similarity, but more data are needed to determine therelationship between these peptidases and other cellularpeptidases.

Cryptons in animals (CryptonA and CryptonI groups)We identified Cryptons in seven metazoan animals belong-ing to five phyla (Table 1 and Additional file 1). CryptonIelements were found only in insects, whereas CryptonAelements were found in various animals, including cnidar-ians. Animal Cryptons (both CryptonA and CryptonI) haveno C-terminal domain (Figure 1). We did not find anyterminal repeats in animal Cryptons. CryptonI-1_RProfrom Rhodnius prolixus hosts a non-autonomous deriva-tive family, CryptonI-1N1_RPro, in which 5’ 438 bp and 3’260 bp are 98% identical to those of CryptonI-1_RPro.This is the first report of non-autonomous Crypton ele-ments. Comparison of 50 copies of CryptonI-1_RPro andCryptonI-1N1_RPro revealed no terminal repeats (neitherdirect nor inverted). In medaka, we also found two familiesof non-autonomous derivatives (CryptonA-1N1_OL andCryptonA-1N2_OL) of CryptonA-1_OL. As in the case ofother DNA transposons, Crypton non-autonomous ele-ments are much more abundant than their autonomouscounterparts.We can safely rule out the theoretically possible contam-

ination of the genomic sequences from medaka used inthis study. First, we identified more than 2,700 copies ofautonomous and non-autonomous Crypton elements withDNA sequence identities to consensus ranging from 59%to 98%. The nucleotide diversity of Cryptons from medakais consistent with their long-term presence in the medakalineage. Second, we found many Crypton sequences in thedatabase of expressed sequence tags (ESTs) from three dif-ferent medaka strains: Hd-rR, CAB and HNI (data notshown). We also found several Cryptons with insertedmedaka-specific transposons such as piggyBac-N1_OL andRTE-1_OL (Table 2).

Crypton-derived sequences in the ATF7IP geneIdentification of Cryptons in three deuterostome species(medaka, sea urchin and acorn worm) prompted us toextend analysis of Cryptons in chordates, including foursequenced actinopterygian species (Fugu rubripes, Tetrao-don nigroviridis, Gasterosteus aculeatus and Danio rerio).Although multiple copies of Crypton elements were foundonly in medaka, sequences similar to Cryptons were foundin various chordate species (Table 3). Most of them do notencode any functional recombinases, owing to frameshifts,deletions and substitutions at catalytically essentialresidues.However, two similar sequences (ABQF01015803 from

the zebra finch Taeniopygia guttata and AAVX01068049from the chimaera (elephant shark) Callorhinchus milii)include an intact open reading frame of YR (Figure 4A).We did not further analyze the sequence from chimaera,because the sequenced region was only 2,661 bp inlength. The Crypton-like sequence in zebra finch is insidean intron of a gene coding for activating transcriptionfactor 7 interacting protein (ATF7IP) (Figure 4B). Thereis a YR sequence at the orthologous locus of chicken Gal-lus gallus, which encodes a protein 97% identical to thatof zebra finch, but it contains a frameshift inside the YRregion. The orthologous YR sequence from the turkeyMeleagris gallopavo contains a frameshift at the sameposition (data not shown). Because the divergencebetween chicken and zebra finch occurred some 107 mil-lion years ago (MYA) [30], this unusually high similarityindicates a strong selection operating on these YRsequences. An exon-intron prediction program wouldpredict alternative splicing in the ATF7IP gene fromzebra finch, although at present there are no mRNA orESTs corresponding to the fusion transcript. It is possiblethat the YR is translated as part of the ATF7IP proteinand retains catalytic activity in some birds.Using the University of California Santa Cruz (UCSC)

Genome Browser http://genome.ucsc.edu/, we foundthat there are partial Crypton sequences at the ortholo-gous positions of the ATF7IP gene from the human,horse, kangaroo and platypus genomes (Figures 4B and4C). There are also closely related sequences present inthe genomes of rhesus macaque and tarsier. Therefore,the insertion of Crypton in the ATF7IP gene must haveoccurred in the common ancestor of amniotes morethan 325 MYA [30]. None of the mammalian ortholo-gous sequences encode intact YR proteins, and manymammalian species are missing the YR sequence. Thisindicates only a slight, if any, selective pressure on thissequence in mammals.

Ancient domestication of Cryptons in animalsMost vertebrate genes similar to Crypton code for pro-teins (Additional file 4). In the human genome, there are


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http://genome.ucsc.edu/

seven proteins similar to Crypton YRs, which are anno-tated as parts of six genes (Figure 5 and Additional file5). The KIAA1958 gene contains two isoforms, both ofwhich include YR-derived sequences. The other genesare potassium channel tetramerization domain contain-ing 1 (KCTD1), zinc finger, myeloproliferative and mentalretardation type 2 (ZMYM2)/zinc finger protein 198(ZNF198), ZMYM3/ZNF261, ZMYM4/ZNF262 and glu-tamine-rich protein 1 (QRICH1) (Figure 5). A PSI-BLAST search of these proteins against the NationalCenter for Biotechnology Information (NCBI) conserveddomain database (CDD) revealed that they share adomain of unknown function (DUF3504 superfamily;E-value ≤ 1e-29). The six genes are widespread amongvertebrates (Figure 6) and are highly conserved amongphylogenetically distant species (Table 4). The phyloge-netic relationship of each gene agreed with that of species(data not shown). The nucleotide sequences corresponding

to all seven DUF3504 domains were present in the NCBIEST database, indicating their expression. The data clearlyshow that they are neither pseudogenes nor defective Cryp-tons (see the accession numbers of DUF3504 genes inAdditional file 5). However, none of them preserve the YRcatalytic site. All of them lost the catalytic tyrosine and thesecond conserved arginine, and all but KCTD1 also lostthe conserved histidine.Although the resolution is low because of high diver-gence and the short length of the YR sequence, animalDUF3504 genes tend to cocluster with animal Cryptons(CryptonA) in the YR phylogenetic tree (Figure 2). Thereare four independent clusters of DUF3504 genes:KCTD1, KIAA1958a, KIAA1958b/KIAA1958L and WOC/ZMYM/QRICH1 (Figure 2, filled circles). KCTD1 coclus-ters with several animal Cryptons, and the clustering issupported by 100% bootstrap value. Cryptons form aparaphyletic cluster, which indicates that the DUF3504

