Characterization of the Runx Gene Family in a Jawless Vertebrate, the Japanese Lamprey (Lethenteron japonicum)

The cyclostomes (jawless vertebrates), comprising lampreys and hagfishes, are the sister group of jawed vertebrates (gnathostomes) and are hence an important group for the study of vertebrate evolution. In mammals, three Runx genes, Runx1, Runx2 and Runx3, encode transcription factors that are essential for cell proliferation and differentiation in major developmental pathways such as haematopoiesis, skeletogenesis and neurogenesis and are frequently associated with diseases. We describe here the characterization of Runx gene family members from a cyclostome, the Japanese lamprey (Lethenteron japonicum). The Japanese lamprey contains three Runx genes, RunxA, RunxB, and RunxC. However, phylogenetic and synteny analyses suggest that they are not one-to-one orthologs of gnathostome Runx1, Runx2 and Runx3. The major protein domains and motifs found in gnathostome Runx proteins are highly conserved in the lamprey Runx proteins. Although all gnathostome Runx genes each contain two alternative promoters, P1 (distal) and P2 (proximal), only lamprey RunxB possesses the alternative promoters; lamprey RunxA and RunxC contain only P2 and P1 promoter, respectively. Furthermore, the three lamprey Runx genes give rise to fewer alternative isoforms than the three gnathostome Runx genes. The promoters of the lamprey Runx genes lack the tandem Runx-binding motifs that are highly conserved among the P1 promoters of gnathostome Runx1, Runx2 and Runx3 genes; instead these promoters contain dispersed single Runx-binding motifs. The 3′UTR of lamprey RunxB contains binding sites for miR-27 and miR-130b/301ab, which are conserved in mammalian Runx1 and Runx3, respectively. Overall, the Runx genes in lamprey seem to have experienced a different evolutionary trajectory from that of gnathostome Runx genes which are highly conserved all the way from cartilaginous fishes to mammals.


Introduction
The polyomavirus enhancer-binding protein 2 (PEBP2) or corebinding factor (CBF) is an ancient Runt domain heterodimeric transcription factor of a and b subunits. In human, the a-subunit is encoded by three Runt gene family members, RUNX1, RUNX2 and RUNX3 that contain an evolutionarily conserved 128 amino acid Runt domain responsible for DNA binding and heterodimerization with the b-subunit. The b-subunit comprises a single protein, RUNXb (also known as PEBP2b or CBFb) that does not directly interact with DNA but serves to allosterically enhance the DNA-binding activity of the a-subunit and regulate its turnover by protecting it from ubiquitin-proteasome-mediated degradation [1,2]. Basally branching chordates such as amphioxus (Branchiostoma floridae) and tunicates (Ciona and Oikopleura) possess a single Runx gene. By contrast, vertebrates such as cartilaginous fishes (e.g. elephant shark, dogfish) and tetrapods contain three Runx genes (Runx1, Runx2 and Runx3) [3,4,5] owing to the two rounds of whole genome duplication at the base of vertebrates [6,7]. Teleost fishes such as zebrafish contain a duplicated Runx2 gene (Runx2b) that resulted from an additional teleost-specific genome duplication event [8]. On the other hand, pufferfishes (fugu and Tetraodon) possess three Runx genes like tetrapods and an additional fourth Runx gene called frRunt [9,10], which is hypothesized to represent an ancestral vertebrate Runx gene that was lost in tetrapods and zebrafish [9]. In contrast to multiple copies of a-subunit encoding Runx genes, Runxb is present as a single copy in vertebrates as well as in invertebrates analysed, with the exception of the fruit fly (Drosophila melanogaster) that possesses two orthologs of Runxb genes, Brother and Big brother [11,12,13].
In mammals, the three Runx genes play pivotal roles in several developmental processes including haematopoiesis, neurogenesis, and skeletogenesis. Runx1 is required for the emergence and maintenance of hematopoietic stem cells [14,15] and is frequently mutated in human leukaemia [16,17,18]. Runx2 is indispensable for osteogenesis as evidenced by the complete lack of ossified skeleton in Runx2-deficient mice [19]. Haploinsufficiency of RUNX2 results in the autosomal dominant bone disease, cleidocranial dysplasia [20]. Runx3 is involved in diverse biological pathways including the regulation of epithelial homeostasis in the gastrointestinal tract [21,22], development of T cells during thymopoiesis [23] and the differentiation of various cell types of the immune system [24,25,26]. Runx3 is also essential for the differentiation of proprioceptive neurons [27] and chondrocyte maturation during skeletogenesis [28].
