BACKGROUND
thanks to the increasing number of available genomic sequences, it has become possible to study the evolutionary history of specific genes or gene families in relation to their co-option in innovations that have punctuated the evolutionary diversification of metazoans.
transcription factors  involved in regulation of gene-specific expression are key players in the evolution of development.
the coe family of transcription factors takes its name from the founding members of the family, collier  and olfactory-1/early-b-cell factor  isolated from drosophila and rodents, respectively  <cit> .
while there was no evidence for coe genes in either fungi, plants, or any of the various phyla of protozoans, identification of a cnidarian coe gene, nvcoe, in the anthozoan sea anemone nemostella vectensis, suggested that coe proteins have appeared with metazoa  <cit> , a conclusion strengthened by the identification of coe members both in other cnidaria and porifera  <cit> .
while up to  <dig> ebf paralogs have been identified in vertebrates  <cit> , a single coe member has been identified in all the other animals for which genome sequences have become available, suggesting that expansion of the coe gene family only occurred at the origin of vertebrates.
expression profiles of coe genes in embryos from various protostomes and deuterostomes and n. vectensis have revealed a common feature, namely, an expression in subsets of sensory neurons  <cit> .
this feature raised the possibility that one ancestral role of coe proteins was to participate in the specification of specialised sensory cells and the ontogeny of an elaborate nervous system  <cit> .
however, genetic analyses performed in mice and, more recently, drosophila raised the possibility that another ancestral function of coe proteins could have been in development of cellular immunity  <cit> .
the diversity of coe protein functions strikingly contrasts with the high degree of primary sequence conservation and lack of expansion of this family of tfs throughout metazoan evolution.
owing to its low complexity, the coe family constitutes a well-suited paradigm for studying evolution of tf structure and function, including the specific question of protein modularity.
pioneering analysis of ebf identified three functional domains  <cit> : an amino-terminal, about  <dig> amino-acid long dbd which is the signature of coe proteins; ii) a helix-helix dimerisation motif made of two tandemly arranged α-helical repeats showing limited sequence similarity to the hlh motif described in basic helix-loop-helix  proteins; iii) a transcription-activating domain without marked specific signature.
the presence of an ig-like/plexin/transcription factor  domain between the dbd and hlh domains was also noticed but the function of this domain remains unknown  <cit> .
comparison between col and ebf showed that the dbd, ipt and hlh domains have been particularly well conserved during evolution.
however, one of the tandemly arranged α-helices noted in ebf/olf- <dig> was missing.
this, and further examination of the col and ebf primary sequences led us to postulate the existence, in all coe proteins, of an hlh motif distinct from and partly overlapping the motif initially identified in ebf and olf- <dig>  <cit> .
this motif is designated below as h1lh <dig> while the vertebrate-specific motif is designated as h2d-h2a, h2d and h2a  corresponding to the duplication of the single h <dig> helix found in drosophila.
to get more insight into the evolutionary history of coe genes, we compared in detail their genomic structure between various metazoan phyla.
this comparison shows that the metazoan ancestor coe dbd was built from at least  <dig> separate exons with no evidence for exon shuffling with other gene families.
detailed analysis of various chordate genomes and ests indicated that the h2d duplication has taken place in the vertebrate lineage prior to the two rounds of whole genome duplication characterising the origin of this taxon  <cit> .
it thus provides an example of a novel vertebrate characteristic, which may have been fixed early in the gnathostome lineage.
it also revealed that the vertebrate-specific h <dig> duplication originated from a two-step tandem exon duplication.
careful inspection of the intron phases leads us to put forward an original scenario that involves the selection of a new splice donor site, resulting in the formation of a "cassette" h2d exon.
we show here that, alike ebf, col does bind to dna as homodimer and that the ancestral h1lh <dig> motif mediates formation of col/ebf homodimers and heterodimers.
incorporation of h2d in all four mammalian ebf proteins reveals an interesting example of compulsory counter-selection of alternative splicing following exon duplication.
methods
search for coe/ebf related sequences in genomic and estdatabases
systematic searches for coe/ebf proteins were conducted in available databases using the blast algorithm and mouse coe sequences  as query.
the databases analysed included current versions of the genomes of monosiga brevicollis, nematostella vectensis, capitella capitata, lottia gigantae, branchiostoma floridae  <cit> , of strongylocentrotus purpuratus  <cit>  as well as the ensembl databases of predicted proteins of ciona intestinalis, homo sapiens, mus musculus, monodelphis domestica, ornithorynchus anatinus, gallus gallus, xenopus tropicalis and danio rerio  <cit> .
in petromyzon marinus, the genomic scaffolds containing coe/ebf coding sequences were retrieved from the pre-ensembl genome version available at  <cit> .
coding sequences were identified in these scaffolds with genewise  <cit>  and assembled using homologous sequences  identified from lampetra fluviatilis ests as templates.
a survey of ests annotated in the databanks for alternative transcript variants included the use of aceview  <cit> .
the sponge amphimedon queenslandica coe sequence was taken from  <cit> .
molecular phylogenetic analysis
the alignment of coe/ebf protein sequences was obtained using muscle  <cit>  and checked by hand under bioedit  <cit> .
only full length sequences and unambiguously aligned segments were retained for the phylogenetic analysis .
neighbor-joining , maximum likelihood  and bayesian  phylogenetic reconstructions were conducted using the mega <dig>  software, phyml  <cit>  and mrbayes  <dig>   <cit> .
in each case, we used the jtt model of sequence evolution with invariant+gamma distribution rates.
bootstrap proportions  were calculated by analysis of  <dig> replicates for nj and by the rell method  <cit>  on the  <dig> top-ranking trees for ml analyses.
in the bi analysis, four chains were run for  <dig> million iterations with default heating parameters and sampled every  <dig> iterations; the first  <dig> trees were discarded as burn-in.
in vitro translation and electrophoretic mobility shift assays
the pethbf <dig>  <cit> , pet15bhis-mebf <dig>  and pet17bhis col plasmids and deletions therein were used for in vitro transcription/translation of ebf, ebf*, ebfΔh <dig>  ebfΔh <dig>  col, col*, colΔh <dig>  colΔh <dig> and colΔh2l.
to generate internal deletions corresponding to the h <dig> and h <dig> helices, we used the four oligonucleotides pcr method  <cit> .
in vitro transcription and translation using rabbit reticulocyte lysate was as described by the manufacturer .
for each protein synthesised, the efficiency of translation was assessed by sds page of parallel translation reactions performed in the presence of 35smethionine.
electrophoretic mobility shift assays  were performed in the conditions described by  <cit> , using either a  <dig> bp dna fragment containing mb- <dig> promoter sequences  which includes the ebf binding site 5'-agactcaagggaat-3' or the pal probe which contains the palindromic site 5'-attcccaagggaat-3'  <cit>  and data not shown.
competition experiments were performed using a 100× molar excess of  <dig> bp oligonucleotides containing either the wild type 5' -ctagagagagactcaagggaattgtggccagccc- 3' or mutated ctagagagagactcaaccgaattgtggccagccc- 3' mb- <dig> recognition site, as described in  <cit> .
fly strains
the p ; col <dig> strain designated in fig.s <dig> as col <dig> and uas-col strains have been described in  <cit> .
the p  transgene rescues the embryonic lethality but not the wing defects of col <dig> mutants.
the uas-mm ebf and uas-mm ebf <dig> constructs were made by cloning the entire ebf/ebf <dig> open reading frame in the puast vector.
three independent lines were used for ectopic expression assays.
all other stocks were obtained from the bloomington stock center and described in flybase  <cit> .
