An enigmatic fourth runt domain gene in the fugu genome: ancestral gene loss versus accelerated evolution

Abstract

Background: The runt domain transcription factors are key regulators of developmental processes in bilaterians, involved both in cell proliferation and differentiation, and their disruption usually leads to disease. Three runt domain genes have been described in each vertebrate genome (the RUNX gene family), but only one in other chordates. Therefore, the common ancestor of vertebrates has been thought to have had a single runt domain gene.

Results: Analysis of the genome draft of the fugu pufferfish (Takifugu rubripes) reveals the existence of a fourth runt domain gene, FrRUNT, in addition to the orthologs of human RUNX1, RUNX2 and RUNX3. The tiny FrRUNT packs six exons and two putative promoters in just 3 kb of genomic sequence. The first exon is located within an intron of FrSUPT3H, the ortholog of human SUPT3H, and the first exon of FrSUPT3H resides within the first intron of FrRUNT. The two gene structures are therefore "interlocked". In the human genome, SUPT3H is instead interlocked with RUNX2. FrRUNT has no detectable ortholog in the genomes of mammals, birds or amphibians. We consider alternative explanations for an apparent contradiction between the phylogenetic data and the comparison of the genomic neighborhoods of human and fugu runt domain genes. We hypothesize that an ancient RUNT locus was lost in the tetrapod lineage, together with FrFSTL6, a member of a novel family of follistatin-like genes.

Conclusions: Our results suggest that the runt domain family may have started expanding in chordates much earlier than previously thought, and exemplify the importance of detailed analysis of whole-genome draft sequence to provide new insights into gene evolution.

Figure 1

The four fugu scaffolds analyzed and visualized using the GESTALT Workbench. The RD genes are aligned by the position of the proximal promoter. For each scaffold, graphs are shown of the CpG contrast values (observed/expected, typically ~0.5 for the fugu genome), G+C percentage (green – below 43%, blue – between 43% and 50%, red – above 50%), interspersed repeats (SINEs in red, LINEs in green, DNA and LTR elements in brown, other repeats in purple), and gene annotations. Genes and repeats displayed above each black midline are in the forward strand, while those displayed under the midline are in the reverse strand of the sequence. All scaffolds are shown at a resolution of 200 bp/pixel, except for scaffold 835, which includes the tiny FrRUNT gene. For clarity, only the last 17 kb of this scaffold are shown at 20 bp/pixel. The much longer scaffold scaffold 183 is also shown truncated at 182 kb; the LCK locus has been studied in detail elsewhere [42].

Figure 2

FrRUNT sequence detail, showing its compact organization and its interlocking with the SUPT3H gene. The two genes are disposed in opposite orientations. The inverted lettering for SUPT3H denotes translation from the reverse strand. The two putative promoters and the extent of the runt domain (RD) are indicated; x1–x6 denote the six exons. The black triangle points at the asparagine residue absent from all previously known runt domain proteins.

Figure 3

Localized high divergence between TnRUNT and FrRUNT. This alignment between the first 171 bp of the predicted TnRUNT coding sequence, with the first 180 bp of FrRUNT, shows a cluster of mutations (curly brackets) around the N-terminus of the runt domain (gray background). Non-conservative mutations (negative BLOSUM scores) are surrounded with thick black frames; thin frames enclose conservative mutations, and synonymous mutations are not indicated. The first sixteen codons derive from exon 1, the rest of the alignment from exon 2. The secondary structures indicated (alpha helix, beta strands and loops) are after [21].

Figure 4

Phylogenetic reconstruction of runt domain evolution. Top left: unrooted tree of RD proteins based on the runt domain; X1, X2 and X3 denote the RUNX clades, BfRUNT, FrRUNT, TnRUNT, CiRUNT, OdRUNT, SpRUNT and CeRNT1 represent RUNT proteins from amphioxus, fugu, Tetraodon, Ciona, Oikopleura dioica, sea urchin and C. elegans, respectively, while taxa named Dm- represent D. melanogaster RD proteins. Top right: unrooted tree of first exons of human and fugu runt domain genes. Bottom: expanded species trees for the three RUNX orthologous groups. For all trees, numbers indicate percent bootstrap support; the horizontal bars indicate 10% divergence along each branch. White and gray backgrounds indicate comparisons at the nucleotide and amino acid level, respectively. The dashed branch in the RUNX2 panel represents the position of zebrafish (A) prior to AsaturA analysis. The arrows indicate the change effected by this correction.

Figure 5

Comparative genomic organization. The genomic neighborhoods of the human and fugu RD genes are compared and contrasted, highlighting synteny conservation (same color between the two species) and gene loss (green features). Feature widths represent rough gene size but are not exactly proportional to gene lengths; likewise intergenic distances are not meant to be precise. Arcs indicate larger genomic distances with one or more intervening genes (not displayed). The numbers associated with the arcs represent the distance in Mb.

Figure 6

Comparison of selected follistatin-like proteins. FST: follistatin. (a) Multiple alignment of the cysteine-rich domain. Darker shading indicates perfect conservation, lighter shading indicates positions that can be explained assuming one or two mutations. (b) Neighbor-joining tree rooted using human osteonectin/SPARC (NP_003109) as outgroup. Numbers on branches indicate the percent support in 1000 bootstrap replicates. The horizontal bar indicates 10% divergence along each branch. The column on the right indicates the scaffold in which each gene is located (fugu) or its genomic location in Mb coordinates and cytogenetic band (human).

Figure 7

Hypothesis for the evolution of vertebrate RD genes. Features with thick edges represent RD genes interlocked with SUPT3H family members. The closed padlock icon represents the interlocking event between the RUNX2 and SUPT3H, and the open padlock represents the degradation of a copy of SUPT3H, releasing RUNX2 from interlocking. Not enough information is yet available to establish which of the zebrafish RUNX2 genes is interlocked with a SUPT3H gene. "dup": duplication. Top right inset: Hypothesis of how the ancestral RD duplication could map to the first vertebrate tetraploidization event. RT and RX represent the ancestral forms of RUNT and RUNX genes.

Figure 8

The hypothesized recombination event between the ancestral RUNX2A and RUNT genes. After the recombination, the RUNT gene is linked to SUPT3H and has a RUNX2-like first exon. The derivative RUNX2A gene has not yet been observed in pufferfishes, and may have been lost in evolution.