The living vertebrates are divided into two broad groups: the jawless vertebrates (cyclostomes) and jawed vertebrates (gnathostomes). While the gnathostomes are represented by cartilaginous fishes, ray-finned fishes, lobe-finned fishes and tetrapods, the cyclostomes are represented by only lampreys and hagfish which constitute a monophyletic group. Cyclostomes diverged from gnathostomes ,500 Mya and are morphologically and physiologically quite distinct from gnathostomes. They possess a single median dorsal ''nostril'' as opposed to ventrally located nostrils in gnathostomes. In addition, they lack mineralized tissues, hinged jaws, paired appendages, pancreas and spleen [29]. Although cyclostomes lack an immunoglobulin-based adaptive immune system, a thymus-like organ called the 'thymoid', has been identified at the tips of gill filaments in the gill basket [30] and is the site of antigen receptor gene assembly of T-cell like lymphocytes [31]. The adaptive immune system of cyclostomes makes use of an alternative immune receptor system different from that of gnathostomes. Instead of T-cell and B-cell receptors generated from the immunoglobulin superfamily, cyclostomes utilize variable lymphocyte receptors (VLR) that are assembled from leucine-rich repeat modules (reviewed in [32]). These distinctive morphological and physiological traits along with the unique phylogenetic position of cyclostomes make them an important group for understanding the evolution and function of vertebrate Runx genes.
Full-length coding sequences of two Runx genes, designated MgRunxA and MgRunxB have been previously cloned in a cyclostome, the Atlantic hagfish (Myxine glutinosa). These Runx genes were found to be expressed in cartilaginous tissues, suggestive of their involvement in early vertebrate skeletogenesis [4]. Mining of the genome assembly of the sea lamprey (Petromyzon marinus) led to the identification of partial sequences for only two Runx genes, PmRunxA and PmRunxB and the conclusion that this is likely to be the full complement of Runx genes in lamprey [33]. However, it should be noted that the sea lamprey genome assembly was generated from DNA extracted from a somatic tissue (liver) that has been shown to lose ,20% of genomic DNA due to developmentally programmed rearrangement during early embryonic development [34] and hence does not contain the full complement of genes present in the germline genome. Recently, the whole genome sequence of another lamprey, the Japanese lamprey (Lethenteron japonicum) was generated using DNA from the testis [35] (http:// jlampreygenome.imcb.a-star.edu.sg/). The Japanese lamprey and sea lamprey are northern hemisphere lampreys that diverged from each other 30-10 Mya ago [36]. Compared to the 2.3 Gb genome of the sea lamprey, the Japanese lamprey has a relatively smaller genome of ,1.6 Gb [37]. To improve our understanding of the evolution, function and regulation of Runx genes, we mined the Japanese lamprey genome assembly for Runx genes and completed the coding sequences for Runx genes by RT-PCR and RACE. Our analyses show that the Japanese lamprey genome codes for three Runx genes like gnathostomes but they are not the exact one-to-one orthologs of gnathostome Runx1, Runx2 and Runx3.

Ethics statement
The Japanese lamprey specimens were collected from commercial fishermen who routinely catch them like other commercial fishes in Ishikari River near Ebetsu in Hokkaido, Japan. The procedure for extraction of DNA and RNA from lamprey tissues was approved by the Institutional Animal Care and Use Committee of the Biological Resource Centre, Agency for Science, Technology and Research (A*STAR), Singapore.
Identification of Runx genes in the Japanese lamprey genome assembly The genome assembly of the Japanese lamprey (http:// jlampreygenome.imcb.a-star.edu.sg/) was searched with human RUNX1, RUNX2, RUNX3 and RUNXb protein sequences using the 'TBLASTN' algorithm. Scaffolds #47, #165, #769 and #850 containing sequences homologous to human and elephant shark Runx1, Runx2, Runx3 and Runxb protein sequences were searched against the non-redundant protein database at NCBI using BlastX to identify the coding exons of lamprey Runx genes. Missing exons in the scaffolds (presumably due to gaps in the scaffolds) were identified by sequencing RACE (Rapid Amplification of cDNA Ends) products as described below.

