Salmonid genomes have a remarkably expanded akirin family, coexpressed with genes from conserved pathways governing skeletal muscle growth and catabolism

Abstract

Metazoan akirin genes regulate innate immunity, myogenesis, and carcinogenesis. Invertebrates typically have one family member, while most tetrapod and teleost vertebrates have one to three. We demonstrate an expanded repertoire of eight family members in genomes of four salmonid fishes, owing to paralog preservation after three tetraploidization events. Retention of paralogs secondarily lost in other teleosts may be related to functional diversification and posttranslational regulation. We hypothesized that salmonid akirins would be transcriptionally regulated in fast-twitch skeletal muscle during activation of conserved pathways governing catabolism and growth. The in vivo nutritional state of Arctic charr (Salvelinus alpinus L.) was experimentally manipulated, and transcript levels for akirin family members and 26 other genes were measured by quantitative real-time PCR (qPCR), allowing the establishment of a similarity network of expression profiles. In fasted muscle, a class of akirins was upregulated, with one family member showing high coexpression with catabolic genes coding the NF-kappaB p65 subunit, E2 ubiquitin-conjugating enzymes, E3 ubiquitin ligases, and IGF-I receptors. Another class of akirin was upregulated with subsequent feeding, coexpressed with 14-3-3 protein genes. There was no similarity between expression profiles of akirins with IGF hormones or binding protein genes. The level of phylogenetic relatedness of akirin family members was not a strong predictor of transcriptional responses to nutritional state, or differences in transcript abundance levels, indicating a complex pattern of regulatory evolution. The salmonid akirins epitomize the complexity linking the genome to physiological phenotypes of vertebrates with a history of tetraploidization.

Fig. 1.

Phylogenetic tree constructed with maximum likelihood (ML), demonstrating the evolutionary relationships of salmonid Akirin proteins in relation to family members from a broader range of vertebrates. A Bayesian inference (BI) analysis produced a very similar topology. All ML bootstrap confidence values >50% are shown, as well as BI posterior probabilities >50% at important nodes (underlined numbers). Three putative genomic duplication events are marked with arrows, the first in a common vertebrate ancestor [duplication 1 (D1)], the second in a common ancestor to teleosts (D2), and the third in a common ancestor to salmonids (D3). D3? indicates that D2-level salmonid Akirin1(1) sequences did not spilt into 2 monophyletic clades as observed elsewhere in the tree (discussed in text; see Supplemental Fig. S1). A consensus nomenclature proposal accounting for the evolutionary relationships of vertebrate akirins is clearly demonstrated. Vertebrate sequences were rooted to the single Akirin ortholog of 3 deuterostome invertebrates. Scale bar shows number of substitutions per site.

Fig. 2.

Sequence logo alignment showing the amino acid sequence of individual salmonid Akirin1 proteins (4 species) compared with nonsalmonid teleost Akirin1(1) proteins (5 species), Akirin1 of tetrapods (3 species), and Akirin1 and 2 across vertebrate lineages (58 species). Amino acids are color coded by biochemical property and are vertically scaled to represent their conservation at that site. Blue and red stars indicate type II sites in salmonid Akirin family members at the D2 and D3 levels, respectively. Residues underlined with black lines indicate putative nuclear localization signals (NLSs). Residues underlined with purple lines indicate putative phosphorylated sites conserved across the 4 salmonid species. A scale bar is shown on the black line splitting the first and second halves of the alignment.

Fig. 3.

Sequence logo alignment showing the amino acid sequence of individual salmonid Akirin2 family members (4 species per family member) compared with nonsalmonid teleost Akirin2(1) and Akirin2(2) (7 and 5 species, respectively), Akirin1 of tetrapods (5 species), and Akirin1 and 2 across vertebrates (58 species). Other details are as described in Fig. 2.

Fig. 4.

Relative transcript abundance levels of Arctic charr akirin family members in fast-twitch skeletal muscle at different nutritional states. Values are means + SD; n = 6 for all days except days −21 and 14 (n = 5 and n = 4, respectively), where data points were removed by an objective criterion (discussed in text). Details of the normalization strategy can be found in experimental procedures. Transcript abundance values for each gene are on a scale ranging from 0 to 1, relative to the lowest expression value for that gene; thus no between-family member comparisons are provided in this figure. Statistical differences between sampling day means at the P < 0.01 level are indicated by different letter groupings: if 2 means are grouped with distinct letters (e.g., a vs. b, ab vs. c, bc vs. a) they are statistically different or if grouped with identical letters (e.g., a vs. a, a vs. ab, bc vs. c) they are statistically equivalent.

Fig. 5.

Differences in relative transcript abundance levels among akirin family members in Arctic charr fast-twitch skeletal muscle after feeding to satiation and at maximal fasting. Values are scaled between family members relative to the lowest expression value among all genes. Other details are as described in Fig. 4.

Fig. 6.

Hierarchical clustering and seriation of Arctic charr akirin expression data (i.e., from Fig. 4) among equivalent profiles for 20 further genes involved in regulating the balance between anabolic and catabolic metabolism in fast-twitch skeletal muscle. Data are represented as a heat map showing changes in relative transcript abundances for each gene across the same experimental model (42 samples, n = 6 per sampling day) with an accompanying dendrogram representing among-gene profile similarities. Numbers identify clusters for discussion purposes. Note the strong upregulation of catabolic genes in 2 samples at 14 days of refeeding and in 1 sample at day −21.

Fig. 7.

Expression profiles of a subset of cluster 1 and 2 genes other than akirin family members that were markedly regulated at the transcript level across the experimental model altering nutritional state in Arctic charr skeletal muscle. Other details are as described in Fig. 4.