Developmental and Comparative Immunology 52 (2015) 17–25
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Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i
Evolution of akirin family in gene and genome levels and coexpressed patterns among family members and rel gene in croaker Tianxing Liu a, Yunhang Gao b, Tianjun Xu a,* a b
Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan 316022, China College of Animal Science and Veterinary Medicine, Jilin Agriculture University, Changchun 130118, China
A R T I C L E
I N F O
Article history: Received 16 January 2015 Revised 17 April 2015 Accepted 19 April 2015 Available online 23 April 2015 Keywords: Akirin NLS Gene structure Evolution Expression patterns
A B S T R A C T
Akirins, which are highly conserved nuclear proteins, are present throughout the metazoan and regulate innate immunity, embryogenesis, myogenesis, and carcinogenesis. This study reports all akirin genes from miiuy croaker and analyzes comprehensively the akirin gene family combined with akirin genes from other species. A second nuclear localization signal (NLS) is observed in akirin2 homologues, which is not in akirin1 homologues in all teleosts and most other vertebrates. Thus, we deduced that the loss of second NLS in akirin1 homologues in teleosts likely occurred in an ancestor to all Osteichthyes after splitting with cartilaginous fish. Significantly, the akirin2(2) gene included six exons interrupted by five introns in the miiuy croaker, which may be caused by the intron insertion event as a novel evidence for the variation of akirin gene structure in some species. In addition, comparison of the genomic neighborhood genes of akirin1, akirin2(1), and akirin2(2) demonstrates a strong level of conserved synteny across the teleost classes, which further proved the deduction of Macqueen and Johnston 2009 that the produce of akirin paralogues can be attributed to whole-genome duplications and the loss of some akirin paralogues after genome duplications. Furthermore, akirin gene family members and relish gene are ubiquitously expressed across all tissues, and their expression levels are increased in three immune tissues after infection with Vibrio anguillarum. Combined with the expression patterns of LEAP-1 and LEAP-2 from miiuy croaker, an intricate network of co-regulation among family members is established. Thus, it is further proved that akirins acted in concert with the relish protein to induce the expression of a subset of downstream pathway elements in the NF-kB dependent signaling pathway. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Akirins, conserved nuclear resident NF-kB signaling pathway molecules, are present throughout the metazoan. To date, akirin genes are identified from eukaryotes, including coelenterates, arthropods, fish, amphibians, birds, reptiles, and mammals (Chen et al., 2012; Dai et al., 2011; Goto et al., 2008; Macqueen et al., 2010a, 2010b; Man et al., 2011; Yang et al., 2011). Furthermore, the akirin gene family consists of two members in amphibians and mammals (akirin1 and akirin2), a single member in birds and reptiles (akirin2), and two to three members in teleosts (akirin1(1) and akirin2(1) and/ or akirin2(2)) (Macqueen et al., 2010a, 2010b). However, teleost species of the Salmonidae family include eight akirin family members (akirin1(1a), 1(1b), 1(2a), 1(2b), 2(1a), 2(1b), 2(2a), and 2(2b)) (Macqueen et al., 2010a). Meanwhile, Macqueen and Johnston (2009)
* Corresponding author. Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan 316022, China. Tel.: +580 2550826; fax: 0086-580-2550826. E-mail address: [email protected] (T. Xu). http://dx.doi.org/10.1016/j.dci.2015.04.010 0145-305X/© 2015 Elsevier Ltd. All rights reserved.