Full-length cDNA cloning by RACE
Total RNA was extracted from various tissues of adult Japanese lamprey using Trizol reagent (Gibco BRL, Grand Island, NY) according to manufacturer's protocol. One mg of total RNA was reverse transcribed into 59RACE-ready or 39RACE-ready singlestrand cDNA by using the SMART RACE cDNA Amplification kit (Clontech, Palo Alto, CA). Primers were designed for representative exons identified in the Japanese lamprey scaffolds and full-length Runx transcripts were obtained by 59 RACE and/or 39 RACE (primer sequences available upon request). 59 and 39 RACE were each performed in a nested PCR. The first round of PCR was performed with the Universal Primer and a gene-specific primer. The resulting PCR product was diluted 206 and 1 ml used for the nested PCR with the Nested Universal Primer and a second genespecific primer. All RACE products were cloned into the pGEM-T Easy Vector (Promega, USA), and sequenced completely using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) on an ABI 37306l capillary sequencer (Applied Biosystems, USA). The genome structures of the Japanese lamprey Runx genes, including the exon-intron structures, UTRs and transcription start sites were deciphered by mapping the cloned full length cDNA sequences to the Japanese lamprey genomic sequence. Sequences for various isoforms of the Japanese lamprey Runx genes have been deposited in GenBank (KJ787775-KJ787788).

Amino acid alignment and phylogenetic analysis
The full-length protein sequences for human and other chordate Runx genes, including the two Runx sequences (RunxA and RunxB) from the Atlantic hagfish were retrieved from the National Center for Biotechnology Information (NCBI) database. RunxA and RunxB from the sea lamprey were not used as they are partial sequences. Multiple sequence alignments were generated using MUSCLE (www.ebi.ac.uk/Tools/msa/muscle/). For phylogenet-ic analysis, the alignments were trimmed using the Gblocks Server (ver. 0.91b) with less stringent selection parameters [38]. MEGA 6.06 (http://www.megasoftware.net/) was used to determine the most appropriate amino acid substitution model for each dataset. Maximum Likelihood (ML) and Bayesian (BI) methods were used for phylogenetic analyses employing the JTT+G+I or JTT+G model. MEGA 6.06 was used for ML analyses and 100 bootstrap replicates were used for node support. BI analyses were carried out using MrBayes 3.2 (http://mrbayes.csit.fsu.edu/). Two independent runs starting from different random trees were run for 1 million generations with sampling done every 100 generations. A consensus tree was built from all sampled trees excluding the first 2,500 trees (burn-in).

Expression profiling by qRT-PCR
Purified total RNA was reverse-transcribed into cDNA with Superscript II (Invitrogen, Carlsbad, CA). The single strand cDNA was used as a template in qRT-PCR reactions with KAPA SYBR FAST qPCR Kit reagents (KAPA Biosystems, Boston, MA). Sequences of primers used in qRT-PCR are given in Table S1. All primer pairs span at least one intron which helps to distinguish cDNA sequences from genomic DNA products. Expression levels of Runx genes were normalized using beta-actin gene as the reference. Quantification of gene expression levels was performed using the comparative C T method [40]. The relative expression levels of each Runx gene in different tissues were estimated in relation to a reference tissue (one that showed the lowest level of expression among the tissues analysed). Note, however, that these relative expression levels are not comparable between different Runx genes.

Results and Discussion
Cloning and orthology of Japanese lamprey a-subunit Runx family members To identify Runx genes in the Japanese lamprey, we BLASTsearched the Japanese lamprey genome assembly [35] using human RUNX protein sequences. We identified three scaffolds (Scaffold_850, 47 and 769) that contained fragments of Runx genes. The missing exons and full-length coding sequences of these genes were identified by RT-PCR and/or RACE using cDNA from gill, intestine or skin ( Fig. 1 and S1).
To determine the relationships of the three lamprey Runx genes to Runx1, Runx2 and Runx3 of gnathostomes, we carried out phylogenetic analysis using two different algorithms, ML and BI. The Runt gene from amphioxus was used as the outgroup. Both ML and BI analyses showed that the Japanese lamprey RunxA and RunxB genes are orthologs of the hagfish RunxA and RunxB genes, respectively ( Fig. 2 and S2). However, the three lamprey and two hagfish genes were found to cluster outside the three gnathostome genes. In the ML tree, cyclostome RunxA formed a sister clade to all three gnathostome Runx genes while RunxB and RunxC genes were represented on separate branches outside the RunxA+gnathostome Runx clade (Fig. 2). In BI analysis, the combined clade of cyclostome RunxA and RunxB genes constituted the sister group to all three gnathostome Runx genes with RunxC being an outgroup (Fig. S2). This pattern of clustering of gnathostome and cyclostome Runx genes suggests that three gnathostome Runx genes are the result of duplications in the gnathostome ancestor after it diverged from the cyclostome lineage. This implies that the three lamprey Runx genes are not the exact one-to-one orthologs of the three gnathostome Runx genes.