Table 2 Crypton copies containing insertions of other transposable elements

Chromosome Starta Enda Element Startb Endb Directionc Identityd

chr1 35100344 35100443 Crypton-1N1_OL 470 573 c 0.8614

35100444 35100648 piggyBAC-N1_OL 1 204 d 0.9512

35100649 35101118 Crypton-1N1_OL 1 474 c 0.8728

chr23 4844226 4844361 Crypton-1N1_OL 1 147 d 0.9489

4844362 4844566 piggyBAC-N1_OL 1 205 d 0.9854

4844567 4844980 Crypton-1N1_OL 144 570 d 0.9294

chr23 7640079 7640510 Crypton-1N1_OL 1 441 d 0.8918

7640511 7640716 piggyBAC-N1_OL 1 205 d 0.9466

7640717 7640832 Crypton-1N1_OL 438 573 d 0.8814

chr5 2033028 2033143 Crypton-1N1_OL 3 116 d 0.8966

2033144 2033348 piggyBAC-N1_OL 1 205 c 0.9659

2033349 2033803 Crypton-1N1_OL 113 573 d 0.9132

chr5 22193852 22194261 Crypton-1N1_OL 144 573 c 0.9030

22194262 22194466 piggyBAC-N1_OL 1 205 d 0.9707

22194467 22194605 Crypton-1N1_OL 1 147 c 0.9078

chr8 7666866 7667209 Crypton-1N1_OL 21 376 d 0.9075

7667210 7667896 RTE-1_OL 2,666 3,352 c 0.9796

7667897 7668065 Crypton-1N1_OL 372 550 d 0.8667aSequence coordinates in the corresponding chromosome. bCorresponding coordinates of the consensus sequences of repeat elements from Repbase. cSequenceorientation relative to consensus: (d)irect and (c)omplementary orientation. dIdentity to the consensus sequences.

Table 3 Molecular fossils of Cryptons in chordates

Species Accession numbers

Danio rerio BX530066*

Xenopus tropicalis NP_001120376, AAMC01135377*, AAMC01082917*

Callorhinchus milii AAVX01521991*, AAVX01068049*, AAVX01132927*

Ciona intestinalis XP_002124034, XP_002125964

Ciona savignyi AACT01002283*, AACT01041791*

Halocynthia roretzi BAB40645

Oikopleura dioica CBY34656

Branchiostoma floridae XP_00260067, XP_002595788, XP_002613958, XP_002613959, XP_002587732, XP_002607491

*Nucleotide sequences including Crypton fragments.


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R

H R Y

A

B

C

ATF7IP_ChickenATF7IP_ZebrafinchAAVX01068049Crypton-1_SPCryptonA-2_SPCryptonA-1_SKCryptonA-2_SKCryptonA-3_SKCryptonA-1_OL




111111111

Q L S E Q K V V R C V P Y T C G I F D G T T L I N C F E K Q L K Q E E T S S E N K E A V E A AQMN F N F P E D V L F G L E E E E E D A K S I K N T H K Q T GWA A N L L K QW L A K N - G K D P - S F E L V P V S E L N D IQ L S D H K V V R C V P Y T C G I F D G T T L I N C F E K Q L K Q E E T P S E N K E A V E A AQ T N F N F P E D V L F G L E E D E E D A K S I K N T H K Q T GWA A N L L K QW L A K N - G K D P - S F E L V P V S E L N D I- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M T E DQ L A G D E L L D A A R R H V S V T E E D L I V L E E E R N E - - - - K N T R K Q T DWA V N I L K QW L I E K - GQ D E - H F E V M S V E E L N G V- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M A E L E T L P R F Q L L D D DQM R D L I D S A D S - - - - K N T K N S V K F S M K I F E D F L Q V I - N T D L D S V N K L S N S E L D N V- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M A A A A F K V P T F E I L S T A QMQ E L L N G T D S - - - - A S T K K S I K F G F A K F E A F L G L K - Q I E L G E I - T T D R L R L D D VN D C H P T A H N K L S F A D F V F V R R R C Y F F V - - - - - - - - - - - - - - - - V M A S K R H A E L S P S E L D N I L L E N D S - - - - K R T R M A T S N A I S T F K Q Y L G S K F G Y G E G A L E T F T A T T L D T AM I P Q H E F I F I F I I T N V T F X F V T E N A S L G E A T AQ T T D S D G A E A G P S R P C R F V P V T D D D V R R T I E T Q Q N - - - - K N T A K K T L Y DM R L L GQ F L K T P E I N E A R E I H E I P P N E L E T I- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M K T K R E V A V F N DWK K S M - T Q E K R D L K D I P P S Q L D A L- - - - - - - - - - - - M S D F V V S F N L F G E F D N F D DWD A A T E K E R Q E HQ K R E R R H K E V S P E E L N F L E D D E D E - - - - V N T K K I T K WA V N I F R D F L A Q K - NMD I - N F E N Y T A T T L N E S