and Macqueen et al. (2010a) proposed that the number of akirin gene family members is closely related with whole genome duplications (WGDs). Akirin1 and akirin2 are derived from basal vertebrate WGD (2R) (Putnam et al., 2008). Akirin1(1) and 1(2) along with akirin2(1) and 2(2) are retained from the teleost WGD (3R) (Jaillon et al., 2004). However, akirin1(2) is lost in most species. Eight members of the akirin gene family in the Salmonidae family are paralogues retained from the salmonid WGD (4R) (Macqueen and Johnston, 2014). The innate immune system, which serves as the first line of defense, shields all metazoans against invasion of pathogens and deals with any foreign material until the adaptive immune system can sufficiently take over (Medzhitov and Janeway, 1997; Sinyakov et al., 2002). As an indispensable component of the innate immune responses, akirins can coordinate with “14-3-3” proteins to promote or to inhibit mRNA transcription (Gonzalez and Baylies, 2005; Komiya et al., 2008) and interact genetically and physically with the basic helix–loop–helix transcription factor (Twist) to facilitate the expression of a number of Twist-regulated genes during embryonic myogenesis (Nowak et al., 2012). Akirins are essential in animal development, owing to the lethal embryonic phenotype of mice
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knockouts as well as the lethal or reduced growth phenotypes demonstrated by targeted knockdown in Drosophila, ticks, and nematodes (de la Fuente et al., 2006; Goto et al., 2008; Maeda et al., 2001). Meanwhile, akirins participate in regulating gene expressions in numerous physiological processes, including the innate immune responses of mammalians and insects (Goto et al., 2008; Tartey et al., 2014), metazoan myogenesis (Marshall et al., 2008; Salerno et al., 2009), mammalian carcinogenesis (Komiya et al., 2008), insect reproduction, and arthropod growth (Almazán et al., 2005; de la Fuente et al., 2006, 2008). Furthermore, the study demonstrated that akirins are required to activate a subset of Relish-dependent genes (Bonnay et al., 2014). Akirins acted in concert with relish (rel) protein in the NFkB dependent signaling pathway, which induces the expression of a subset of downstream pathway elements (e.g., LEAP-1 and LEAP2) (Goto et al., 2008; Hou et al., 2013; Xue et al., 2014). In this study, we extended the comparative genomics and phylogenetics work by Macqueen and Johnston (2009) and analyzed the akirin gene structure and the genomic neighborhood surrounding of akirin genes to better understand akirin genes in miiuy croaker (Miichthys miiuy) and to establish the chromosomal locations of akirin genes in teleosts. Furthermore, we conducted realtime quantitative PCR (qRT-PCR) assays by using akirin genes and relish (rel) gene of the miiuy croaker to investigate the regulation relationship among akirin gene family members, along with rel gene.
release Ensembl genome assemblies for teleost and lamprey (Petromyzon marinus). The miiuy croaker corresponding data were obtained from the whole-genome sequences of miiuy croaker (unpublished).
2. Materials and methods
2.5. RNA isolation and cDNA synthesis
2.1. Sequence analysis
Total RNA was extracted from diverse tissues from individuals following the manufacturer’s instructions by using RNAiso Reagent (Takara). The cDNA templates were synthesized through reverse transcriptase M-MLV (Takara) according to the manufacturer’s protocol. Extracted RNA samples were stored at −80 °C until its use, whereas cDNA samples were diluted 10-fold and stored at −20 °C until further analysis.
To identify the akirin1, akirin2(1), and akirin2(2) genes from the miiuy croaker, we used the available akirin homologues reported in other organisms as queries to seek for the transcriptome (Che et al., 2014) and whole-genome sequences of the miiuy croaker (unpublished) by using local BLASTN program. Three corresponding scaffolds were identified. The cDNA sequences were aligned with the corresponding scaffolds by using MAFFT (http://mafft.cbrc .jp/alignment/software/) to determine the exon–intron structure of akirin genes from the miiuy croaker. Open Reading Frame Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to predict the open reading frame (ORF) and to translate nucleotide into protein. Nuclear localization signal (NLS) was forecasted using PSORT II server (http://psort.hgc.jp/form2.html), and SignalP 4.1 server was used to predict the presence and location of signal peptide cleavage sites (Nielsen et al., 1997). 2.2. Sequence alignment and phylogenetic analyses The akirin genes used in this study were derived from GenBank (http://www.ncbi.nlm.nih.gov) and Ensemble database (http:// www.ensembl.org) (Supporting information, Table S1). Multiple alignments of the akirin sequences from different species were performed under codon model by using MUSCLE software (Edgar, 2004). Before constructing the phylogenetic tree, jModeltest software was used to select an optimal substitution model (Posada, 2008), through which GTR + I + G was considered the best-fit model based on Bayesian information criterion (Posada and Buckley, 2004). Phylogenetic analysis was performed using Bayesian approach in MrBayes v3.2 (Ronquist and Huelsenbeck, 2003), which was ran for 5,000,000 generation with the first 25% of trees burned.