In addition, we generated an ML tree that included single Runx sequences from two more well characterized invertebrate species, Ciona intestinalis an urochordate and Nematostella vectensis a cnidarian ( Fig. S4 and S5). In this tree, the relationships of gnathostome Runx genes are essentially similar to that depicted in Fig. 2, and the cyclostome genes cluster outside the gnathostome clade. However, some differences in topology are observed among the cyclostome branches (Fig. S5). These branches, though, are not well supported (bootstrap values of 40 and 37), suggesting that these relationships are not reliable. We believe that such changes to the topology are due to the highly divergent sequences of Ciona and Nematostella (see alignment in Fig. S4).
The three Runx genes in the gnathostomes are likely to be the result of two rounds of whole genome duplication (2R) that occurred early during the evolution of vertebrates [6,41,42]. Consistent with this hypothesis, synteny of sets of paralogous genes are conserved in the gnathostome Runx1, Runx2 and Runx3 gene loci. For example, synteny of paralogs of Runx, Clic and Rcan are conserved in the Runx1, Runx2 and Runx3 loci of all gnathostomes analysed so far (Fig. 3). Conserved synteny of blocks of genes between genomes provides additional support for the orthologous relationship of genes in the syntenic block. For instance, orthologs of several genes in the human RUNX2 locus (e.g. CDC5L, SUPT3H, CLIC5) are conserved in the Runx2 locus of chicken, zebrafish and elephant shark indicating that the Runx gene in this locus of these gnathostomes is indeed an ortholog of human RUNX2 (Fig. 3). An interesting pair of syntenic genes in the gnathostome Runx2 locus is the tightly linked RUNX2 and SUPT3H genes whose first exons overlap. The intertwined organization of these two genes is conserved in all gnathostomes examined, with the exception of the duplicate locus of zebrafish Runx2b in which the duplicate Supt3hb gene has been lost (Fig. 3). The close linkage between Runx and Supt3h genes seems to be an ancestral state, because in invertebrates such as amphioxus, sea anemone (Nematostella vectensis) and demosponge (Amphimedon queenslandica), the Supt3h gene is located next to the single Runt gene (Fig. 3). It can therefore be inferred that after the duplication of the ancestral vertebrate Runx locus in the gnathostome ancestor, the paralogs of Supt3h linked to Runx1 and Runx3 loci were lost whereas the paralog in Runx2 locus was retained.
In order to verify the orthology relationships of the three lamprey Runx genes to gnathostome Runx genes, we analysed the synteny of genes at the lamprey Runx gene loci. Interestingly, none of the lamprey Runx genes is linked to a Supt3h gene. A TBLASTN search of the Japanese lamprey genome assembly using human and elephant shark Supt3h protein sequences identified a single Supt3h gene located on a scaffold (Scaf_1) distinct from those containing Runx genes. Either this gene previously located next to a Runx gene was translocated to a new location in the lamprey genome or the Runx gene linked to it has been lost. Of the three Japanese lamprey Runx genes, the most extensive synteny information is available for RunxB. The syntenic genes of the lamprey RunxB include three genes (Son, Donson and Cdc5l) whose orthologs are conserved in the Runx loci of gnathostomes. However, while the ortholog of Son and Donson are found in the gnathostome Runx1 locus, the ortholog of Cdc5l is found linked to gnathostome Runx2. The synteny of genes in the lamprey RunxB locus therefore suggests that RunxB is not an exact ortholog of either Runx1 or Runx2 of gnathostomes. This provides further support to the inference of the phylogenetic analysis that the three lamprey Runx genes are not one-to-one orthologs of the three gnathostome Runx genes.

Genomic organization of Japanese lamprey a-subunit Runx genes
The exon-intron organization of the three gnathostome Runx genes is largely similar except for Runx3 which characteristically lacks an exon (exon 5.1) at the C-terminal end, that is present in both Runx1 and Runx2 (Fig. S1). The three lamprey a-subunit Runx genes share several structural similarities with those of gnathostomes, including three exons that encode the highly conserved Runt domain ( Fig. 1 and S1). Interestingly, of the three lamprey Runx genes, only RunxB contains the exon equivalent of exon 5.1 of gnathostome Runx1 and Runx2 genes and is therefore structurally more similar to these two gnathostome Runx genes. In addition, RunxB contains an extra exon (exon 4.1) that is not equivalent to any of the gnathostome exons including exon 4.1 in elephant shark Runx1 ( Fig. 1 and S1). This feature distinguishes the lamprey RunxB gene from gnathostome Runx1 and Runx2. Lamprey RunxA and RunxC both lack the exon equivalent to exon 5.1 and are thus more similar to the gnathostome Runx3 gene (Fig. 1A, C and S1). Since phylogenetic and synteny analyses do not support an orthology relationship between these two lamprey genes and the gnathostome Runx3 gene, their similar structure appears to be the result of convergent evolution.