11011074676792

1083694

L R E F Y C K I R N - H D G N T Y S V A S Y K S M R A G L N R H L K M P P Y N R Q I C L M K D K E F A S A NM V F V S M L R M L R I Q G K D E T H H H P - P I A A D D L R K I K L S G V L G L H S P V A L V N K V W F D L Q LL R E F Y Y T I R N - H D G N T Y S V A S Y K S M R A G L N R H L K M P P Y N R Q I C L M K D K E F A S A NM V F M S V L K M L R MQ G K D E T H H H P - P I A A E D L R K I K Q S G V L G L H S P L A L V N K V W F D L Q LL R E F Y G T V R N - H D G N T Y S I S S Y K S I R A G L N R H L K M P P H L R Q I C L MQ D K E F T S A N N V F L G V L K I L R K Q G K D E T N H H P - P I N A A D L R K I R T S G V L G L H T P L A L V N K V W F D L Q LL Q K F Y A G A R R - K N G S Y Y M K K S M L A I R F G L Q R H F L N - - - C N K I D I I K H E D F S N S T R V F K C F S AM L K Q E G K G V V T H K P - A I S A E DMD K I Q - - G S L D L D D P V G L Q D K V F MD V M LL C T F Y S S L R R - E D G SWY S K K T MQ V I R Y G V Q R H F L D - - - L H N F D I R D K A T F S Q S V R M F K A V L V K L K K E G K G S T K H K P - A I S A S DM E I I Q N S S V L D C D T P A G L Q N K V F V D F M TL R T F Y A E V R T - Q K G E V Y T T K S L Q S L R Y G I Q R Y F NQ P P M R R N F D L V N G D E F A D S R K M F A A V S K Q L K S Q G K G N V C H K P - A I L P G D L S K L T T Y F S R Y S T S A P V L L H K V W F S I M LL C K F F I S V R K - P S G D D Y E P T S L R S M L S S F E R H L K R - - H K Y M Y S L I S S I E F N E V R E T I K A K Q K K L K K D G K G N L P R T A E A A T D E E I E I L W E S GQ F G S H S P E A I I N T MW F Y N T VL S Q F Y L G I R K - K N G D E Y E P D S I S L Y Q K S I D R Y L R D - - N D Y G V S I I R G D A F A S S R R T L A A K R K H S K A S G F G N L P N K S Q P I T K DQ E E A L L K A GQ L G V D S A T A I I N T V WY N N T KL R L F Y A S V Q S T K E G G E Y S V A S L R S L R A G I N R H L - - - - - - R D V N I I S D T V F K S S N A V F K A I M K R Y R K S G K D T S S H H P - R I P E S D L E K I R C S S A L S P D T P L G L V R K V W F D I Q L

219219183171173201216144198

H F T K R G R E I L R D L A P D A F V V E K D K N G R R Y A V F - R Y P G K G K N G E - - - D P H K M - - - G K M Y DM P G D P N - C P V F S L E L Y L S K L P - - - - - P E P P A F Y L H P L K L T A E Q - - - - - M K E QH F A K R G R E I L R D L A P D A F V V E K D K N G R R Y AM F - R Y P G K G K N G E - - - D P Q K M - - - G K M Y DM P G D P N - C P V F S L E L Y L S K L P - - - - - P E P P A F Y L H P L K L T S E Q - - - - - MQ E QH F A K R G R E I L R D L P P D A F V I K R D P N G R R Y AM L - K Y T G K G R N R E - - - D P L K L - - - G R M Y DM P G D I N - C P V T S L D V Y L S K L P - - - - - P D P P A F Y L H P L K L T P E Q - - - - - I Q E QY F C N R G R E N L R E M T L D S F D I - C D E G G K C S I T L - - K D T L T K N N R A D K L E K S Q - - G G V M I P T N G - P R - C P V A S F L R Y K D K L N - - - - - P Q C K S F WQ R P A N AQ A K R E L K S N P S S SY F C N R G R E N L R E L K P D D F R L E T D E D G L R Y I T - - K R DQ L T K N N R E D D D E V S N - - N G V M Y E I P G S S K - C P V E S F MQ F V S K L N - - - - - K D C P F L WQ K P K A K K - - - - - - - - P E D GY F C R R G R E GQ R D L R G S H F M V K S D D N G S K Y V I Q - V G S E V S K N HQ C D D D G V S - - - G G I M Y A N N S N P R H C P V R A F E L Y L S K A N - - - - - R R C D A L F Q R P R D N Y - - - - - - - - S A D DH F G L R G N T E H R NM CWG D V S L C T D S S G R E Y L E F S E R Q T K T R T G E N P R D T R K V - - K P K MWD V P A N P R R C P V A I F K K Y L S L R P A G Y T N S D D P Y Y I A T H S R G L - - - - - - - - P R P GL F G L R G N N E H R QM EWG D V V L Q S D E N G - E F L V Y N E R L T K T R H G E - P G N T R S F - - A P K A Y A T P K T P D I C P V R A Y K A Y A A H R P D R MN C S S A P F Y L G I E Y V P C - - - - - - - - T I A KC L A R R G R E G C R E L T M A S F S I H R D E A G A E Y L S L - S H N P D T K N H K T P N D P H K Q N L R G F M F A R P G D P L - C P I Q S F K K Y I S K C P - - - - - P D A K S F Y L H P K R S V - - - - - - - - T A A A

312312276270266295317243294

P V WY K R E P MG V N Y L G AMM P R I S V A A R L - - S Q R Y T N H S L R T T T I Q L L C E A G L G P G E - - - - - - - - - - - - - - - - -P V WY K R E P MG V N Y L G T MM P R I S V A A R L - - S Q R Y T N H S L R T T T I Q L L C E A G L G P R E I M A V T G H R S E S A I R H Y WS V WY K R E P MG V N Y L G S MM P R I S I A A R L - - S Q R Y T N H S L R T T T V R L L C D A G L G A R E I M A L T G H R S E S S I R N Y WDQWY C N A P L G K N S I G D K M K T I S S R A G - - - T K A Y T N H C L R A T S I S T L Q N A G F R D R E I M S V S G H K A E T S L K H Y AD NWY C N A P V G K N T MG N K M K Q I S Q K A G C - - S K L Y T N H C L R A T C I T T L D R A G F E S R D I Q S V S G H H S E Q S L R N Y CD V W F E N K P I G K N T L S E MMA L I S K C A S L - - S Q R Y T N H C I R A T S I T I L S E A G F N N R H T M S V S R H R X E L R A G Y X NE QW F R HQ P I G I N K I G S L M K NMA T A A N L P P N K R L T N H S A R K H L I Q K L S DQ N I P P T Q I MQ I S G H R N I Q S V N A Y SG I W F R NQ P MG I N K L T T I M K S M S K A A D L - - T G K L S N H S A R K T C V Q R L L D A G V P P N T A AQ L S G H K N V S S L N R Y SE V WY S R E P MG V N Y L G AM L K K I S E E V G L - - S Q I Y T N H S L R S T A V G R L S D A G L E S R Q I M S V T G H R C E S S L Q A Y W

ATF7IP chicken

zebrafinchplatypushorsehuman

YRExon 4 5 6 7 8 9 10

10 kb

Figure 4 Crypton-derived sequence in an intron of ATF7IP gene. (A) Alignment of proteins coded by deuterostome Cryptons and Crypton-derived sequences. Catalytically essential residues are shown below the alignment. (B) Illustration of the conservation of ATF7IP loci. The positionof the YR sequence is indicated by the open box. Black boxes represent exons of the chicken ATF7IP gene. Gray boxes indicate conserved blocksbetween chicken and respective species based on the Net Tracks of the UCSC Genome Browser http://genome.ucsc.edu/. Lines between grayboxes indicate that boxes are connected by unalignable sequences. (C) Alignment of nucleotide sequences of Crypton-derived sequences.