RESULTS
the coe gene family
the recent identification of coe sequences in cnidarians and poriferans  <cit> , together with the absence of evidence for coe genes outside metazoans suggests that coe proteins have appeared with this taxon.
in line with this conclusion, no coe-related sequence could be identified in the genome of the choanoflagellate monosiga brevicollis, while coe sequences have been reported in the sponge amphimedon queenslandica and the sea anemone nematostella vectensis  <cit> .
in order to obtain an exhaustive characterisation of coe proteins and their relationships in metazoans, we first updated the phylogenetic analysis available for this family  <cit> , taking advantage of the wide range of genomes now available .
systematic searches for coe/ebf related sequences were carried out in the genome of a diploblast, two ecdysozoans , two lophochotrozoans , an echinoderm , the cephalochordate branchiostoma floridae, the ascidian ciona intestinalis and eight vertebrates, including the lamprey petromyzon marinus, the platypus ornithorhynchus anatinus and the oppossum monodelphis domestica in addition to the mouse, human, chick, xenopus  and zebrafish .
a single coe gene was found in all metazoans studied, except in vertebrates, with evidence for three genes in xenopus, and a fourth one in the four mammals studied as previously reported in the mouse and human  <cit> , but also in the zebrafish.
in p. marinus, two distinct clusters, spanning the 5' part of the coding region and including the hlh region were reconstructed from the genome, thus pointing to the presence of at least two coe genes in lampreys.
the phylogenetic analysis was conducted using nj, ml and bayesian algorithms, excluding highly divergent sequences as well as heavily truncated ones but retaining representatives of ecdysozoans, lophotrochozoans and of the major chordate taxa .
in the resulting trees , protostome, as well as lophotrochozoan coe sequences were found clustered in monophyletic groups in nj, ml and bi, albeit with low statistical supports except in nj .
similarly, the monophyly of the vertebrate sequences retained in the reconstruction was retrieved whatever the algorithm used, with moderate to good statistical supports in bi and ml , thus supporting and extending the results obtained by pang et al.,  <dig>  inside this group, all three reconstruction methods also confirmed the previously reported presence of four additional monophyletic groups.
three of them contain at least one zebrafish or one xenopus sequence in addition to one mammalian sequence .
these groups were named accordingly coe <dig>  coe <dig>  both strongly supported , and coe <dig>  less well supported in bi .
the fourth group  only contains mammalian sequences, clustering with mouse ebf <dig>  together with the identification of coe <dig>  coe <dig>  coe <dig> and ebf <dig> partial sequences in the platypus o. anatinus , this clearly indicates that the emergence of the ebf <dig> class has predated the mammalian radiation.
the branching order observed for this group, always found as a sister group of all other vertebrate sequences , may be taken as evidence for an ancient origin in the vertebrate lineage, with subsequent losses in actinopterygians, amphibians and archosaurs .
however, a reconstruction artefact possibly related to the relatively long branches observed in this group among mammals remains difficult to exclude.
finally, the relative branching orders of the lamprey sequence included in the analyses  and of one of the zebrafish sequences   were found to vary depending on the algorithm and could not be resolved.
altogether, the phylogenomic analysis supports the conclusion that all vertebrate coe genes included in the reconstruction are derived from a single ancestral coe gene, present in the vertebrate lineage prior to the splitting between gnathostomes and cyclostomes.
it also confirms the presence of four coe classes in gnathostomes.
the emergence of three of them  is likely to have been linked to the two rounds of whole genome duplication that have occurred in the vertebrate lineage prior to the gnathostome radiation, while the origin of the fourth one , which only contains mammalian sequences, is less clear.
finally, the chronology of the corresponding gene duplications relative to the cyclostome-gnathostome divergence, as well as the relationships of the lamprey genes with the four gnathostome classes, remained unresolved.
even though the number of genes identified in the lamprey and mammals  is suggestive of the occurrence of a first round of duplication prior to the cyclostome-gnathostome splitting and a second one after their divergence, the phylogeny does not allow firm conclusions on this point.
the metazoan ancestor coe dbd was built from multiple "unique" exons
in vitro functional dissection of ebf, when ebf was a pioneer protein, delineated the coe dbd which turned out to be unrelated to other, previously characterised dbds, except for the presence of a zinc coordination motif  <cit> .
sequence conservation between ebf <dig> and drosophila col then showed that this dbd constituted the molecular trademark of a new family of transcription factors designated as coe proteins  <cit> .
sequence alignments between representative members of different phyla highlight the high degree of evolutionary conservation of each of the three coe-specific domains, the dbd, ipt and atypical hlh domains as well as scattered blocks of sequence similarity in the carboxy-terminal transactivator domain  .
fig.
2b shows a diagrammatic comparison of the exon-intron structure of coe genes between representatives of deuterostomes, including the chordate  mus musculus, the urochordate ascidian c. intestinalis and the echinoderm stongylocentrus purpuratus and representatives of protostomes, including the insect d. melanogaster and the nematode caenorhabditis elegans and the cnidarian n. vectensis.
an immediate outcome from this comparison was the remarkable conservation in number and positions of introns, independent of the overall size of the coe trancription units which ranges from around  <dig> kb in c. elegans  <cit>  to around  <dig> kb in mouse ebf <dig>  <cit> .
this showed that the ancestor coe gene was built from a complex set of multiple exons, and that this highly fragmented structure was maintained throughout metazoan evolution.
functional domains in proteins are, on a large scale, associated with protein coding exons in the genome  <cit> .
it therefore came to us as a surprise to find that the coe dbd was constructed from at least seven separate exons, since introns are generally thought not have functions  <cit>  although more recent reports tend to suggest that intron accumulation in conserved genes might be an adaptive process  <cit> .
since each of the  <dig> introns interrupting the dbd is found at the same position and in the same phase in deuterostomes, protostomes and cnidarians, we conclude that this splice structure is ancestral with the variation of exon number observed, for example in drosophila melanogaster and c. elegans reflecting secondary lineage-specific loss of introns  contrasting with the conservation of length and primary sequence of the dbd and the ipt+hlh domains, significant primary sequence variation between different phyla is observed at the junction between the dbd and ipt domains .
this correlates with a variable position of the intron separating these two domains  within a "linker" region whose sequence and length is itself variable .
further sequence variation in this region is conferred by the use of two different e <dig> splice donor sites, resulting in ebf isoforms differing by the inclusion or not of a  <dig> to  <dig> amino acids, the possible functional consequences of which remains to be addressed.
domain exchange and/or accretion between proteins resulting from exon shuffling is believed to be one of the driving forces behind protein evolution.
during this process, symmetrical exons,i.e.
exons flanked by same-phase introns, can be either deleted, duplicated or inserted, without disrupting the downstream protein reading frame  <cit> .
since, except for exon e <dig>  all the exons contributing the coe dbd are asymmetrical , this domain could not be constructed from accretion of subdomains present in other bilaterian proteins through exon shuffling.
consistent with this conclusion, systematic blast-search analyses with individual dbd exons failed to retrieve proteins other than coe proteins from databanks.
a comprehensive theory to explain intron abundance and high level of position conservation among species is still missing  <cit> .
how the coe-specific dbd structure was put together in first place remains therefore a fascinating question.
the modified hlh motif of ebf proteins is a vertebrate innovation
a noticeable difference between ebf and drosophila col is the specific duplication of a short α-helical region in ebf .
because of remote sequence similarity, this h <dig> tandem repetition was originally proposed to constitute a dimerisation similar to that present in b-hlh proteins found in fungi, plants and metazoans  <cit> .
the absence of h2d in drosophila col led us, however, to propose the existence of an alternative helix-linker-helix  domain  <cit> .
sequence comparison of a wide range of metazoans shows that the h1lh <dig> motif is an ancestral character .
the predicted primary sequence of coe proteins in cephalochordates and urochordates, which are considered as the closest living relatives of the vertebrate ancestor  <cit> , suggested that the h2d-h2a duplication was a vertebrate-specific feature.
to confirm a conclusion mostly based on ests analysis, we retrieved the intronic sequences comprised between the h <dig> and h2a coding exons in representatives of the major chordate groups outside vertebrates, c. intestinalis, b. floridae and s. purpuratus and verified the absence of h2d-related coding sequence .
in contrast, all the gnathostome sequences retrieved from the genomes analysed, including not only the four coe1- <dig> and ebf <dig> classes but also the unassigned zebrafish drcoe sequence, exhibit the h2d addition, strongly suggesting that the h2a-h2d duplication predated the gnathostome radiation .