All gnathostome Runx genes are transcribed from two alternative promoters, P1 (distal) and P2 (proximal) that are separated by a characteristically large intron (Fig. S1). Of the three lamprey Runx genes, only RunxB contains both P1 and P2 promoters (Fig. 1). Lamprey RunxA contains only the proximal (P2) promoter. Our efforts to identify a distal promoter by 59RACE using cDNA from several tissues failed to yield transcripts originating from a distal promoter and led us to conclude that the P1 promoter is absent in RunxA (Fig. 1A). By contrast, lamprey RunxC contains the P1 promoter but lacks the P2 promoter (Fig. 1C). The translation initiation codon (ATG) associated with P2 promoter has been mutated to another codon in this gene and the open reading frame which is devoid of an ATG codon runs all the way to the 59end of exon 2. Thus, RunxC has lost the potential to generate transcripts from the proximal promoter. The presence of two alternative promoters in all gnathostome Runx genes and the lamprey RunxB gene suggests that the two alternative promoters had already emerged in the single ancestral Runx gene in the common ancestor of gnathostomes and cyclostomes. Following the duplication of the ancestral Runx gene, lamprey RunxA and RunxC genes secondarily lost P1 and P2 promoters, respectively.
Each of the gnathostome Runx genes express a diverse repertoire of isoforms arising from two alternative promoters (P1 and P2) as well as by alternative splicing of exons (Fig. 1). These isoforms are differentially expressed during development and exert a variety of biological functions [43,44]. Of the three lamprey Runx genes, only RunxB has the potential to generate isoforms originating from two alternative promoters. In addition, it gives rise to multiple isoforms resulting from alternative splicing of exons similar to gnathostome Runx genes (Fig. 1B). By contrast, we could identify only one transcript for lamprey RunxA and two isoforms for RunxC ( Fig. 1A and C). Thus, these lamprey genes give rise to fewer isoforms than the lamprey RunxB gene and the three gnathostome Runx genes.
The P1 and P2 promoter regions of gnathostome Runx genes harbour binding sites for transcription factors that mediate transcriptional regulation of Runx genes. Notably, the P1 promoters of gnathostome Runx1, 2 and 3 contain two tandem binding sites for Runx (Pu/TACCPuCA) at similar locations in the 59UTRs [3]. These highly conserved binding sites have been implicated in the auto and cross-regulation of the three gnathostome Runx genes [45,46]. The presence of these sites in the three gnathostome Runx genes suggests that they were present in their single ancestral Runx gene. In contrast to gnathostome Runx genes, the P1 promoters of the lamprey RunxB and RunxC genes lack these characteristic tandem Runx binding sites. Instead, the P1 promoter of RunxB contains dispersed single Runx binding sites at positions 2506, 2282 and +131. In addition, its P2 promoter contains one Runx binding site at position 2328 (data not shown). However, no such single Runx binding site is found in the P1 promoter of RunxC or in the P2 promoter of RunxA. It remains to be seen if these dispersed Runx motifs in the lamprey RunxB promoters are involved in its auto-regulation and/or in its cross-regulation by RunxA and RunxC.
The expression of gnathostome Runx1-3 genes is posttranscriptionally regulated by miRNAs that target highly conserved miRNA binding sites in the 39UTR of their transcripts [47,48,49]. To verify if the lamprey Runx genes are also regulated by miRNAs, we searched the 39UTRs of the lamprey Runx genes for seed miRNA binding sites that are well characterized in the mammalian Runx genes. While no such binding sites were found in RunxA and RunxC, RunxB was found to contain binding sites for miR-27 and miR-130b/301ab (Fig. 4). In mammals, the binding site for miR-27 is present in Runx1 whereas that for miR-130b/301ab is present in Runx3 (Fig. 4). It is therefore possible that the single ancestral vertebrate Runx gene contained sites for both miRNAs and that they were differentially lost in mammalian Runx1 and Runx3 genes. Murine miR-27 has been shown to be involved in the regulation of Runx1 in megakaryocytic and granulocytic differentiation [48] whereas human miR-130b and miR-301a have been implicated in the down-regulation of RUNX3 in gastric cancer [50,51]. These three miRNAs have been previously shown to be expressed in cyclostomes such as the sea lamprey and the Atlantic hagfish [52]. It is therefore likely that the predicted binding sites for these miRNAs in Japanese lamprey RunxB are functional and mediate regulation of RunxB.