2.4. Fish sampling and challenge experiment Healthy miiuy croakers (mean weight: 800g) were collected from Zhoushan Fisheries Research Institute (Zhejiang, China). Fish were acclimatized in a recirculating seawater system at an ambient temperature (25 °C) for one week. Challenge experiments, as previously described by Zhu et al. (2013), were performed on the miiuy croaker. Fish were randomly divided into two groups, namely, injection and control groups. In the injection group, fish were challenged with 1 mL of Vibrio anguillarum suspension (approximately 3.0 × 107 CFU/mL) by intraperitoneal injection as the control fish were injected with 1 mL of phosphate-buffered saline. The infected and control fish samples were killed at 6, 12, 24, 36, 48, and 72 h after the challenge. Three immune tissues (head kidney, liver, and spleen) from three individuals were sampled. Furthermore, 10 tissues (head kidney, liver, spleen, intestines, heart, muscle, gill, brain, eye, and skin) from healthy miiuy croaker were removed. All tissues were immediately frozen in liquid nitrogen after dissection and then separately stored at −80 °C until RNA extraction.
2.6. Expression analysis of akirin genes and rel gene To examine the tissue distribution of akirin mRNAs in different tissues of healthy miiuy croaker and to investigate the regulation relationship among akirin and rel genes after immune challenges, real-time qPCR was conducted on a 7300 Real-time PCR system (Applied Biosystems, USA) by using a RealMaster Mix kit (TIANGEN) following the manufacturer’s instructions. Reaction was carried out without the template used as blank control. Five pairs of qPCR primers were used to amplify akirin, rel, and β-actin genes as normalizer gene fragments (Supporting information, Table S2). Realtime qPCR reactions were carried out as described by Liu et al. (2014). The reaction for each sample was conducted in triplicate, and the cycling conditions were as follows: 15 min at 95 °C, followed by 45 cycles of 15 s at 95 °C, 60 s at 60 °C, and 31 s at 72 °C. Dissociation curve analysis was performed after each assay to determine target specificity. The PCR efficiency was determined using LinRegPCR (Karlen et al., 2007; Ruijter et al., 2014). Pfaffl (2001) method was selected as the relative quantification calculation method. Statistical analysis was performed using one-way analysis of variance statistical test followed by Duncan’s multiple comparison tests. P-values of less than 0.05 were considered statistically significant. All data were expressed as mean ± standard error. 3. Results
2.3. Comparative analysis of gene synteny To obtain insight on whether the genomic neighborhoods surrounding akirin genes are evolutionary conserved, synteny maps for the genomic neighborhoods surrounding akirin1, akirin2(1), and akirin2(2) were constructed. Data were manually obtained from
3.1. Characterization and structure of akirin genes from miiuy croaker The molecular characteristics of akirin1, akirin2(1), and akirin2(2) from the miiuy croaker are compiled in Table 1. The complete cDNA
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Table 1 Summary of characteristic features of akirin gene family members from miiuy croaker. Features
akirin1
akirin2(1)
akirin2(2)
GenBank accession number Gene size Length of cDNA Open reading frame 5’-UTR 3’-UTR Polyadenylation signals RNA instability motif Amino acids Molecular mass of protein Isoelectric point of protein Signal peptide Nuclear localization signal (NLS) Second NLS Number of exons Number of introns
KP340435 12267 1337 564 56 717 AATAAA ATTTA 187 21.2KDa 9.2 Nil 19PQSPKRRRCN28 Nil 5 4
KP340436 3805 1515 540 204 771 AATAAA ATTTA 179 20.4 9.01 Nil 19PASPKRRRCA28 73KRRH76 5 4
KP340437 2736 1498 555 138 805 AATAAA ATTTA 184 20.8 8.79 Nil 19PTSPKRRRCI28 75KRKH78 6 5
sequence of akirin1 consisted of 1337 bp, which contained a 5’terminal untranslated region (UTR) of 56 bp; an ORF of 564 bp; a 3’-UTR of 717 bp; and encoded a protein with 187 amino acids (Supporting information, Fig. S1). The full length of akirin2(1) cDNA was 1515 bp, with an ORF of 540 bp encoding for 179 amino acids. The 5’-UTR and 3’-UTR were 204 and 771 bp, respectively (Supporting information, Fig. S2). The complete cDNA sequence of akirin2(2) was 1498 bp, consisting of a 5’-UTR of 138 bp, an ORF of 555 bp encoding for 184 amino acids, and a 3’-UTR of 805 bp (Supporting information, Fig. S3). The instability index (II) of akirin1, akirin2(1), and akirin2(2) was computed as 90.61, 94.98, and 92.21, respectively, which indicated that akirins are unstable. The protein sequence analysis for the motifs, domains, and signal peptide revealed