Evolutionary divergence of the vertebrate TNFAIP8 gene family: Applying the spotted gar orthology bridge to understand ohnolog loss in teleosts

Abstract

Comparative functional genomic studies require the proper identification of gene orthologs to properly exploit animal biomedical research models. To identify gene orthologs, comprehensive, conserved gene synteny analyses are necessary to unwind gene histories that are convoluted by two rounds of early vertebrate genome duplication, and in the case of the teleosts, a third round, the teleost genome duplication (TGD). Recently, the genome of the spotted gar, a holostean outgroup to the teleosts that did not undergo this third genome duplication, was sequenced and applied as an orthology bridge to facilitate the identification of teleost orthologs to human genes and to enhance the power of teleosts as biomedical models. In this study, we apply the spotted gar orthology bridge to help describe the gene history of the vertebrate TNFAIP8 family. Members of the TNFAIP8 gene family have been linked to regulation of immune function and homeostasis and the development of multiple cancer types. Through a conserved gene synteny analysis, we identified zebrafish orthologs to human TNFAIP8L1 and TNFAIP8L3 genes and two co-orthologs to human TNFAIP8L2, but failed to identify an ortholog to human TNFAIP8. Through the application of the orthology bridge, we determined that teleost orthologs to human TNFAIP8 genes were likely lost in a genome inversion event after their divergence from their common ancestor with spotted gar. These findings demonstrate the value of this enhanced approach to gene history analysis and support the development of teleost models to study complex questions related to an array of biomedical issues, including immunity and cancer.

Fig 1. Genomic distribution of human TNFAIP8-family genes.

Grey dots along Hsa5 represent genes whose paralogs are plotted directly above or below the dot on the human chromosomes on which they occur. Note that chromosomes 1, 15, and 19 share many paralogs with the TNFAIP8-containing region of Hsa5. This result suggests that these four genes originated as ohnologs in rounds 1 and 2 of the vertebrate genome duplication events (VGD1 and VGD2).

Fig 2. Zebrafish possesses orthologs to human TNFAIP8L1.

(A) Grey dots along Dre22 represent genes whose orthologs are plotted on the human chromosomes on which they occur. Red dots represent regions of orthology between zebrafish and human chromosomal segments. Zebrafish (Danio rerio) chromosome 22 (Dre22), which contains the tnfaip8 gene incorrectly annotated in genome assembly Zv9, shares conserved synteny with Hsa19, which contains the human TNFAIP8L1 gene. An ortholog to human TNFAIP8 could not be located in the zebrafish genome. (B) Grey dots along Hsa19 represent genes whose orthologs are plotted above the dot on the zebrafish (Dre) chromosomes on which they occur. Red dots represent regions of orthology between Hsa19 and each zebrafish chromosome. Between 0 and 10 Mb, Hsa19 has conserved syntenies with Dre2 and Dre22, which contains the tnfaip8l1 ortholog. (C) Composite cluster mapping shows conserved synteny with the regions surrounding zebrafish tnfaip8l1 (red letters) and human TNFAIP8L1 (red letters), with several local inversions (as illustrated by the crossed lines).

Fig 3. Zebrafish possesses co-orthologs to human TNFAIP8L2.

(A) Dre19, which contains the zebrafish tnfaip8l2a gene, and (B) Dre16, which contains the tnfaip8l2b gene, each share conserved synteny with Hsa1, which contains the human TNFAIP8L2 gene. Gray dots represent genes on Dre 19 (A) and Dre16 (B) with human (Hsa) orthologs, represented as red dots. (C) Dre16 and Dre19 show conserved synteny (red dots that are aligned) [39]; tnfaip8l2a and tnfaip8l2b are TGD co-orthologs of the human TNFAIP8L2 gene.

Fig 4. Zebrafish possesses an ortholog of human TNFAIP8L3.

(A) Red dots represent regions of orthology between zebrafish and human chromosomal segments. Zebrafish (Danio rerio) chromosome 18 (Dre18) (gray dots), which contains the tnfaip8l3 gene, shares orthology with Hsa15, which contains the human TNFAIP8L3 gene. (B) Red dots represent regions of orthology between Hsa15 (gray dots) and the zebrafish genome. Evidence of two co-orthologous regions in zebrafish, likely arising from the TGD, are present in the region on Dre18 and Dre25. The tnfaip8l3 gene is present on Dre18. Additional co-orthologons are present on Dre17 and Dre20 and Dre7 and Dre25. The presence of the tnfaip8l3 orthologon on Dre18 is likely caused by a translocation event that occurred with Dre7. (C) Composite cluster mapping showing conserved synteny between Hsa15 and Dre18 in the regions surrounding human TNFAIP8L3 (red letters) and zebrafish tnfaip8l3 (red letters).

Fig 5. The tnfaip8 gene was lost in a genome inversion event after the divergence of the teleost and spotted gar lineages.

TNFAIP8 is flanked by DMXL1 and HSD17B4 in (A) humans and rayfin fishes that diverged before the teleost genome duplication like the (D) spotted gar. The three genes are transcribed in the same direction in both human and gar genomes. In teleosts including (B) zebrafish and (C) stickleback, tnfaip8 is missing and dmxl1 and hsd17b4 are transcribed in opposite directions. (E, F) Dre8 and Hsa5 share conserved synteny in the region surrounding TNFAIP8. Crossing lines indicate shifts in gene order consistent with a chromosome inversion event. The other breakpoint for the inversion occurred between hsd17b4 and prr16, because they transcribe in opposing directions in the zebrafish (E) and in the same direction in humans (F).

Fig 6. After the TGD, the chromosome region surrounding the ancestral tnfaip8 gene reverted to a single copy.

Grey dots represent regions of orthology between zebrafish chromosomes and human Hsa5. (A) The region surrounding human TNFAIP8 has two sets of co-orthologous paralogons in the zebrafish genome on Dre5 and Dre10 (left) and Dre14 and Dre21 (right). (B) No obvious paralogons are evident in the left tip of Dre8 (containing tnfaip8-neighboring gene hsd17b4). Dre5 and Dre10 show evidence of distant homology.

Fig 7. Adjacent regions of Hsa5 show evidence of orthology with Dre8 and Dre10.

Only a single copy of the region lacking the TNFAIP8 ortholog is present in the zebrafish on Dre8.

Fig 8. Phylogeny of the TNFAIP8 family of proteins.

Sequences were collected from Ensembl (www.ensembl.org) and GenPept (http://www.ncbi.nlm.nih.gov/protein) and aligned using Clustal Omega (S2 Fig) [30]. Sequence identifiers are provided in S2 Fig. Drosophila sequence CG4091-RB was used as an outgroup. The unrooted consensus tree with % bootstrap support for each node (above 50%) is presented. The common and binomial names of animals comprising the alignment (S2 Fig) and cladogram are as follows: zebrafish (Danio rerio), mouse (Mus musculus), human (Homo sapiens), frog (Xenopus tropicalis), chicken (Gallus gallus) and turkey (Meleagris gallopavo), stickleback (Gasterosteus aculeatus), spotted gar (Lepisosteus oculatus), coelacanth (Latimeria chalumnae), Chinese softshell turtle (Pelodiscus sinensis) and anole lizard (Anolis carolinensis).