in the lamprey p. marinus, we found no evidence for the presence of h2d in the pmcoe-a locus, but a definitive conclusion could not be obtained in this case due to sequence gaps between the h <dig> and h2a coding regions in the current genome version.
in contrast, the presence of h2d could be unambiguously recognised, and at the expected position, in the deduced pmcoe-b amino acid sequence .
preliminary est analyses of a closely related lamprey species, lampetra fluviatilis, confirmed the presence of h2d in transcripts of the orthologous lfcoe-b gene but also highlighted the presence of alternatively spliced forms, devoid of the duplicated h <dig> sequence .
the presence of h2d in lamprey lcoe-b may thus be subject to alternative splicing, while no indication for a similar process has been obtained thus far in gnathostomes.
taken together, these data indicate that the h <dig> duplication occurred early in the vertebrate lineage, prior to splitting between gnathostomes and cyclostomes, in a single copy ancestral gene from which all gnathostome and at least the lamprey coe-a genes are derived.
they also suggest that this additional protein domain may have been fixed early in the gnathostome lineage.
the coe-hlh dimerisation motif revisited
ebf/olf- <dig> was initially isolated as a nuclear factor recognising functionally important cis-regulatory dna sequences in the promoter of mb- <dig>  an early b-lymphocyte specific gene and olfactory marker protein genes  <cit> .
further characterisation showed that ebf/olf- <dig> recognises variations on the palindromic sequence ttcccnngggaat and binds dna as a homodimer  <cit> .
it was thus proposed that homodimer formation was mediated by the h2d-h2a α-helical repetition  <cit>  .
the absence of h2d in coe proteins outside vertebrates  raised, however, the question of whether all coe proteins could bind dna as dimers and, if so, which motif was involved in dimer formation.
we therefore proposed that the h1lh2a motif that is found in all coe proteins was playing this role.
to experimentally address this question, we compared the dimerization properties of drosophila col and ebf and modified versions of these two proteins , using gel-shift assays with the vertebrate mb- <dig> promoter dna.
we first found that col forms complexes with mb- <dig> dna that migrate at the same position than ebf/mb- <dig> complexes, without evidence of fast migrating complexes which would correspond to monomers .
we could therefore conclude that, similar to ebf, col binds to dna as homodimer.
truncated forms of ebf and col, that lack the transactivation domain  form complexes of higher mobility than the full-length proteins .
we took advantage of this higher mobility to assay col ability to form heterodimers with ebf, using a mixture of full length col or ebf and ebf*.
the formation of three types of dna/protein complexes  indicated that col is able to form heterodimers with ebf.
interestingly, col/ebf heterodimer formation was favoured over homodimer formation .
since binding to mb- <dig> dna is an indirect assay for dimer formation, this observation which could indicate either favoured heterodimerization or higher dna binding affinity of heterodimers needs to be further investigated.
above all, these data indicated that h2d is not required for dimerisation of coe proteins.
conversely, a truncated ebf- <dig> protein containing h2d but lacking h2a  was reported to bind to dna as homodimer  <cit> .
together, these data allow to conclude that the presence of a single copy of h <dig> is sufficient for binding of coe proteins to bind dna as dimers.
we then tested the specific requirement both for h <dig> and h <dig> , by precisely removing either helical domain in col or ebf* .
neither deleted protein form was able to bind to dna, when co-expressed with either ebf* or col* .
this led us to conclude that the presence of both h <dig> and at least one h <dig> are essential for coe dimer formation and that the ancestral h1-l-h2a mediate dimerisation of coe proteins.
a two-step evolutionary scenario for inclusion of h2d in vertebrate ebf
all gnathostome coe/ebf cdnas analysed to date include h2d.
to investigate the possible mechanisms behind this inclusion, we compared in detail the genomic structure of the hlh region between gnathostomes and their closest relatives, the urochordates  <cit> , for which genomic sequences are available.
each of the  <dig> α-helical repeats present in human ebf  is encoded by a separate exon, .
an intron also found between h <dig> and h2a in c. intestinalis ci-coe, designated i11- <dig> must have predated the separation between urochordates and vertebrates.
the presence of this intron in other deuterostomes, the cephalochordate b. floridae and the echinoderm strongylocentrus purpuratus but also the cnidarian n. vectensis  confirmed its ancestral character .
both i <dig>  i <dig> and i11/ <dig> are phase  <dig> introns, which could indicate a simple scenario whereby a tandem duplication of the exon encoding ancestral h2a would be at the origin of vertebrate h2d.
such a straightforward scenario is not compatible, however, with the position and phase of the 3'-next intron  which, both in c. intestinalis and mammals, is neither situated immediately downstream of the h <dig> coding sequence nor a phase  <dig> but a phase  <dig> intron .
since only symmetrical exons can be inserted into introns, of the same phase, without disrupting the downstream reading frame, this observation rules out a simple exon e <dig> duplication and implies a secondary event.
we therefore propose the following two-steps model: first, a duplication of exon e <dig>  led to a situation where either one of the ancestral or duplicated exon, but not both, could be incorporated in the mature mrna, a classical case of mandatory alternative splicing  <cit> .
second, selection of a new splice donor site, a few nucleotides downstream of the h2d coding region, restored a phase  <dig> intron , allowing both h2a and h2d to be inserted into the reading frame .
other possible scenarii were envisaged, using different intronic recombination events, but none appeared to be more parsimonious than the two-step scenario that we put forward here.
of the two vertebrate h <dig>  the c-terminal is the more closely related to the single h <dig> of invertebrates , indicating that the duplicated helix has started to diverge.
the "cassette" h2d exon can theoretically be inserted or removed from the transcript without affecting the rest of the protein.
removal of the h2d exon from the mature ebf mrnas through exon-skipping  would restore an invertebrate-like protein.
however, we could not find evidence for h2d exon skipping in gnathostome coe proteins, either by surveying ests annotated in the databanks  or by pcr amplification of this coding region in mouse coe <dig>  coe <dig> and coe <dig>  using specifically designed primers with mrna from several different tissues .
therefore we conclude that in gnathostomes, the prevalent form of coe proteins results from the compulsory inclusion of h2d.
this inclusion therefore represents an interesting case of counter-selection of exon-skipping, a mechanism widely used in vertebrates to amplify the register of protein products and their differential expression during development  <cit> .
discussion
the coe family of transcription factors was first defined by the sequence similarity between rodent ebf/olf- <dig> and drosophila col  <cit> .
cloning of a coe cdna from the cnidarian n. vectensis and identification of coe sequences in another cnidarian, hydra magnipapillata and a poriferan, the sponge amphimedon queenslandica  <cit>  strengthened the conclusion that coe genes are metazoan-specific genes.
our systematic blast-search for coe orthologs in dna sequence databanks confirmed that coe genes are metazoan genes present at a single copy per genome, except for vertebrates.
it further showed a remarkable degree of conservation of the coe genomic structure throughout metazoan evolution, except for one exon duplication in the vertebrate lineage.
the scattered structure of coe genes
all introns found in the cnidaria n. vectensis nvcoe gene are also found, at the same position, in deuterostomes and at least one of the protostomes examined, suggesting that this scattered organisation was already present in the metazoan ancestral coe gene.
in case of the dbd, which is both specific of coe proteins and conserved to the same degree over its entire length, a split structure into  <dig> exons was rather unexpected.
moreover, we could not find evidence for exon shuffling with other gene families, consistent with the conserved asymmetric intron phases, but leaving intact the question of the genomic building up of this unique dna binding domain.
the hccc zinc finger structure proposed to be an essential feature of the ebf dna-binding domain  <cit>  is itself encoded by two exons, already in the last common cnidarian/bilaterian ancestor , suggesting a bipartite origin.
since exon e <dig> is symmetrical, it can possibly be subject to regulated exon-skipping, allowing for the production of different protein isoforms, with putatively different functions.
whereas there is some preliminary evidence for it, as a subclass of human ebf <dig> cdnas may differ from the main class by the loss of exon e <dig> .