Comparison of Japanese lamprey Runx a-subunit protein sequences
The domain structures and motifs of Runx proteins are highly conserved among gnathostome Runx1-3. These include the 128 amino acid Runt domain that is required for their DNA binding and heterodimerization with the b-subunit, as well as the transactivation domain (TAD) and inhibitory domain (ID) located at their carboxy termini. In addition, gnathostome Runx proteins contain a nuclear localization signal (NLS) located contiguous to the Runt domain that mediates their nuclear import as well as a nuclear matrix targeting signal (NMTS) in the TAD that directs Runx transcription factors to specific nuclear-matrix associated sites involved in the regulation of gene expression. Other distinctive features of gnathostome Runx proteins include the PY and VWPRY motifs that mediate the transcriptional activity of Runx proteins by recruiting different interacting partners. The proline-rich PY motif in the TAD mediates the binding of Runx proteins to WW domain-containing proteins, such as Yesassociated protein (YAP), transcriptional co-activator with PDZbinding motif (TAZ), and Smurf [53,54,55]; while the C-terminal VWRPY motif recruits the Groucho/Transducin-like enhancer Figure 5. Runx a-subunit proteins in human, elephant shark and Japanese lamprey. Alignment of human, elephant shark and Japanese lamprey Runx a-subunit proteins. The first block shows the amino-terminal part of the protein derived from the P1 promoter that differs from that derived from the P2 promoter. The highly conserved Runt domain is highlighted in pink. Within the Runt domain, surfaces involved in DNA contact and interaction with the b-subunit are denoted by black and blue lines, respectively. Cysteine residues involved in the redox regulation of DNA-binding activity are indicated with asterisks. Nuclear localization signal (NLS) is demarcated by a green dashed box. The PY and VWRPY motifs are indicated in red and blue, respectively. The transactivation domain (TAD) is highlighted in blue and the inhibitory domain (ID) is boxed in blue. The nuclear matrix targeting signal (NMTS) is boxed in black. Minimal consensus sequences for phosphorylation by Erk are boxed by dashed black lines. The consensus phosphorylation site for Cdc2 is indicated in green. The residue targeted for phosophorylation is indicated by . Hs, Homo sapiens; Cm, Callorhinchus milii; Lj, Lethenteron japonicum. doi:10.1371/journal.pone.0113445.g005 (TLE) transcriptional co-repressor for transcriptional repression [56].
All of these domains and motifs are conserved in the Japanese lamprey RunxA, B and C proteins (Fig. 5). All three proteins contain the characteristic Runt domain, within which, residues for DNA-binding and interaction with the b-subunit [57,58] are remarkably well conserved with those of gnathostome Runx proteins. The NLS, NMTS, PY and VWPRY motifs are also conserved in lamprey Runx proteins albeit with slight variations: while gnathostome Runx proteins terminate at the VWPRY motif, the open reading frame of lamprey RunxC continues beyond the VWPRY motif to include four additional amino acids (Fig. 5). Among all known Runx proteins, this peculiar feature is seen only in the single Runt protein of Ciona. The biological significance of these extra residues is unknown. Additionally, lamprey RunxB contains a unique stretch of 47 amino acids within the NLS domain of RunxB (Fig. 5, boxed in green dotted lines). This sequence is encoded by the lamprey-specific extra exon 4.1 that is alternatively spliced in two of the isoforms of RunxB (Fig. 1B). Whether these intervening amino acids affect the nuclear translocation of these RunxB isoforms remains to be verified. The N-termini of the lamprey Runx P1 isoforms beginning with MAS(N/D)S are highly conserved with gnathostome Runx proteins. However, the N-termini of the P2 isoforms of the lamprey proteins vary from those of gnathostomes which comprise MR(I/V)PV. The N-terminal sequences of the lamprey RunxA and RunxB P2 isoforms contain MHIPV and MRTLL, respectively. Similar divergent N-terminal sequences have also been noted in the P2 promoters of Runx1 (MVFLW), Runx2 (MRPIV) and Runx3 (MHIPV) genes of teleost fishes such as fugu and zebrafish [10]. The implications of such divergent N-terminal sequences on the functions of Runx proteins are not known.