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http://genome.ucsc.edu/

domain of KCTD1 was derived from a Crypton YR. Theposition of KIAA1958a is distinct from either CryptonAor CryptonI, and WOC/ZMYM/QRICH1 is clustered as asister group of all animal CryptonA elements. Therefore,phylogeny alone does not support the domestication ofanimal Cryptons leading to WOC/ZMYM/QRICH1 andKIAA1958a.The DUF3504 domain was derived from YR, not vice

versa, because DUF3504 lacks the complete catalytic tetradessential for YR activity. YR is essential for transposition,and repeated generation of active YRs from defective YRsis highly improbable. The distributions of WOC/ZMYM/QRICH1 and KIAA1958a are restricted to bilaterians andjawed vertebrates, respectively. Apart from Cryptons, theonly other possible sources of YRs in animal genomes arethe retrotransposon families DIRS and Ngaro [23,24].However, all searches of the YRs from CryptonA-1_OL,Crypton-1_SP and CryptonA-1_SK matched the DUF3504sequence with E-values ≤ 8e-12, whereas YRs of DIRS andNgaro did not match the DUF3504 sequence at all (evenwhen the threshold E-value was set at 100). Several repre-sentatives of DUF3504 are actually Crypton sequences; forexample, XP_001639277 is the protein coded by Crypton-1_NV. The patchy distribution of Cryptons and the incon-sistency between Crypton and host phylogenies indicateancient amplification and extinction events in Cryptonevolution. The ancient amplifications would have gener-ated many lineages of Cryptons, and it is likely that WOC/ZMYM/QRICH1 and KIAA1958a derived from lineages of

Cryptons that are now extinct. We cannot completely ruleout the possibility that the two genes and CryptonA ele-ments were independently derived from DIRS-like retro-transposons or some as yet uncharacterized types ofmobile elements, but this implies independent origins ofCryptonA and other Crypton groups (CryptonF, CryptonSand CryptonI). Therefore, four independent domesticationevents of animal Cryptons are the most parsimoniousexplanation for the origins of animal DUF3504 genes.A representative of DUF3504 from Halocynthia roretzi

(BAB40645) has orthologs in other tunicates: Ciona intes-tinalis (XP_002125964), Ciona savignyi (AACT01002283and AACT10141791) and Oikopleura dioica (CBY34656).They could also represent a domestication event of Cryp-ton. Another representative (YP_025778) is coded in themitochondrion of the green alga Pseudendoclonium akine-tum. It could also be a candidate Crypton-derived gene;owing to the lack of related sequences, however, we didnot analyze it further.All DUF3504 genes encode much longer proteins than

their DUF3504 domains, and it is possible that the pre-existing genes captured entire Crypton protein-codingsequences. However, the only recognizable domainencoded by animal Cryptons is YR (DUF3504), andthere is little sequence similarity beyond YRs amongCryptons themselves. Therefore, it is unlikely that anysignificant sequence similarity was preserved beyond theDUF3504 domains between the DUF3504 and Cryptonproteins.

DUF3504

BTB/POZ Isoform b KCTD1

WOC

P-rich Zinc finger/MYM-type

ZMYM4

ZMYM3

ZMYM2

QRICH1

Crypton-1_SP

Q-rich

Isoform a

Isoform b KIAA1958

200 aa

CryptonA-1_OL

Figure 5 Schematic structures of DUF3504 proteins. KIAA1958 gene has two isoforms, each of which encodes a DUF3504 domain. Thestructures of KCTD1, KIAA1958, QRICH1, ZMYM2, ZMYM3 and ZMYM4 are from humans. The structure of WOC is from Drosophila melanogaster.


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KCTD1 geneThe KCTD1 gene contains the DUF3504 domain con-fined within a single exon. Among the vertebrate genes,

the KCTD1 DUF3504 domain is the closest to the Cryp-ton YRs in terms of protein sequence similarity. Thesequence identity between the KCTD1 DUF3504 domain

200My

WO

C

KC

TD1

KIA

A19

58a

KIA

A19

58b

ATF7

IP

Species

Cry

pton

KC

TD1b

KIA

A19

58a

KIA

A19

58b

KIA

A19

58L

QR

ICH

1

ZMY

M2

ZMY

M3

ZMY

M4

ATF7

IP

Human - + + + - + + + + *

Mouse - + + + - + + + +

Dog - + + + - + + + +

Opossum - + + + - + + + +

Platypus - + + + + + + + *

Chicken - + * + + + + + + +

Zebrafinch - + * + + + + + + +

Lizard - + + + - + + + +

Frog * + + + - + + + +

Zebrafish * + - + - + + - ++Stickleback - + - - - ++ + - ++

Pufferfish - + - - - ++ + - +

Medaka + + - - - ++ + - +++

Chimaera * + + - - + + + +

Lamprey +

Lancelet * - - - - +

Sea squirt * - - - - +

Sea urchin + - - - - +

Acorn worm + +

Sea hare * +

Bloodfluke - +

Fruit fly - - - - - +

Sea anemone + - - - - -

WOC

Figure 6 Distribution of Cryptons and Crypton-derived genes. Each gene identified in the haploid genome is represented by a plus symbol.Minus symbols indicate the absence of Cryptons or Crypton-derived genes. Asterisks indicate the presence of their disrupted fragments. Thebranch ages are based on TimeTree [30]. The unit of time is indicated. Crypton-derived genes listed at nodes of the tree indicate the times oftheir domestication based on their distribution in different species. KIAA1958L, QRICH1, ZMYM2, ZMYM3 and ZMYM4 are not shown, because theywere likely derived by gene duplications.