systematic genome sequencing programs should soon give access to the coe gene structure in many additional phyla, including sister clades of bilateriae.
it offers the exciting prospect of deeper insight into the evolutionary roots of the coe gene family and their scattered genomic organisation.
the ancestral coe hlh motif revisited
sequence similarity of the ancestral coe h1lh <dig> motif with the hlh motif of basic-hlh proteins  <cit>  has led to classify coe proteins as one distant subgroup in this superfamily of proteins, despite displaying distinctive dbd and additional protein domains  <cit> .
in vitro dna binding assays show that the h1lh <dig> motif is required for binding of coe proteins to dna as dimers.
this conclusion differs from the initial report that the ebf dimerisation motif was h2d-h2a, a conclusion supported by the analysis of two different deletions in ebf.
indeed, an internal deletion of ebf removing amino acids  <dig> to  <dig> , namely h <dig> and part of the ipt domain, was reported to lower but not prevent dimer formation  <cit> .
since we found that removal of h <dig> alone in either ebf or col abolished dimer formation, one possibility is that the presence of the ipt domain interferes with the ability of the h2d-h2a repeat to mediate homophilic interactions.
in support of this possibility, the h2d-h2a repeat, when taken out of its normal context, is able to promote formation of dimers, as shown by using a truncated nuclear hormone receptor lacking its own dimerization domain  <cit> ).
the high degree of sequence conservation of the coe ipt domain  suggests that this domain is subject to very stringent structural and functional constraints.
together, our results from dna-binding assays and those reported by  <cit> , further suggest that the positioning of the ipt and hlh domains in relation to one another is a critical aspect of coe dimer formation.
hagman et al;  <dig>  <cit>  also reported that a modified ebf protein lacking amino acids  <dig> to  <dig> , i.e., part of h2d, leaving intact h2a , showed a drastically reduced level of binding to mb <dig> dna, suggesting that h2d was essential for forming ebf homodimers.
yet, the  <dig> to  <dig> a.a. deletion does not only remove part of h2d but also part of the linker separating h <dig> and h <dig> .
our data suggest that it is removal of this linker rather than h2d itself which prevents ebf dimer formation.
the conservation of sequence and genomic structure of this proline-rich linker throughout metazoan evolution  supports a key role in positioning h <dig> and h <dig> relative to each other and contribution to the dna-binding specificity of coe dimers.
while efficient in vitro binding to dna of either col dimers, col/ebf heterodimers or dimers of ebf isoforms lacking either h2a  or h2d  <cit>  indicates that the h <dig> duplication is not essential for ebf dimer formation, inclusion of a duplicated helix <dig> raises the interesting possibility that it could result in an increased partnership flexibility and functional versatility of the vertebrate coe proteins.
the observation that col/ebf heterodimers more efficiently form and/or bind to dna, at least in vitro, raises the speculative hypothesis that it could have been the initial force behind the selection of h <dig> exon inclusion.
counter-selection of alternative splicing
together, the compared structures of vertebrate and urochordate coe genes between echinoderms, cephalochordates, urochordates and a wide range of vertebrates, including cyclostomes suggest that the duplication of h <dig> occurred in the vertebrate ancestor and resulted from an exon-duplication event.
this is the only major change in the modular structure of coe proteins that appears to have been fixed throughout metazoan evolution.
exon duplication is one widely used mechanism for adding a coding region within an existing gene.
alternative splicing of duplicated exons has been postulated to favor protein diversification, since each exon can, in principle, evolve independently of the other  <cit> .
recent genomic studies have suggested that 40–60% of human genes are alternatively spliced and comparative analysis of close to  <dig>  orthologous genes in human and mouse has shown that alternative splicing is frequently associated with recent exon creation and/or loss  <cit> .
however, other studies suggest that the contribution of gene duplication, followed by sequence divergence and alternative splicing to the diversification of the protein repertoire could be substantially different  <cit> .
in the case of the vertebrate coe genes, alternative splicing was not selected by evolution following exon h2d duplication, since both h <dig> repeats are incorporated in the ebf proteins.
taking into account the splice frame rules, we put forward here an original two-step model to account for the inclusion of h2d in vertebrate coe proteins .
the first step in our model is a classical tandem duplication of an "ancestral" h2a coding exon.
however, this exon was probably not symmetrical  and, due the splice frame rule, only the ancestral or the duplicated exon could be incorporated in the coding transcript without disrupting the open reading frame, a classical case of mandatory alternative splicing  <cit> .
we believe that inclusion of the duplicated exon occured via the activation of a phase  <dig> splice donor site, 3' to h <dig> in the duplicated exon this allowed the incorporating of h2d, while preserving the open reading frame .
to our knowledge such a two-step selection of a cassette exon has not yet been invoked for other proteins.
while our data underline the conservation of coe protein structure throughout evolution, the molecular mechanisms underlying the cell-context dependence of coe regulatory targets remains unknown.
for example, mouse ebf/coe <dig> or ebf2/coe <dig> can substitute for col activity in uas-gal <dig> transgenic assays  <cit> , using as a paradigm col function in patterning of the wing  <cit> , indicating that col and ebf are able to regulate similar set of genes in a tissue-dependent manner .
so far, little insight was obtained from systematic searches for ebf or col directly protein interactors  <cit> .
this remains a pre-eminent question in view of the evolutionary diversification of the biological functions of coe proteins revealed by mutant analyses in both mouse, c. elegans and drosophila  <cit> .
within this context, more extensive analysis of genomic structure, expression and function of coe proteins in other phyla could be of primary interest.
CONCLUSIONS
our systematic blast-search for coe  orthologs in dna sequence databanks confirmed that coe genes are metazoan genes present at a single copy per genome, except for vertebrates.
it further showed a remarkable degree of conservation of the coe genomic structure throughout metazoan evolution, except for one exon duplication in the vertebrate lineage, leading to a modified dimerisation domain of structure h1lh2dh2a in vertebrates and hlh2a in all other metazoans.
taking into account the splice frame rules, we put forward here an original two-step duplication model to account for h2d inclusion in vertebrate coe proteins the vertebrate gene configuration is such that it remains possible to remove h2d through alternative splicing, through exon-skipping.
however, the presence of both h2d and h2a in all gnathostome coe/ebf transcripts characterised to date both indicates that, in this case, exon-skipping is highly counter-selected.
while in vitro experiments indicate that the h <dig> duplication is not essential for binding of coe proteins to dna as dimers, it raises the interesting possibility that it could result in an increased partnership flexibility and functional versatility of the vertebrate coe proteins.
authors' contributions
vd and sem carried out the experimental work, sem, j–lp and sym carried our the phylogenic analyses, sem, mc and av contributed the conceptual framework and sym and av wrote the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
supplementary table.
identification of the metazoan coe sequences used in this study.
click here for file
additional file 2
sequence alignments used in the phylogenetic analysis of metazoan coe proteins.
click here for file
additional file 3
alignment of the dbd, ipt and hlh domains of the coe proteins.
click here for file
additional file 4
structure of p. marinus and l. fluviatilis coe-b transcripts at the level of the h2d coding region.
click here for file
additional file 5
diagrammatic alignment of ebf and col protein amino-acid sequences.
the sequence alignment provided shows the position of the h <dig> and h <dig> deletions introduced in modified versions of ebf and col used in dna-binding assays  and ebf internal deletion described in hagman et al.,  <dig>  <cit> .
click here for file
additional file 6
ebf can substitute for col in patterning of the drosophila wing.
this figure compares the phenotypes of adult drosophila wings subject to targeted expression of either mouse ebf <dig> or ebf <dig> or drosophila col.
click here for file
acknowledgements
we are grateful to patrick wincker and corinne da silva  for help with lamprey ests sequencing.
we also thank julian smith and jean deutsch for critical reading of early versions of the manuscript and serge plaza and members of our laboratory for discussion.
this research was supported by cnrs and ministère de la recherche  and cnrg.
s. mella was supported by a doctoral fellowship from ministère de la recherche.