that no signal peptide was present in akirin1, akirin2(1), and akirin2(2), which indicated that they were non-secretory proteins. A putative 10-peptide nuclear localization signal (NLS) was found in the N-terminal region in akirin1 (19PQSPKRRRCN28), akirin2(1) (19PASPKRRRCA28), and akirin2(2) (19PTSPKRRRCI28), and their presence was approved in the nucleus. Furthermore, a putative second NLS was presented in akirin2(1) (73KRRH76) and akirin2(2) (75KRKH78), but was absent in akirin1 in miiuy croaker. One NLS was observed in akirin1 homologues, whereas two NLSs were present in akirin2 homologues. This phenomenon was similarly found in all teleosts. However, two NLSs in akirin1 of elephant shark (Callorhinchus milii) were derived. The genomic DNA sequences of akirin1, akirin2(1), and akirin2(2) from the miiuy croaker contained 12267, 3805, and 2736 bp, respectively (GenBank accession no. KP340435, KP340436, and KP340437). The alignment results of the cDNA sequences and the genomic DNA sequences of akirin1, akirin2(1), and akirin2(2) genes revealed that akirin1 and akirin2(1) contained five exons and four introns (Fig. 1A), which is consistent with the akirin homologues from other teleost species. The exon–intron structure of akirin2(2) from the miiuy croaker indicated the presence of six exons interrupted by five introns (Fig. 1A), which is different from the akirin homologues from other teleost species (except for akirin2(1) of rock bream (Oplegnathus fasciatus). It should be named akirin2(2) according to the analyses of phylogenies and conserved synteny. Thus, it was named akirin2(2) in this paper.). All introns of akirin genes in the miiuy croaker completely conformed with the GT-AG rule (Supporting information, Figs. S1, S2, and S3). 3.2. Evolution of akirin gene structure Akirin1 and akirin2(1) genes of the miiuy croaker consisted of five exons and four variable introns, whereas the akirin2(2) gene in the miiuy croaker comprised six exons, and its exon 1 failed to
translate nucleotides (Figs. 1A, B). However, Macqueen and Johnston (2009) demonstrated that the akirin gene structure was from a single exon into the present-day gene structure of five exons and four introns (Fig. 1A,B). 3.3. Comparison of gene synteny with other vertebrates Synteny analysis results revealed that the miiuy croaker akirin1 gene located between rhbdl2 and cnr2 genes was consistent with most teleosts although different from lamprey, elephant shark, coelacanth (Latimeria chalumnae), and platyfish (Xiphophorus maculatus), in which rhbdl2 or cnr2 was missing. The genomic surrounding genes of akirin1 only had one homologous gene in the lamprey, elephant shark, and coelacanth, whereas a number of genes surrounding akirin1 included two homologous genes on different chromosomes or scaffolds in the other teleosts. Furthermore, the akirin1 gene was not found in the atlantic cod (Gadus morhua) and the green spotted puffer (Tetraodon nigroviridis). However, a single akirin1 gene was identified in the others examined (Fig. 2). However, akirin2(1) and akirin2(2) were present in the Acanthopterygian taxa (i.e., atlantic cod and green spotted puffer), which were located on different chromosomes or scaffolds, as well as in most other teleosts (Fig. 3). Furthermore, the akirin2(1) located between rars2 and cnr1(1) genes, as well as the akirin2(2) located between znf292(2) and cnr1(2) in the miiuy croaker, were consonant with other teleosts (Fig. 3). Most genomic neighborhood genes of akirin1 and akirin2 in the teleosts included two homologues (e.g., cnr2, fhl3, fabp10, rragca, znf292, gabrr1, gabrr2, syncrip, begain, nt5e, cnr1, and pm20d2), which were positioned on different chromosomes or scaffolds. In addition, comparing the genes in the genomic neighborhood of akirin1, akirin2(1), and akirin2(2) indicated a strong level of conserved synteny across teleost classes, with limited intra- or inter-chromosomal rearrangements, which is consistent with the results obtained by Macqueen and Johnston (2009). 3.4. Phylogenetic analysis of akirin genes Phylogenetic analysis of akirin gene family members from different species indicated sequences as they completely clustered in two groups, namely, akirin1 and akirin2 (Fig. 4). In the akirin2 clade, teleost samples split into two subclades, and akirin2(1) and akirin2(2) of the miiuy croaker were divided into two subclades, respectively. In the subclades, salmonid sequences were further split into two groups, each containing four sequence homologues. A similar phenomenon in the akirin2 group was observed in the akirin1 clade, whereas akirin1 included only a single sequence in teleosts (except for Salmonid).
