Expression patterns of Japanese lamprey α-subunit <i>Runx</i> genes.

<p>Relative expression levels of (A) <i>LjRunxA</i>, (B) <i>LjRunxB</i> and (C) <i>LjRunxC</i> in various tissues of the Japanese lamprey determined by qRT-PCR. Note that the relative expression levels of each <i>Runx</i> gene between different tissues are estimated in relation to the expression level of the tissue showing the lowest expression, and hence the expression levels are not comparable between different <i>Runx</i> genes.</p

Synteny of genes in the <i>Runx</i> loci of Japanese lamprey and selected metazoans.

<p>Genes are represented by block arrows. Genes with conserved synteny are coloured. Clusters of some non-syntenic genes are represented as white boxes. Grey circles indicate the end of scaffolds.</p

miRNA binding sites in the 3′UTR of human <i>Runx1</i> and <i>Runx3</i> and Japanese lamprey <i>RunxB</i> genes.

<p>The last coding region of <i>Runx</i> gene is represented by a rectangle and 3′UTR by a grey line. Positions of miRNA binding sites are indicated by vertical lines. Binding sites conserved in human and Japanese lamprey are shown in red.</p

Exon-intron organization of lamprey (<i>Lj</i>) <i>Runx</i> genes.

<p>Schematic representation of the gene structures and transcript isoforms of (A) <i>LjRunxA</i>, (B) <i>LjRunxB</i> and (C) <i>LjRunxC</i>. Exons are indicated by boxes. The vertical dashed lines indicate internal splice sites located within the coding exon. Exons constituting the Runt domain are indicated in grey. The two alternative promoters are denoted as P1 and P2. Crosshatched boxes indicate 5′- and 3′-UTRs. The asterisk (*) indicates an exon in <i>LjRunxB</i> that is absent in gnathostome <i>Runx</i>. Not drawn to scale.</p

Phylogenetic analysis of chordate Runx sequences.

<p>Protein sequences of Japanese lamprey <i>Runx</i> genes were aligned with homologous sequences from selected chordates. The gaps in the alignments were trimmed using the Gblocks Server. The resulting protein alignment used for phylogenetic analyses is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113445#pone.0113445.s004" target="_blank">Fig. S3</a>. Maximum Likelihood (ML) trees were generated for the alignments. Statistical support values for the nodes are shown as ML bootstrap percentages. Hagfish and Japanese lamprey Runx proteins are highlighted in red. Lancelet (<i>Branchiostoma floridae</i>) Runt (BfRunt) was used as the outgroup. Hs, <i>Homo sapiens</i>; Gg, <i>Gallus gallus</i>; Dr, <i>Danio rerio</i>; Cm, <i>Callorhinchus milii</i>; Sc, <i>Scyliorhinus canicula</i>; Mg, <i>Myxine glutinosa</i>; Lj, <i>Lethenteron japonicum</i>.</p

Phylogenetic tree (Bayesian inference) of chordate Runx sequences.

<p>Values adjacent to the nodes represent branch support (Bayesian posterior probability). Lancelet (<i>Branchiostoma floridae</i>) Runt (BfRunt) was used as the outgroup. Hs, human; Dr, <i>Danio rerio</i>; Fr, <i>Fugu</i> (<i>Takifugu</i>) <i>rubripes</i>; Cm, <i>Callorhinchus milii</i>; Sc, <i>Scyliorhinus canicula</i>; Ci, <i>Ciona intestinalis</i>.</p

Runx α-subunit proteins Runx1, Runx2 and Runx3 in elephant shark and human.

<p>(A) Schematic representation of Runx α-subunit proteins. The characteristic domains found in all Runx proteins are indicated to show their relative positions along the protein. (B) Alignment of elephant shark and human Runx α-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 boxed with a red line. Within the Runt domain, surfaces involved in DNA contact and interaction with the β-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 by orange and blue boxes, respectively. The transactivation domain (TAD) is highlighted in blue and the inhibitory domain (ID) is boxed by purple dotted lines. The nuclear matrix translocalization signal (NMTS) is boxed with a brown line. Minimal consensus sequences for phosphorylation by Erk or cdc2 are boxed by dashed and solid black lines, respectively. The residue targeted for phosophorylation is indicated by ℗. Cm, <i>Callorhinchus milii</i>; Hs, <i>Homo sapiens.</i></p

Runx α-subunit proteins in human, elephant shark and Japanese lamprey. Alignment of human, elephant shark and Japanese lamprey Runx α-subunit proteins.

<p>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 β-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, <i>Homo sapiens</i>; Cm, <i>Callorhinchus milii</i>; Lj, <i>Lethenteron japonicum</i>.</p

Exon-intron organization and protein sequence encoded by the Japanese lamprey <i>Runxβ</i>.

<p>(A) Schematic representation of the genomic structure and the four transcripts cloned (<i>LjRunxb</i> types 1, 3, 4 and 5). Exons are indicated by boxes. The 5′- and 3′-UTRs are represented as crosshatched boxes. (B) Alignment of Japanese lamprey, elephant shark and human RUNXβ amino acid sequences using ClustalW. Conserved residues are shaded grey. Hs, <i>Homo sapiens</i>; Cm, <i>Callorhinchus milii</i>; Lj, <i>Lethenteron japonicum</i>.</p

A model depicting the evolution of <i>Runx</i> genes in vertebrates.

<p>The phylogenetic analysis and synteny maps suggest that the three <i>Runx</i> genes in lamprey are not one-to-one orthologs of the three <i>Runx</i> genes in gnathostomes.</p