 thanks to the increasing number of available genomic sequences, it has become possible to study the evolutionary history of specific genes or gene families in relation to their co-option in innovations that have punctuated the evolutionary diversification of metazoans. transcription factors involved in regulation of gene-specific expression are key players in the evolution of development. the coe family of transcription factors takes its name from the founding members of the family, collier and olfactory-1/early-b-cell factor isolated from drosophila and rodents, respectively. while there was no evidence for coe genes in either fungi, plants, or any of the various phyla of protozoans, identification of a cnidarian coe gene, nvcoe, in the anthozoan sea anemone nemostella vectensis, suggested that coe proteins have appeared with metazoa, a conclusion strengthened by the identification of coe members both in other cnidaria and porifera. while up to ebf paralogs have been identified in vertebrates, a single coe member has been identified in all the other animals for which genome sequences have become available, suggesting that expansion of the coe gene family only occurred at the origin of vertebrates. expression profiles of coe genes in embryos from various protostomes and deuterostomes and n. vectensis have revealed a common feature, namely, an expression in subsets of sensory neurons. this feature raised the possibility that one ancestral role of coe proteins was to participate in the specification of specialised sensory cells and the ontogeny of an elaborate nervous system. however, genetic analyses performed in mice and, more recently, drosophila raised the possibility that another ancestral function of coe proteins could have been in development of cellular immunity. the diversity of coe protein functions strikingly contrasts with the high degree of primary sequence conservation and lack of expansion of this family of tfs throughout metazoan evolution. owing to its low complexity, the coe family constitutes a well-suited paradigm for studying evolution of tf structure and function, including the specific question of protein modularity. pioneering analysis of ebf identified three functional domains: an amino-terminal, about amino-acid long dbd which is the signature of coe proteins; ii) a helix-helix dimerisation motif made of two tandemly arranged α-helical repeats showing limited sequence similarity to the hlh motif described in basic helix-loop-helix proteins; iii) a transcription-activating domain without marked specific signature. the presence of an ig-like/plexin/transcription factor domain between the dbd and hlh domains was also noticed but the function of this domain remains unknown. comparison between col and ebf showed that the dbd, ipt and hlh domains have been particularly well conserved during evolution. however, one of the tandemly arranged α-helices noted in ebf/olf- was missing. this, and further examination of the col and ebf primary sequences led us to postulate the existence, in all coe proteins, of an hlh motif distinct from and partly overlapping the motif initially identified in ebf and olf-. this motif is designated below as h1lh while the vertebrate-specific motif is designated as h2d-h2a, h2d and h2a corresponding to the duplication of the single h helix found in drosophila. to get more insight into the evolutionary history of coe genes, we compared in detail their genomic structure between various metazoan phyla. this comparison shows that the metazoan ancestor coe dbd was built from at least separate exons with no evidence for exon shuffling with other gene families. detailed analysis of various chordate genomes and ests indicated that the h2d duplication has taken place in the vertebrate lineage prior to the two rounds of whole genome duplication characterising the origin of this taxon. it thus provides an example of a novel vertebrate characteristic, which may have been fixed early in the gnathostome lineage. it also revealed that the vertebrate-specific h duplication originated from a two-step tandem exon duplication. careful inspection of the intron phases leads us to put forward an original scenario that involves the selection of a new splice donor site, resulting in the formation of a "cassette" h2d exon. we show here that, alike ebf, col does bind to dna as homodimer and that the ancestral h1lh motif mediates formation of col/ebf homodimers and heterodimers. incorporation of h2d in all four mammalian ebf proteins reveals an interesting example of compulsory counter-selection of alternative splicing following exon duplication. methods search for coe/ebf related sequences in genomic and estdatabases systematic searches for coe/ebf proteins were conducted in available databases using the blast algorithm and mouse coe sequences as query. the databases analysed included current versions of the genomes of monosiga brevicollis, nematostella vectensis, capitella capitata, lottia gigantae, branchiostoma floridae, of strongylocentrotus purpuratus as well as the ensembl databases of predicted proteins of ciona intestinalis, homo sapiens, mus musculus, monodelphis domestica, ornithorynchus anatinus, gallus gallus, xenopus tropicalis and danio rerio. in petromyzon marinus, the genomic scaffolds containing coe/ebf coding sequences were retrieved from the pre-ensembl genome version available at. coding sequences were identified in these scaffolds with genewise and assembled using homologous sequences identified from lampetra fluviatilis ests as templates. a survey of ests annotated in the databanks for alternative transcript variants included the use of aceview. the sponge amphimedon queenslandica coe sequence was taken from. molecular phylogenetic analysis the alignment of coe/ebf protein sequences was obtained using muscle and checked by hand under bioedit. only full length sequences and unambiguously aligned segments were retained for the phylogenetic analysis. neighbor-joining, maximum likelihood and bayesian phylogenetic reconstructions were conducted using the mega software, phyml and mrbayes. in each case, we used the jtt model of sequence evolution with invariant+gamma distribution rates. bootstrap proportions were calculated by analysis of replicates for nj and by the rell method on the top-ranking trees for ml analyses. in the bi analysis, four chains were run for million iterations with default heating parameters and sampled every iterations; the first trees were discarded as burn-in. in vitro translation and electrophoretic mobility shift assays the pethbf, pet15bhis-mebf and pet17bhis col plasmids and deletions therein were used for in vitro transcription/translation of ebf, ebf*, ebfΔh ebfΔh col, col*, colΔh colΔh and colΔh2l. to generate internal deletions corresponding to the h and h helices, we used the four oligonucleotides pcr method. in vitro transcription and translation using rabbit reticulocyte lysate was as described by the manufacturer. for each protein synthesised, the efficiency of translation was assessed by sds page of parallel translation reactions performed in the presence of 35smethionine. electrophoretic mobility shift assays were performed in the conditions described by, using either a bp dna fragment containing mb- promoter sequences which includes the ebf binding site 5'-agactcaagggaat-3' or the pal probe which contains the palindromic site 5'-attcccaagggaat-3' and data not shown. competition experiments were performed using a 100× molar excess of bp oligonucleotides containing either the wild type 5' -ctagagagagactcaagggaattgtggccagccc- 3' or mutated ctagagagagactcaaccgaattgtggccagccc- 3' mb- recognition site, as described in. fly strains the p; col strain designated in fig.s as col and uas-col strains have been described in. the p transgene rescues the embryonic lethality but not the wing defects of col mutants. the uas-mm ebf and uas-mm ebf constructs were made by cloning the entire ebf/ebf open reading frame in the puast vector. three independent lines were used for ectopic expression assays. all other stocks were obtained from the bloomington stock center and described in flybase.  the coe gene family the recent identification of coe sequences in cnidarians and poriferans, together with the absence of evidence for coe genes outside metazoans suggests that coe proteins have appeared with this taxon. in line with this conclusion, no coe-related sequence could be identified in the genome of the choanoflagellate monosiga brevicollis, while coe sequences have been reported in the sponge amphimedon queenslandica and the sea anemone nematostella vectensis. in order to obtain an exhaustive characterisation of coe proteins and their relationships in metazoans, we first updated the phylogenetic analysis available for this family, taking advantage of the wide range of genomes now available. systematic searches for coe/ebf related sequences were carried out in the genome of a diploblast, two ecdysozoans, two lophochotrozoans, an echinoderm, the cephalochordate branchiostoma floridae, the ascidian ciona intestinalis and eight vertebrates, including the lamprey petromyzon marinus, the platypus ornithorhynchus anatinus and the oppossum monodelphis domestica in addition to the mouse, human, chick, xenopus and zebrafish. a single coe gene was found in all metazoans studied, except in vertebrates, with evidence for three genes in xenopus, and a fourth one in the four mammals studied as previously reported in the mouse and human, but also in the zebrafish. in p. marinus, two distinct clusters, spanning the 5' part of the coding region and including the hlh region were reconstructed from the genome, thus pointing to the presence of at least two coe genes in lampreys. the phylogenetic analysis was conducted using nj, ml and bayesian algorithms, excluding highly divergent sequences as well as heavily truncated ones but retaining representatives of ecdysozoans, lophotrochozoans and of the major chordate taxa. in the resulting trees, protostome, as well as lophotrochozoan coe sequences were