Table 4 Protein identities between DUF3504 domains in two species

Comparison KCTD1 KIAA1958b QRICH1 ZMYM2 ZMYM3 ZMYM4

Human-chicken 98% (268 of 274) 99% (282 of 287) 97% (299 of 309) 92% (287 of 313) 85% (265 of 315) 88% (302 of 347)

Human-zebrafish 90% (273 of 304) 86% (245 of 287) 75% (229 of 306) 47% (145 of 310) - 52% (ZMYM4a)(176 of 345) 52% (ZMYM4b)(169 of 331)

Numbers in parentheses represent the number of identical residues (left) and the aligned domain length (right). Gaps are excluded in the comparison.


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and the YR of Crypton-1_SP is 32%, which exceeds theanalogous identity among different lineages of Cryptons.For example, Crypton-1_SP in the CryptonA lineage andCrypton-1_TC in the CryptonI lineage show less than30% sequence identity to each other. KCTD1 encodestwo protein isoforms of different lengths. The longerisoform (isoform b) contains both an N-terminalDUF3504 domain and a C-terminal BTB/POZ (Broad-complex, Tramtrack and Bric-a-brac/poxvirus and zincfinger) domain (Figure 5), whereas the shorter one (iso-form a) contains only the BTB/POZ domain. Theshorter isoform is approximately 80% identical to theKCTD15 gene at the protein level, and related genes arefound in various organisms, including lancelet, seaurchin and insects (Additional file 6). The KCTD15gene does not have any DUF3504 domain and is foundin gnathostomes from mammals to chimaera. We inferthat KCTD1 and KCTD15 were duplicated from a singlegene in the early evolution of vertebrates and after thata Crypton copy was inserted upstream of the KCTD1gene, which generated a new transcriptional variantencoding the isoform b. This insertion should haveoccurred before the branching of the Chondrichthyes(sharks, rays, skates and chimaeras) about 527 MYA[30].

KIAA1958 geneThe KIAA1958 gene, of unknown function, is present intwo isoforms (a and b) which contain different DUF3504domains encoded by different exons (Figure 5). DUF3504domains in isoforms a and b are only 26% identical toeach other in the human genome. Although alternativesplicing has not been confirmed experimentally, the highconservation of both DUF3504-coding sequences indi-cates that both encode functional proteins (Table 4 andAdditional file 4). Neither of the two DUF3504-codingsequences is interrupted by introns. We found both iso-forms in a wide range of tetrapods (Figure 6). Zebrafishlacks isoform a, whereas Chondrichthyes (chimaera) lackisoform b. Some Actinopterygii (medaka, stickleback,fugu and pufferfish) lack both isoforms. Chicken, zebrafinch and platypus have an additional gene similar toKIAA1958 isoform b, designated KIAA1958L. The exonsencoding isoform-specific regions of KIAA1958b arepositioned upstream from those of KIAA1958a. TheKIAA1958L gene likely originated from a duplication ofthe segment including KIAA1958b-specific exons but notincluding KIAA1958a-specific exons, and this duplicationevent predated the branching between mammals andbirds. KIAA1958L is less conserved than KIAA1958 iso-forms a and b. The DUF3504 domains of KIAA1958Lproteins from platypus and chicken are only 44% identi-cal, whereas those of the KIAA1958b are 97% identicalbetween the species. KIAA1958a is nonfunctional in

chicken and zebra finch, but it is intact in lizard. Isoformb was not found in Chondrichthyes, and it is possiblethat it originated later in the lineages which branched offChondrichthyes. KIAA1958a might have originated inthe common ancestor of gnathostomes. Another possibi-lity is that both isoforms were acquired in the commonancestor of gnathostomes and isoforms a and b had beenlost in the lineages of Actinopterygii and Chondrichthyes,respectively.

ZMYM2, ZMYM3, ZMYM4 and QRICH1 genesThe ZMYM2, ZMYM3 and ZMYM4 genes are present indiverse gnathostomes from human to chimaera (Figure 6).ZMYM2, ZMYM3 and ZMYM4 are similar to arthropodWOC in terms of their sequence and structure [31,32].The DUF3504 domains from the Drosophila melanogasterWOC gene and the human ZMYM2 gene are 41% identi-cal at the protein level. Some introns are also positioned atthe corresponding sites of ZMYM2 and WOC (Figure 7A).In addition to chordates and arthropods, we foundsequence fragments related to WOC in echinoderms(Strongylocentrotus purpuratus), hemichordates (Saccoglos-sus kowalevskii), mollusks (Pinctada maxima and Aplysiacalifornica) and platyhelminthes (Schistosoma mansoni,S. japonicum and Schmidtea mediterranea) (Additionalfile 5). There is no evidence that WOC forms multiplegene families in invertebrates. The ZMYM2, ZMYM3 andZMYM4 genes are listed in the data set of ohnologsreported recently by Makino and McLysaght [33], whichmeans that they were duplicated from a single gene duringtwo rounds of whole-genome duplication in the early evo-lution of vertebrates before the split between jawed verte-brates and agnathans [34,35]. The synteny blocks ofZMYM2, ZMYM3 and ZMYM4 share several genes inaddition to ZMYM genes (Figure 7B). The most parsimo-nious scenario is that the WOC/ZMYM gene family origi-nated from the domestication of Crypton in the commonancestor of bilaterians.There are three other ZMYM genes (ZMYM1,

ZMYM5 and ZMYM6) in the synteny blocks (Figure7B), but they have no DUF3504 domain. The N-term-inal part of ZMYM5 is similar to that of ZMYM2,whereas those of ZMYM1 and ZMYM6 are similar tothat of ZMYM4. These three genes are present onlyamong eutherian mammals. These data support inde-pendent gene duplication events inside each syntenyblock. It is noteworthy that the C-terminal parts ofZMYM1, ZMYM5 and ZMYM6 derived from transpo-sases of hAT-type DNA transposons, but these hAT-derived sequences are not close to each other. TheC-terminal part of ZMYM6 is close to Charlie elementsin the human genome, whereas that of ZMYM1 is closerto plant hAT elements such as HAT-1_Mad from apple(data not shown).