The stability and activity of gnathostome Runx proteins are affected by several post-translational modifications including phosphorylation. In human RUNX1, the serine and threonine residues S249, S266, S276 and T273 followed by a proline residue act as phosphorylation sites for ERK [59,60]. Among these sites, S249 and S276 are conserved in lamprey RunxB but not in the other two lamprey Runx proteins (Fig. 5). Phosphorylation at serine residue S104 within the Runt domain of human RUNX2 has been shown to negatively regulate RUNX2 activity by inhibiting its heterodimerization with RUNXb [61]. This serine residue is invariably conserved among all gnathostome Runx. It is also conserved in all three lamprey Runx proteins. Additionally, the consensus phosphorylation site for CDC2, (S/T)PX(R/K), at which serine residue S451 of human RUNX2 was reported to be phosphorylated [62], is highly conserved in gnathostome Runx1-3 proteins. However, it is conserved only in lamprey RunxC and not in the other two lamprey Runx proteins (Fig. 5).

Expression profile of Japanese lamprey a-subunit Runx genes
We investigated the expression patterns of Runx genes in various tissues of adult Japanese lamprey by quantitative RT-PCR. RunxA and RunxB are highly expressed in the gills and intestine-tissues that comprise the primary lymphoid organs of the lamprey (Fig. 6A and B). In the lamprey, lymphocytes develop in the typhlosole, an invaginated spiral valve spanning the length of the intestine as well in the ''thymoid'', the lamprey thymusequivalent that is located at the tips of gill filaments [30]. Runx expression in the gills is reminiscent of that seen in the hagfish and amphioxus, where the gills and associated blood vessels are enriched with lymphocyte-like cells [4,63]. Expression of Runx in the lymphoid compartments of these phylogenetically ancient chordates is consistent with its integral function in the specification of immune-related cells in gnathostomes, and may indicate a possible role for Runx in the primordial immune system of the chordate ancestor. Furthermore, significant Runx expression in the intestine of the adult lamprey appears to support the prevailing view of an ancestral function of Runx genes in the gut, since sea urchin [64], nematode (C.elegans) [65], amphioxus [4] and gnathostomes all express Runx in the developing gut.
Particularly striking is the significant expression of RunxB and RunxC in the skin of the lamprey (Fig. 6B and C). Apart from the primary lymphoid organs such as the gill and intestine, the lamprey epidermis is also home to abundant numbers of lymphocytes, in particular, the VLRC+ lymphocytes that display dendritic morphology and which have been posited to phenotypically resemble mammalian dendritic epidermal T cells (DETC) [31]. Though yet to be verified, based on the significant role Runx3 plays in the development of DETC [66], it may be postulated that the lamprey RunxC similarly contributes to the specification of these immune cell types in the lamprey. In addition, the lamprey brain is another domain of significant RunxB expression (Fig. 6B). Runx expression in the brain and cranial ganglia has been previously demonstrated in the early larvae of the sea lamprey [33]. These expression patterns are consistent with those observed in the neuronal tissues of gnathostomes [27,67,68] as well as a basal metazoan, the sea anemone, Nematostella [11], suggestive of an evolutionarily ancient function in neural development.
Prominent expression of RunxC was also observed in the Japanese lamprey ovary (Fig. 6C). This expression pattern is concordant with Runx function in the female reproductive system of mammals [69,70] and Drosophila [71] and may therefore hint at a possible role of Runx in conserved pathways of ovarian function. In gnathostomes, Runx proteins are central regulators in the development of cellular cartilage and ossification of endochondral bone [28,72]. Expression of Runx has been described in the cartilage of vertebrates that lack mineralized bone, such as the hagfish and cartilaginous fishes [4], suggesting that Runx genes have a conserved functional role in cartilage formation in stem vertebrates. In the sea lamprey, Cattell et. al [33] detected RunxA and RunxB expression in the mucocartilage of larvae but noted the apparent absence of Runx expression in the branchial basket cartilage, skeletal elements that are believed to be homologous to the cellular cartilage of gnathostomes. This led to the speculation that Runx genes may have lost their ancestral function in cartilage development in the lamprey lineage or are not engaged in gene regulatory networks of the ancestral vertebrate cartilage [33]. Here, we have reported the presence of three Runx genes in the Japanese lamprey, as compared to only two previously identified in the sea lamprey. The characterization of the expression of all three Runx genes in the lamprey may shed new light into the involvement of Runx in early vertebrate skeletal development.

Evolution of Runx family genes in vertebrates
Invertebrate chordates such as the amphioxus, Branchiostoma floridae (cephalochordate) and Ciona intestinalis (urochordate) contain a single Runx gene, known as Runt genes. The functions of these genes are not well characterized, although reports documenting Runx expression in the developing gut, pharyngeal endoderm and regenerating oral cirral skeletal cells of the amphioxus [4,73] reflect functions in endodermal specification and roles in the rudimentary skeletogenic programs of these phylogenetically ancient chordates.