found clustered in monophyletic groups in nj, ml and bi, albeit with low statistical supports except in nj. similarly, the monophyly of the vertebrate sequences retained in the reconstruction was retrieved whatever the algorithm used, with moderate to good statistical supports in bi and ml, thus supporting and extending the results obtained by pang et al., inside this group, all three reconstruction methods also confirmed the previously reported presence of four additional monophyletic groups. three of them contain at least one zebrafish or one xenopus sequence in addition to one mammalian sequence. these groups were named accordingly coe coe both strongly supported, and coe less well supported in bi. the fourth group only contains mammalian sequences, clustering with mouse ebf together with the identification of coe coe coe and ebf partial sequences in the platypus o. anatinus, this clearly indicates that the emergence of the ebf class has predated the mammalian radiation. the branching order observed for this group, always found as a sister group of all other vertebrate sequences, may be taken as evidence for an ancient origin in the vertebrate lineage, with subsequent losses in actinopterygians, amphibians and archosaurs. however, a reconstruction artefact possibly related to the relatively long branches observed in this group among mammals remains difficult to exclude. finally, the relative branching orders of the lamprey sequence included in the analyses and of one of the zebrafish sequences were found to vary depending on the algorithm and could not be resolved. altogether, the phylogenomic analysis supports the conclusion that all vertebrate coe genes included in the reconstruction are derived from a single ancestral coe gene, present in the vertebrate lineage prior to the splitting between gnathostomes and cyclostomes. it also confirms the presence of four coe classes in gnathostomes. the emergence of three of them is likely to have been linked to the two rounds of whole genome duplication that have occurred in the vertebrate lineage prior to the gnathostome radiation, while the origin of the fourth one, which only contains mammalian sequences, is less clear. finally, the chronology of the corresponding gene duplications relative to the cyclostome-gnathostome divergence, as well as the relationships of the lamprey genes with the four gnathostome classes, remained unresolved. even though the number of genes identified in the lamprey and mammals is suggestive of the occurrence of a first round of duplication prior to the cyclostome-gnathostome splitting and a second one after their divergence, the phylogeny does not allow firm conclusions on this point. the metazoan ancestor coe dbd was built from multiple "unique" exons in vitro functional dissection of ebf, when ebf was a pioneer protein, delineated the coe dbd which turned out to be unrelated to other, previously characterised dbds, except for the presence of a zinc coordination motif. sequence conservation between ebf and drosophila col then showed that this dbd constituted the molecular trademark of a new family of transcription factors designated as coe proteins. sequence alignments between representative members of different phyla highlight the high degree of evolutionary conservation of each of the three coe-specific domains, the dbd, ipt and atypical hlh domains as well as scattered blocks of sequence similarity in the carboxy-terminal transactivator domain. fig. 2b shows a diagrammatic comparison of the exon-intron structure of coe genes between representatives of deuterostomes, including the chordate mus musculus, the urochordate ascidian c. intestinalis and the echinoderm stongylocentrus purpuratus and representatives of protostomes, including the insect d. melanogaster and the nematode caenorhabditis elegans and the cnidarian n. vectensis. an immediate outcome from this comparison was the remarkable conservation in number and positions of introns, independent of the overall size of the coe trancription units which ranges from around kb in c. elegans to around kb in mouse ebf. this showed that the ancestor coe gene was built from a complex set of multiple exons, and that this highly fragmented structure was maintained throughout metazoan evolution. functional domains in proteins are, on a large scale, associated with protein coding exons in the genome. it therefore came to us as a surprise to find that the coe dbd was constructed from at least seven separate exons, since introns are generally thought not have functions although more recent reports tend to suggest that intron accumulation in conserved genes might be an adaptive process. since each of the introns interrupting the dbd is found at the same position and in the same phase in deuterostomes, protostomes and cnidarians, we conclude that this splice structure is ancestral with the variation of exon number observed, for example in drosophila melanogaster and c. elegans reflecting secondary lineage-specific loss of introns contrasting with the conservation of length and primary sequence of the dbd and the ipt+hlh domains, significant primary sequence variation between different phyla is observed at the junction between the dbd and ipt domains. this correlates with a variable position of the intron separating these two domains within a "linker" region whose sequence and length is itself variable. further sequence variation in this region is conferred by the use of two different e splice donor sites, resulting in ebf isoforms differing by the inclusion or not of a to amino acids, the possible functional consequences of which remains to be addressed. domain exchange and/or accretion between proteins resulting from exon shuffling is believed to be one of the driving forces behind protein evolution. during this process, symmetrical exons,i.e. exons flanked by same-phase introns, can be either deleted, duplicated or inserted, without disrupting the downstream protein reading frame. since, except for exon e all the exons contributing the coe dbd are asymmetrical, this domain could not be constructed from accretion of subdomains present in other bilaterian proteins through exon shuffling. consistent with this conclusion, systematic blast-search analyses with individual dbd exons failed to retrieve proteins other than coe proteins from databanks. a comprehensive theory to explain intron abundance and high level of position conservation among species is still missing. how the coe-specific dbd structure was put together in first place remains therefore a fascinating question. the modified hlh motif of ebf proteins is a vertebrate innovation a noticeable difference between ebf and drosophila col is the specific duplication of a short α-helical region in ebf. because of remote sequence similarity, this h tandem repetition was originally proposed to constitute a dimerisation similar to that present in b-hlh proteins found in fungi, plants and metazoans. the absence of h2d in drosophila col led us, however, to propose the existence of an alternative helix-linker-helix domain. sequence comparison of a wide range of metazoans shows that the h1lh motif is an ancestral character. the predicted primary sequence of coe proteins in cephalochordates and urochordates, which are considered as the closest living relatives of the vertebrate ancestor, suggested that the h2d-h2a duplication was a vertebrate-specific feature. to confirm a conclusion mostly based on ests analysis, we retrieved the intronic sequences comprised between the h and h2a coding exons in representatives of the major chordate groups outside vertebrates, c. intestinalis, b. floridae and s. purpuratus and verified the absence of h2d-related coding sequence. in contrast, all the gnathostome sequences retrieved from the genomes analysed, including not only the four coe1- and ebf classes but also the unassigned zebrafish drcoe sequence, exhibit the h2d addition, strongly suggesting that the h2a-h2d duplication predated the gnathostome radiation. in the lamprey p. marinus, we found no evidence for the presence of h2d in the pmcoe-a locus, but a definitive conclusion could not be obtained in this case due to sequence gaps between the h and h2a coding regions in the current genome version. in contrast, the presence of h2d could be unambiguously recognised, and at the expected position, in the deduced pmcoe-b amino acid sequence. preliminary est analyses of a closely related lamprey species, lampetra fluviatilis, confirmed the presence of h2d in transcripts of the orthologous lfcoe-b gene but also highlighted the presence of alternatively spliced forms, devoid of the duplicated h sequence. the presence of h2d in lamprey lcoe-b may thus be subject to alternative splicing, while no indication for a similar process has been obtained thus far in gnathostomes. taken together, these data indicate that the h duplication occurred early in the vertebrate lineage, prior to splitting between gnathostomes and cyclostomes, in a single copy ancestral gene from which all gnathostome and at least the lamprey coe-a genes are derived. they also suggest that this additional protein domain may have been fixed early in the gnathostome lineage. the coe-hlh dimerisation motif revisited ebf/olf- was initially isolated as a nuclear factor recognising functionally important cis-regulatory dna sequences in the promoter of mb- an early b-lymphocyte specific gene and olfactory marker protein genes. further characterisation showed that ebf/olf- recognises variations on the palindromic sequence ttcccnngggaat and binds dna as a homodimer. it was thus proposed that homodimer formation was mediated by the h2d-h2a α-helical repetition. the absence of h2d in coe proteins outside vertebrates raised, however, the question of whether all coe proteins could bind dna as dimers and, if so, which motif was involved in dimer formation. we therefore proposed that the h1lh2a motif that is found in all coe proteins was playing this role. to experimentally address this question, we compared the dimerization properties