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The QRICH1 gene was found in diverse vertebrates,including lamprey (Figure 6). The DUF3504 domain inQRICH1 is quite similar to those of ZMYM2, ZMYM3and ZMYM4. Besides, five of eight introns of QRICH1were at the sites corresponding to those of ZMYM2,ZMYM3 and ZMYM4 (Figure 7A and data not shown).The high structural and sequence similarity betweenWOC, ZMYM2/3/4 and QRICH1 indicates that QRICH1originated from either WOC or ZMYM genes. In theneighborhood of QRICH1, we could not find any genesparalogous to genes in the synteny blocks of ZMYM2,

ZMYM3 and ZMYM4. However, because QRICH1 ispresent in the lamprey genome, it must have originatedat the time close to the whole-genome duplicationevents.

DiscussionEvolution of WOC: the third-oldest event of transposondomesticationThe most ancient transposon-derived gene known to dateis TERT, which was generated by the domestication of aPenelope-like retroelement [4], and Prp8, a spliceosomal

ZMYM2_human AAGCATACTTCCAGATGgt..agGGTCAATATTCTCTCGA S I L P D G S I F S R

ZMYM3_human CACACTCCTCCCCAACAgt..agATACGGTGTTCTCTCGA T L L P N N T V F S R

ZMYM4_human TACAATACTTCCTAATGgt..agGTTACATGTTCTCTCGC T I L P N G Y M F S R

QRICH1_human CCGGGTCACTCCACTTGgt..agGCTATGTCTTGCCCAGC R V T P L G Y V L P S

WOC_sea_urchin GACCGTGCACCCCACCA------CCGGTCTCCTCCTGAAC T V H P T T G L L L N

WOC_sea_hare TAAAGTCAACAGCCAAGgt..agGTCAGACACTTTGCCGA K V N S Q G Q T L C R

WOC_bloodfluke TATCAGTCCTGAAG---------GTCTTCTTATCTGTCGT I S P E G L L I C R

WOC_fluitfly GCTTTACAACGATTCGCgt..agAATACATTGTCACTCGC L Y N D S Q Y I V T R

ZMYM2_human TGCTACTTGTCTAAAAGgt..agTCCACAGAATCTTAAT C Y L S K S P Q N L N

ZMYM3_human TTCTATCTCTCAAAATGgt..agTCCTGAAAGCCTCCGG F Y L S K C P E S L R

ZMYM4_human TTTTACCTGTCAAAATGgt..agTTCTGAAAGTGTGAAG F Y L S K C S E S V K

QRICH1_human TTCTACCTCTTCAAATGgt..agCCCCCAGAGTGTGAAA F Y L F K C P Q S V K

WOC_sea_urchin TTTTACCTCTCCAAATGgt..agCCCTGAGAGCATCAAG F Y L S K C P E S I K

WOC_sea_hare TTTTTCCTTTCCAAATGgt..agTCCGGAGAGCATCAAA F F L S K C P E S I K

WOC_bloodfluke GCATACTTCGCCCGTTGgt..agTCCCCCAGAACTACTA A Y F A R C P P E L L

WOC_fluitfly TTTTATCTCTCCAAATGgt..agCCCGGAGAGCGTGAAG F Y L S K C P E S V K

Ggt..agGG Agt..agAN Ggt..agGG Ggt..agGG A C

Ggt..agG Agt..agAN Ggt..agG Ggt..agG

Ggt..agT

Ggt..agT

Ggt..agT

Ggt..agC

Ggt..agC

Ggt..agT

Ggt..agT

Ggt..agC

gt..agT

gt..agT

gt..agT

gt..agC gt..agC

gt..agT gt..agT

gt..agC

Ggt..agGG -------G

Ggt..agG

Cgt..agAQ gt..agA ..

A

B

GJB6

GJB2

GJA3

ZMYM2 PSPC1

ZMYM5

GJB1

ZMYM3

NONO

ZMYM6 SFPQ

ZMYM4ZMYM1DLGAP3

C1orf212

GJA4

GJB3

GJB4

GJB5

P3

ZMYM6 SFPQ

Chr 13

Chr 1

Chr X

Figure 7 Paralogous relationships of WOC/ZMYM/QRICH1 genes. (A) Two conserved intron positions among WOC, ZMYM2, ZMYM3, ZMYM4and QRICH1. Introns are printed in lowercase letters and shaded. Protein sequences are shown below nucleotide sequences. The upper andlower intron positions correspond to the 20th and 22nd introns of human ZMYM2, respectively. (B) The synteny blocks of ZMYM2, ZMYM3 andZMYM4. Ohnologous relationships reported by Makino and McLysaght [33] are indicated by dotted lines. GJB = gap junction protein b; GJA =gap junction protein a; DLGAP3 = discs large homolog-associated protein 3; C1orf212 = chromosome 1 open reading frame 212. Other genenames are described in the text.


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component derived from a retrointron (group II self-spli-cing intron) [36]. TERT retains the catalytic activity of RT,but Prp8 does not. These two genes are shared by almostall eukaryotes. Another example of an ancient domestica-tion event is the RAG1 gene [5]. It is distributed widelyamong gnathostomes, but no RAG1 ortholog was found inagnathans, including lamprey and hagfish. Given thatagnathans have a different type of adaptive immune sys-tem called “variable lymphocyte receptors” [37], thedomestication of RAG1 likely occurred in the last commonancestor of gnathostomes after their branching fromagnathans. Other transposons domesticated in the distantpast are in HARBI1 and PBDG5 genes, both of which arepresent in vertebrates from humans to actinopterygian fish[38,39]. The KCTD1b, KIAA1958a and KIAA1958b genesare as old as or older than the HARBI1 and PBDG5 genes(Figure 6). A transposon-derived CENP-B, a highly con-served mammalian centromere, and three CENP-B-likeproteins (Abp1, Cbh1 and Cbh2) in fission yeast resembleeach other in terms of their sequences and functions, butthey derived independently from different pogo-like trans-posases [40]. The human genome harbors a significantnumber of genes derived from transposons [6]. Some ofthem were domesticated in the distant past, and there areno traces of related repetitive sequences or TEs fromwhich they were derived. For example, the HARBI1 genewas derived from PIF/Harbinger and PHSA (THAPdomain-containing protein 9, or THAP9) from a P-likeelement [38,41]. Both HARBI1 and PHSA were found byscreening mammalian genes against DNA transposonsfrom zebrafish. Similarly, the key to our findings of Cryp-ton-derived genes was screening of genes against Cryptonspreserved in medaka, because there are only a few rem-nants of Cryptons left in vertebrate genomes sequenced todate, except in medaka.The ancestral gene for WOC/ZMYM probably origi-

nated in the common ancestor of all bilaterians more than910 MYA [30]. This is the third-oldest transposon domes-tication event known to date, following the two domestica-tion events of RT [4,36]. Our study indicates thatdomestication of Crypton-like elements in eukaryotes wasrelatively common in the distant past. This implies thatCryptons are very ancient and, given their rare occurrencein the genomic fossil record and their great diversity, theywere probably much more active in the distant past thanin more recent evolutionary history.