Based on the identification of the three Runx genes in the lamprey and their comparative analysis with Runx genes known in gnathostomes, we propose that the stem vertebrate ancestor contained a single Runx gene. As part of the two rounds of whole genome duplications, this gene locus underwent duplications (Fig. 7) in the lineage that gave rise to gnathostomes such as cartilaginous fishes, lobe-finned fishes and tetrapods giving rise to four Runx paralogs [6,7]. Since these vertebrates contain only three Runx paralogs each (Runx1, Runx2 and Runx3), we infer that the fourth paralog was lost in the common ancestor of gnathostomes. The remaining three Runx genes in the gnathostome ancestor acquired specialized and distinct functions such that Runx1 is involved mainly in hematopoiesis [18], Runx2 in skeletogenesis [19] and Runx3 in neurodevelopment and immunity [24,25,27].
The whole-genome duplication history in the lamprey lineage is not well resolved. Although previous studies suggested that lampreys shared the two rounds of duplication with gnathostomes, a more recent study suggested that the two rounds of duplications may have occurred independently in the lamprey lineage followed by an additional round of whole genome duplication [35]. In any case, the presence of six Hox clusters in the Japanese lamprey and the relative age of paralogous genes in the lamprey genome as measured by the transversion rate of four-fold degenerate sites [35] suggests that its lineage has experienced three rounds of genome duplication. According to this hypothesis, the genome duplications would have given rise to eight paralogs of Runx genes in the lamprey lineage. Since only three Runx genes are present in the Japanese lamprey, we infer that five of the eight Runx genes have been lost (Fig. 7). Although the exact functions of the lamprey Runx genes have not been investigated, the differential expression patterns of Runx genes in various tissues of the Japanese lamprey ( Fig. 6), sea lamprey [33] and the Atlantic hagfish [4] are suggestive of distinct functions of each of the cyclostome Runx genes. However, further investigations will be required to understand the specific functions of the Runx gene family in cyclostomes and how they differ from those in gnathostomes. Since lampreys lack mineralized tissues and an Ig-based adaptive immune system, some differences in the functions of gnathostome and lamprey Runx genes are expected.

Characterization of a Japanese lamprey b-subunit Runx gene
Gnathostome a-subunit Runx proteins form heterodimeric complexes with their b-subunit partner. This association not only enhances the DNA binding affinity of the complex by stabilizing the interaction of the a-subunit Runt domain with DNA, it also protects against the ubiquitin-mediated degradation of the a-subunit [74]. In addition to a-subunit Runx genes in the Japanese lamprey, we also cloned full-length coding sequence and various isoforms of the b-unit encoding Runxb. The genomic organization of the Japanese lamprey Runxb gene is largely similar to that of its gnathostome ortholog except for the presence of an additional exon 4.1 (Fig. 8A). Through alternative splicing, the gnathostome Runxb gene gives rise to three isoforms, Runxb Type 1, 2 and 3. Type 1 and Type 2 differ in several amino acids at their carboxyl termini while Type 1 and Type 3 isoforms are almost identical, except for the absence of exon 5 in Type 3 owing to exon skipping [1]. The Japanese lamprey Runxb is also transcribed into these three isoforms (Fig. 8A). In addition, we identified two isoforms (Type 4 and 5) that are unique to the Japanese lamprey (Fig. 8A). Runxb Type 4 terminates at the alternative exon 4.1, while Runxb Type 5 appears to be a truncated form of Type 1, terminating prematurely in exon 5 (Fig. 6A). The Runxb type 1, 3, 4 and 5 isoforms encode proteins of 189, 157, 137 and 168 amino acids, respectively (Fig. 6B). At the protein level, lamprey and gnathostome Runxb show high conservation of amino acid residues 1-165 (Fig. 8B). Of these, the N-terminal 135 amino acids required for its heterodimerization with the asubunit and stimulation of DNA-binding activity [2] as well as essential residues 68-93 for its interaction with filamin A in the cytoplasm [75] are almost perfectly conserved Functional significance of the various isoforms of gnathostomes Runxb are not well documented, though one might speculate that Runxb Type 3, which is missing the domain required for the heterodimerization with the a-subunit, may perform functions that are independent of its interaction with a-subunit Runx proteins. Such a function is supported by the recent observation that the retention of Runxb in the cellular midbody during cytokinesis was not seen to be compromised in the absence of asubunit Runx [76]. It remains to be demonstrated whether the various isoforms of lamprey Runxb exhibit different functions.

Supporting Information
Table S1 Primers used for qRT-PCR of Japanese lamprey Runx genes.