of drosophila col and ebf and modified versions of these two proteins, using gel-shift assays with the vertebrate mb- promoter dna. we first found that col forms complexes with mb- dna that migrate at the same position than ebf/mb- complexes, without evidence of fast migrating complexes which would correspond to monomers. we could therefore conclude that, similar to ebf, col binds to dna as homodimer. truncated forms of ebf and col, that lack the transactivation domain form complexes of higher mobility than the full-length proteins. we took advantage of this higher mobility to assay col ability to form heterodimers with ebf, using a mixture of full length col or ebf and ebf*. the formation of three types of dna/protein complexes indicated that col is able to form heterodimers with ebf. interestingly, col/ebf heterodimer formation was favoured over homodimer formation. since binding to mb- dna is an indirect assay for dimer formation, this observation which could indicate either favoured heterodimerization or higher dna binding affinity of heterodimers needs to be further investigated. above all, these data indicated that h2d is not required for dimerisation of coe proteins. conversely, a truncated ebf- protein containing h2d but lacking h2a was reported to bind to dna as homodimer. together, these data allow to conclude that the presence of a single copy of h is sufficient for binding of coe proteins to bind dna as dimers. we then tested the specific requirement both for h and h, by precisely removing either helical domain in col or ebf*. neither deleted protein form was able to bind to dna, when co-expressed with either ebf* or col*. this led us to conclude that the presence of both h and at least one h are essential for coe dimer formation and that the ancestral h1-l-h2a mediate dimerisation of coe proteins. a two-step evolutionary scenario for inclusion of h2d in vertebrate ebf all gnathostome coe/ebf cdnas analysed to date include h2d. to investigate the possible mechanisms behind this inclusion, we compared in detail the genomic structure of the hlh region between gnathostomes and their closest relatives, the urochordates, for which genomic sequences are available. each of the α-helical repeats present in human ebf is encoded by a separate exon,. an intron also found between h and h2a in c. intestinalis ci-coe, designated i11- must have predated the separation between urochordates and vertebrates. the presence of this intron in other deuterostomes, the cephalochordate b. floridae and the echinoderm strongylocentrus purpuratus but also the cnidarian n. vectensis confirmed its ancestral character. both i i and i11/ are phase introns, which could indicate a simple scenario whereby a tandem duplication of the exon encoding ancestral h2a would be at the origin of vertebrate h2d. such a straightforward scenario is not compatible, however, with the position and phase of the 3'-next intron which, both in c. intestinalis and mammals, is neither situated immediately downstream of the h coding sequence nor a phase but a phase intron. since only symmetrical exons can be inserted into introns, of the same phase, without disrupting the downstream reading frame, this observation rules out a simple exon e duplication and implies a secondary event. we therefore propose the following two-steps model: first, a duplication of exon e led to a situation where either one of the ancestral or duplicated exon, but not both, could be incorporated in the mature mrna, a classical case of mandatory alternative splicing. second, selection of a new splice donor site, a few nucleotides downstream of the h2d coding region, restored a phase intron, allowing both h2a and h2d to be inserted into the reading frame. other possible scenarii were envisaged, using different intronic recombination events, but none appeared to be more parsimonious than the two-step scenario that we put forward here. of the two vertebrate h the c-terminal is the more closely related to the single h of invertebrates, indicating that the duplicated helix has started to diverge. the "cassette" h2d exon can theoretically be inserted or removed from the transcript without affecting the rest of the protein. removal of the h2d exon from the mature ebf mrnas through exon-skipping would restore an invertebrate-like protein. however, we could not find evidence for h2d exon skipping in gnathostome coe proteins, either by surveying ests annotated in the databanks or by pcr amplification of this coding region in mouse coe coe and coe using specifically designed primers with mrna from several different tissues. therefore we conclude that in gnathostomes, the prevalent form of coe proteins results from the compulsory inclusion of h2d. this inclusion therefore represents an interesting case of counter-selection of exon-skipping, a mechanism widely used in vertebrates to amplify the register of protein products and their differential expression during development. discussion the coe family of transcription factors was first defined by the sequence similarity between rodent ebf/olf- and drosophila col. cloning of a coe cdna from the cnidarian n. vectensis and identification of coe sequences in another cnidarian, hydra magnipapillata and a poriferan, the sponge amphimedon queenslandica strengthened the conclusion that coe genes are metazoan-specific genes. our systematic blast-search for coe orthologs in dna sequence databanks confirmed that coe genes are metazoan genes present at a single copy per genome, except for vertebrates. it further showed a remarkable degree of conservation of the coe genomic structure throughout metazoan evolution, except for one exon duplication in the vertebrate lineage. the scattered structure of coe genes all introns found in the cnidaria n. vectensis nvcoe gene are also found, at the same position, in deuterostomes and at least one of the protostomes examined, suggesting that this scattered organisation was already present in the metazoan ancestral coe gene. in case of the dbd, which is both specific of coe proteins and conserved to the same degree over its entire length, a split structure into exons was rather unexpected. moreover, we could not find evidence for exon shuffling with other gene families, consistent with the conserved asymmetric intron phases, but leaving intact the question of the genomic building up of this unique dna binding domain. the hccc zinc finger structure proposed to be an essential feature of the ebf dna-binding domain is itself encoded by two exons, already in the last common cnidarian/bilaterian ancestor, suggesting a bipartite origin. since exon e is symmetrical, it can possibly be subject to regulated exon-skipping, allowing for the production of different protein isoforms, with putatively different functions. whereas there is some preliminary evidence for it, as a subclass of human ebf cdnas may differ from the main class by the loss of exon e. systematic genome sequencing programs should soon give access to the coe gene structure in many additional phyla, including sister clades of bilateriae. it offers the exciting prospect of deeper insight into the evolutionary roots of the coe gene family and their scattered genomic organisation. the ancestral coe hlh motif revisited sequence similarity of the ancestral coe h1lh motif with the hlh motif of basic-hlh proteins has led to classify coe proteins as one distant subgroup in this superfamily of proteins, despite displaying distinctive dbd and additional protein domains. in vitro dna binding assays show that the h1lh motif is required for binding of coe proteins to dna as dimers. this conclusion differs from the initial report that the ebf dimerisation motif was h2d-h2a, a conclusion supported by the analysis of two different deletions in ebf. indeed, an internal deletion of ebf removing amino acids to, namely h and part of the ipt domain, was reported to lower but not prevent dimer formation. since we found that removal of h alone in either ebf or col abolished dimer formation, one possibility is that the presence of the ipt domain interferes with the ability of the h2d-h2a repeat to mediate homophilic interactions. in support of this possibility, the h2d-h2a repeat, when taken out of its normal context, is able to promote formation of dimers, as shown by using a truncated nuclear hormone receptor lacking its own dimerization domain ). the high degree of sequence conservation of the coe ipt domain suggests that this domain is subject to very stringent structural and functional constraints. together, our results from dna-binding assays and those reported by, further suggest that the positioning of the ipt and hlh domains in relation to one another is a critical aspect of coe dimer formation. hagman et al; also reported that a modified ebf protein lacking amino acids to, i.e., part of h2d, leaving intact h2a, showed a drastically reduced level of binding to mb dna, suggesting that h2d was essential for forming ebf homodimers. yet, the to a.a. deletion does not only remove part of h2d but also part of the linker separating h and h. our data suggest that it is removal of this linker rather than h2d itself which prevents ebf dimer formation. the conservation of sequence and genomic structure of this proline-rich linker throughout metazoan evolution supports a key role in positioning h and h relative to each other and contribution to the dna-binding specificity of coe dimers. while efficient in vitro binding to dna of either col dimers, col/ebf heterodimers or dimers of ebf isoforms lacking either h2a or h2d indicates that the h duplication is not essential for ebf dimer formation, inclusion of a duplicated helix raises the interesting possibility that it could result in an increased partnership flexibility and functional versatility of the vertebrate coe proteins. the observation that col/ebf heterodimers more efficiently form and/or bind to dna, at least in vitro, raises the speculative hypothesis that it could have been the initial force behind the selection of h exon inclusion. counter-selection