Functional implications for domesticated Crypton YRsNo function of DUF3504 domains has been reported todate. Even so, the YR origin of DUF3504 domains impliestheir functions to some extent. YR forms a multimer whenit binds substrate DNA during recombination [42]. Onthat basis, we can envision two possible functions derivedfrom YRs: DNA binding and protein-protein interaction.

There are several indications for functions of domesticatedYRs. First, many genes derived from YRs are transcrip-tional regulators. Gcr1, KCTD1, WOC, ZMYM2, ZMYM3ZMYM4, and ATF7IP are either transcriptional activatorsor repressors [43-48]. Cbf2 acts as a centromeric proteindirectly binding to centromere-specific sequences and isessential for spindle pole body formation [49,50]. Althoughthese proteins usually contain a DNA-binding domainother than DUF3504, exemplified by the GCR1_C domainof Gcr1 and Cbf2, the DUF3504 domain could also workas a DNA-binding domain. Second, there is an interestingresemblance between functions of domesticated DDE-transposases and YRs. Daysleeper is a transcription factorderived from a hAT DDE-transposase and binds a specificmotif for transcription regulation [12]. CENP-B is a cen-tromere protein derived from the DDE-transposase of apogo-like transposon [13]. In these genes, transposase-derived domains act as DNA-binding domains. Third, alarge family of prokaryotic transcriptional activators,AraC/XylS, shows structural similarity to YRs. The overallfold of the 129-amino acid protein MarA, a member ofthe AraC/XylS family, almost entirely recapitulates the YRdomain of Cre recombinase [51]. MarA can simulta-neously bind RNA polymerase II and DNA to form a tern-ary complex [52]. These data support the putative functionof DUF3504 to be DNA or protein binding.To date relatively little is known about Cryptons. There

have been no studies of their transposition, transcription,translation or regulation. The sequence similarity betweenCryptons is very low, especially in their non-protein-cod-ing regions. We compared DNA sequences of Cryptonsfrom different species, but we could not find any con-served nucleotide sequences among them. Furthermore,all Crypton domestication events are very old. Therefore, itis very difficult to propose any specific functions ofDUF3504 domains. Instead, herein we propose potentialpathways in which DUF3504 domains could be involved.KCTD1 and KCTD15 are paralogs that have diverged

during the early evolution of vertebrates (Additionalfile 6). KCTD1 isoform b, generated by an insertion ofCrypton upstream of the original KCTD1 gene, is widelyconserved among jawed vertebrates (Figure 6), although itis unclear whether the agnathans carry the KCTD1b gene.The high conservation of KCTD1b (Table 4) indicates itsessential function shared among jawed vertebrates.KCTD1 represses the activity of the AP-2a transcriptionfactor, and the BTB/POZ domain is responsible for theinteraction [46]. AP-2a plays an essential role in neuralborder (NB) and neural crest (NC) formations duringembryonic development [53]. NB is the precursor of NC.KCTD15 is expressed in NB and inhibits NC induction[54]. The NC cells are a transient, multipotent, migratorycell population unique to vertebrates. They give rise todiverse cell lineages. We can speculate that by adding a


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new protein-protein or protein-DNA interaction KCTD1bcan contribute to the network of NC formation throughthe regulation of AP-2a.Among DUF3504 genes, the function of WOC/ZMYM is

of special interest because two of the genes in this group,ZMYM2 and ZMYM3, are linked to human diseases. Achromosomal translocation between ZMYM2 and fibro-blast growth factor receptor 1 (FGFR1) causes lymphoblas-tic lymphoma and a myeloproliferative disorder [55]. Atranslocation involving ZMYM3 is associated withX-linked mental retardation [56]. Mutations of theirortholog, WOC, cause larval lethality in D. melanogaster[31].WOC/ZMYM gene-encoded proteins are involved in

various processes, including transcription, DNA repair andsplicing. WOC is a transcriptional regulator that coloca-lizes with the initiating forms of RNA polymerase II[31,32]. The WOC proteins also colocalize with all telo-meres, and mutants of WOC are associated with frequenttelomeric fusions [31,32]. ZMYM2, ZMYM3 and ZMYM4are components of a multiprotein corepressor complex,including histone deacetylase 1 (HDAC1) and HDAC2[47,48]. ZMYM2 binds to various transcriptional regula-tors including Smad proteins [57]. It also binds to proteinsinvolved in homologous recombination, such as RAD18,HHR6A and HHR6B, which are human orthologs of theyeast RAD proteins [58], and to spliceosomal componentsincluding SFPQ (splicing factor, proline- and glutamine-rich) [59].Interestingly, the SFPQ gene is a component of the syn-

tenic cluster of ZMYM4 (Figure 7B). The paralog of SFPQin the cluster of ZMYM3 is NONO (non-POU domain-containing, octamer-binding), which is a partner of SFPQin heteromers [60]. PSPC1 (paraspeckle component 1)present in the cluster of ZMYM2 also shows similarity toSFPQ and NONO genes. In addition to their involvementin splicing, the SFPQ proteins contribute to DNA repairby interacting with RAD51 [61]. They are also recruitingHDAC1 to the STAT6 transcription complex [62]. There-fore, it is likely that WOC/ZMYM and SFPQ/NONO/PSPC1 proteins cooperatively act in transcription regula-tion, splicing and DNA repair, and that they have coe-volved by maintaining their functional relationships. TheirDUF3504 domains may contribute to some of the protein-protein interactions.

Evolution of CryptonsTo date Cryptons have been identified in a limited num-ber of fungi and animal species. Herein we report thepresence of Cryptons in new species, but informationregarding their overall distribution continues to be pat-chy (Table 1). CryptonF elements are present in threephyla of fungi (Ascomycota, Basidiomycota and

Zygomycota) and two orders of oomycetes (Peronospor-ales and Saprolegniales). Our phylogenetic analysis sup-ports the horizontal transfer of CryptonF elementsbetween fungi and oomycetes (Figure 2), which is consis-tent with frequent horizontal transfer of genes betweenthem [63]. CryptonS elements are also present in twooomycete orders and one species of diatoms. Both oomy-cetes and diatoms are lineages of stramenopiles, and theorigin of CryptonS elements could date back to theircommon ancestor.Animal Cryptons (CryptonA and CryptonI) were found

in six phyla: Chordata, Echinodermata, Hemichordata,Arthropoda, Mollusca and Cnidaria. CryptonI elementshave the same overall structure as CryptonA elementsand were observed only in insect genomes. It is possiblethat CryptonI elements constitute a branch of CryptonAbut they have evolved more rapidly in insects. The over-all distribution in fungi, oomycetes and animals indi-cates that Cryptons were long present in these threeeukaryotic groups, probably with some contribution of ahorizontal transfer. It is likely that Cryptons originatedin the common ancestor of these three groups, althoughbecause of the low resolution of the YR phylogeny, wecannot rule out the possibility of their independentorigins.The identification of Crypton elements in medaka is

surprising. The nucleotide diversity of Cryptons in themedaka genome clearly shows that Cryptons were main-tained in the lineage leading to medaka for a long time.It is possible that Cryptons invaded the medaka popula-tion after the split of medaka from the three actinopter-ygian fish species (Gasterosteus, Takifugu andTetraodon), whose genomes have been sequenced. Thevertical transfer of Cryptons in the lineage leading tomedaka is a preferable scenario because of the domesti-cation of Crypton in the common ancestor of bilateriananimals, which led to the origin of WOC genes. In mostidentified host organisms, Cryptons are preserved invery low copy numbers (Additional file 1). We foundseveral fragments of Cryptons in various vertebrates,including zebrafish (Table 3). The origin of Crypton-derived genes took place at different times during theevolution of vertebrates (Figure 6). This is consistentwith the hypothesis that Cryptons continued to maintainvery low copy numbers in the vertebrate genomes andwere occasionally amplified in certain lineages.

ConclusionsThis study has revealed the diversity of a unique class ofDNA transposons, Cryptons, and their repeated domesti-cation events. The DUF3504 domains are domesticatedYRs of animal Crypton elements. Our findings add anew repertoire of domesticated proteins and provide


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further evidence for an important role of transposableelements as a reservoir for new cellular functions.

MethodsData sourceGenome sequences of various species were obtainedmostly from GenBank, and sequences of known Cryp-tons, DIRS and Ngaro were obtained from Repbasehttp://www.girinst.org/repbase/. All characterized Cryp-tons have been deposited in Repbase.

Sequence analysisCharacterization of new Cryptons was achieved byrepeated BLAST [64] and CENSOR [65] searches usinggenome sequences of various species with Cryptons asqueries. All analyses were done with default settings.The consensus sequences of elements were derivedusing the majority rule applied to the correspondingsets of multiple aligned copies of Cryptons. Alignmentgaps were manually adjusted to maximize similarity toother related elements. Characterization of DUF3504genes was performed by BLAST searches against bothprotein and nucleotide databases with known DUF3504genes as queries. We predicted exon-intron boundarieswith the aid of SoftBerry FGENESH:http://linux1.softberry.com/berry.phtml?topic=fgen-

esh&group=programs&subgroup=gfind and manuallyadjusted them through the comparison to orthologoussequences in other species.

Sequence alignment and phylogenetic analysisWe used MAFFT [66] with the linsi option or MUSCLE[67] with default settings to align both nucleotide and pro-tein sequences of various Cryptons and Crypton-derivedproteins. We constructed maximum likelihood trees byusing PhyML [68,69] with 100 bootstrap replicates [70] forthe amino acid substitution model LG. We also con-structed trees with other substitution models, WAG,RtREV and DCMut, and with the Neighbor-joiningmethod, but the resolution did not improve. The treetopology search method was Nearest Neighbor Inter-change (NNI), and the initial tree was BIONJ. The phylo-genetic trees were drawn with FigTree 1.3.1 softwarehttp://tree.bio.ed.ac.uk/software/figtree/.

Additional material

Additional file 1: PDF file listing Crypton elements found in this study.

Additional file 2: PDF file listing Crypton-derived genes in fungi.

Additional file 3: PDF file showing alignment of Cryptons andCrypton-derived genes in Saccharomycetaceae fungi in fasta format.

Additional file 4: PDF file showing alignment of Cryptons andCrypton-derived genes in animals in fasta format.

Additional file 5: PDF file listing the accession numbers for DUF3504genes.

Additional file 6: PDF file showing alignment of KCTD1, KCTD15and related protein sequences in fasta format.

Abbreviationsbp: base pair; EN: endonuclease; MYA: million years ago; RT: reversetranscriptase; TE: transposable element; TERT: telomerase reversetranscriptase; TIR: terminal inverted repeat; TSD: target site duplication; YR:tyrosine recombinase.

AcknowledgementsThis work was supported by National Institutes of Health grant 5 P41LM006252. The content is solely the responsibility of the authors and doesnot necessarily represent the official views of the National Library ofMedicine or the National Institutes of Health.

Authors’ contributionsKKK initiated the research. KKK and JJ performed the research and wrote themanuscript. Both authors read and approved the final manuscript.

Competing interestsThe authors declare that they have no competing interests.

Received: 19 August 2011 Accepted: 19 October 2011Published: 19 October 2011

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doi:10.1186/1759-8753-2-12Cite this article as: Kojima and Jurka: Crypton transposons: identificationof new diverse families and ancient domestication events. Mobile DNA2011 2:12.

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