of alternative splicing together, the compared structures of vertebrate and urochordate coe genes between echinoderms, cephalochordates, urochordates and a wide range of vertebrates, including cyclostomes suggest that the duplication of h occurred in the vertebrate ancestor and resulted from an exon-duplication event. this is the only major change in the modular structure of coe proteins that appears to have been fixed throughout metazoan evolution. exon duplication is one widely used mechanism for adding a coding region within an existing gene. alternative splicing of duplicated exons has been postulated to favor protein diversification, since each exon can, in principle, evolve independently of the other. recent genomic studies have suggested that 40–60% of human genes are alternatively spliced and comparative analysis of close to orthologous genes in human and mouse has shown that alternative splicing is frequently associated with recent exon creation and/or loss. however, other studies suggest that the contribution of gene duplication, followed by sequence divergence and alternative splicing to the diversification of the protein repertoire could be substantially different. in the case of the vertebrate coe genes, alternative splicing was not selected by evolution following exon h2d duplication, since both h repeats are incorporated in the ebf proteins. taking into account the splice frame rules, we put forward here an original two-step model to account for the inclusion of h2d in vertebrate coe proteins. the first step in our model is a classical tandem duplication of an "ancestral" h2a coding exon. however, this exon was probably not symmetrical and, due the splice frame rule, only the ancestral or the duplicated exon could be incorporated in the coding transcript without disrupting the open reading frame, a classical case of mandatory alternative splicing. we believe that inclusion of the duplicated exon occured via the activation of a phase splice donor site, 3' to h in the duplicated exon this allowed the incorporating of h2d, while preserving the open reading frame. to our knowledge such a two-step selection of a cassette exon has not yet been invoked for other proteins. while our data underline the conservation of coe protein structure throughout evolution, the molecular mechanisms underlying the cell-context dependence of coe regulatory targets remains unknown. for example, mouse ebf/coe or ebf2/coe can substitute for col activity in uas-gal transgenic assays, using as a paradigm col function in patterning of the wing, indicating that col and ebf are able to regulate similar set of genes in a tissue-dependent manner. so far, little insight was obtained from systematic searches for ebf or col directly protein interactors. this remains a pre-eminent question in view of the evolutionary diversification of the biological functions of coe proteins revealed by mutant analyses in both mouse, c. elegans and drosophila. within this context, more extensive analysis of genomic structure, expression and function of coe proteins in other phyla could be of primary interest.  our systematic blast-search for coe orthologs in dna sequence databanks confirmed that coe genes are metazoan genes present at a single copy per genome, except for vertebrates. it further showed a remarkable degree of conservation of the coe genomic structure throughout metazoan evolution, except for one exon duplication in the vertebrate lineage, leading to a modified dimerisation domain of structure h1lh2dh2a in vertebrates and hlh2a in all other metazoans. taking into account the splice frame rules, we put forward here an original two-step duplication model to account for h2d inclusion in vertebrate coe proteins the vertebrate gene configuration is such that it remains possible to remove h2d through alternative splicing, through exon-skipping. however, the presence of both h2d and h2a in all gnathostome coe/ebf transcripts characterised to date both indicates that, in this case, exon-skipping is highly counter-selected. while in vitro experiments indicate that the h duplication is not essential for binding of coe proteins to dna as dimers, it raises the interesting possibility that it could result in an increased partnership flexibility and functional versatility of the vertebrate coe proteins. authors' contributions vd and sem carried out the experimental work, sem, j–lp and sym carried our the phylogenic analyses, sem, mc and av contributed the conceptual framework and sym and av wrote the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 supplementary table. identification of the metazoan coe sequences used in this study. click here for file additional file 2 sequence alignments used in the phylogenetic analysis of metazoan coe proteins. click here for file additional file 3 alignment of the dbd, ipt and hlh domains of the coe proteins. click here for file additional file 4 structure of p. marinus and l. fluviatilis coe-b transcripts at the level of the h2d coding region. click here for file additional file 5 diagrammatic alignment of ebf and col protein amino-acid sequences. the sequence alignment provided shows the position of the h and h deletions introduced in modified versions of ebf and col used in dna-binding assays and ebf internal deletion described in hagman et al.,. click here for file additional file 6 ebf can substitute for col in patterning of the drosophila wing. this figure compares the phenotypes of adult drosophila wings subject to targeted expression of either mouse ebf or ebf or drosophila col. click here for file acknowledgements we are grateful to patrick wincker and corinne da silva for help with lamprey ests sequencing. we also thank julian smith and jean deutsch for critical reading of early versions of the manuscript and serge plaza and members of our laboratory for discussion. this research was supported by cnrs and ministère de la recherche and cnrg. s. mella was supported by a doctoral fellowship from ministère de la recherche.
the increasing number of available genomic sequences makes it now possible to study the evolutionary history of specific genes or gene families. comparison of the splice structure of coe genes between cnidariae and bilateriae shows that the ancestral coe dbd was built from separate exons, with no evidence for exon shuffling with other metazoan gene families. coe proteins display a modular organisation, with three highly conserved domains: a coe-specific dna-binding domain, an immunoglobulin/plexin/transcription domain and an atypical helix-loop-helix motif. it also confirms the presence of an ancestral h1lh motif present in all coe proteins which partly overlaps the repeated h2d-h2a motif first identified in rodent ebf. the low complexity coe family of transcription factors constitutes a well-suited paradigm for studying evolution of tf structure and function, including the specific question of protein modularity. here, we compare the structure of coe genes within the metazoan kingdom and report on the mechanism behind a vertebrate-specific exon duplication. three main features in the history of the coe gene family can be inferred from these analyses: each conserved domain of the ancestral coe gene was built from multiple exons and the same scattered structure has been maintained throughout metazoan evolution. transcription factors involved in regulation of gene-specific expression are key players in the evolution of metazoan development. this duplication provides an interesting example of counter-selection of alternative splicing. electrophoretic mobility shift assays show that formation of coe dimers is mediated by this ancestral motif. the h2a-h2d duplication that is specific to vertebrate proteins provides an example of a novel vertebrate characteristic, which may have been fixed early in the gnathostome lineage.
 the increasing number of available genomic sequences makes it now possible to study the evolutionary history of specific genes or gene families. transcription factors involved in regulation of gene-specific expression are key players in the evolution of metazoan development. the low complexity coe family of transcription factors constitutes a well-suited paradigm for studying evolution of tf structure and function, including the specific question of protein modularity. here, we compare the structure of coe genes within the metazoan kingdom and report on the mechanism behind a vertebrate-specific exon duplication.  coe proteins display a modular organisation, with three highly conserved domains: a coe-specific dna-binding domain, an immunoglobulin/plexin/transcription domain and an atypical helix-loop-helix motif. comparison of the splice structure of coe genes between cnidariae and bilateriae shows that the ancestral coe dbd was built from separate exons, with no evidence for exon shuffling with other metazoan gene families. it also confirms the presence of an ancestral h1lh motif present in all coe proteins which partly overlaps the repeated h2d-h2a motif first identified in rodent ebf. electrophoretic mobility shift assays show that formation of coe dimers is mediated by this ancestral motif. the h2d-h2a α-helical repetition appears to be a vertebrate characteristic that originated from a tandem exon duplication having taken place prior to the splitting between gnathostomes and cyclostomes. we put-forward a two-step model for the inclusion of this exon in the vertebrate transcripts.  three main features in the history of the coe gene family can be inferred from these analyses: each conserved domain of the ancestral coe gene was built from multiple exons and the same scattered structure has been maintained throughout metazoan evolution. there exists a single coe gene copy per metazoan genome except in vertebrates. the h2a-h2d duplication that is specific to vertebrate proteins provides an example of a novel vertebrate characteristic, which may have been fixed early in the gnathostome lineage. this duplication provides an interesting example of counter-selection of alternative splicing. 