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Fig. 1. The genomic organization of akirin proto-orthologues across eukaryotic lineages. A. Comparison of the miiuy croaker akirin gene structure with the previously published gene structures of other species. The lines stand for introns and exons are shown as boxes. The boxes with slashes represent the untranslated exons. The genes covered by the shadow represent that this gene had been lost after genome duplication. B. Evolution of gene structure and gene duplication of akirin gene family members. The question mark (?) symbol represents the exon gain events or the intron insertion events. The genes covered by the shadow represent that this gene had been lost after genome duplication. (This figure is an extension to Macqueen and Johnston, 2009.)
3.5. Comparative transcript of akirin gene family members and rel gene We examined the tissue expression patterns of akirin1, akirin2(1), akirin2(2), and rel in the miiuy croaker through real-time qPCR. Expression results showed that the akirin genes and the rel gene from the miiuy croaker, as seen in most akirin genes in other teleost species, were ubiquitously expressed across all 10 tissues (head kidney, liver, spleen, intestines, heart, muscle, gill, brain, eye, and skin). However, the expression levels were obviously different in each tissue (Fig. 5A). Within the examined tissues, the high expression level of akirin genes appeared in the liver, spleen, head kidney, and muscle, and the high-level expressions of rel were detected in the liver and spleen. In addition, the lowest expression levels of akirin1, akirin2(2), and rel appeared in the intestine, whereas the heart indicated the lowest expression level for akirin2(1) (Fig. 5A). To determine the potential roles of akirin genes (akirin1, akirin2(1), and akirin2(2)) from the miiuy croaker after injection with the pathogenic bacteria V. anguillarum and to understand the regulating relationship among akirin genes, along with the rel gene, the relative quantity changes of the miiuy croaker in the liver, spleen, and head kidney during 72 h of induction were assessed. In the liver, the relative expressions of akirin and rel genes increased sharply at 6 h, and maximum induction after challenge also occurred at this time point. Subsequently, the expression level gradually decreased at 12, 24, 36, and 48 h but moderately increased at 72 h (Fig. 5B). In the spleen and head kidney, the expressions of akirin and rel genes were gradually increased after infection where the highest expression level was recorded at 24 h and then gradually decreased (Fig. 5C,D). However, the expression of rel gene in the spleen and akirin2(2) gene in the head kidney moderately increased
at 72 h (Fig. 5C,D). To sum up, the expression levels of akirin and rel genes in the liver, spleen, and head kidney were found first upregulated and then downregulated, and finally recovered closer to a normal level (ignoring 72 h) after infection with V. anguillarum.
4. Discussion In this study, we identified first the full length of akirin1, akirin2(1), and akirin2(2) cDNA sequences and genomic DNA sequences from the miiuy croaker and further investigated the evolution of the akirin gene family in gene structure and the duplication of akirin at the genome level. Meanwhile, the transcript relationships of each member from the akirin gene family and rel gene were analyzed using the corresponding genes from miiuy croaker, and the co-expressed patterns among akirin and rel genes were established. The analysis suggested that a typical NLS motif was present in the N-terminal of miiuy croaker akirin1, akirin2(1), and akirin2(2). A putative second NLS was observed in akirin2(1) and akirin2(2). However, this NLS was not identified in akirin1 of miiuy croaker. Furthermore, Macqueen and Johnston (2009) demonstrated that a second NLS was present in akirin and akirin2 but not in akirin1, and the earliest second NLS in akirin could date back to Placozoans. Confusingly, we found that two NLSs exist in akirin1 and akirin2 from other vertebrates (e.g., elephant shark). In all teleosts, second NLS was present only in akirin2 homologues but not in akirin1 homologues. Thus, we deduced that the loss of the second NLS of akirin1 homologues in all teleosts likely occurred in an ancestor to all Osteichthyes after the split with cartilaginous fish for some unclear reason. However, this inference needs further research in the future.
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Fig. 2. The genomic neighborhood surroundings akirin1 of fish and lamprey. The arrows indicate the transcriptional direction. Single, double and triple diagonal lines indicate a genomic distance respectively spanning 1, 2 and >3 genes. Two genes with red shades connected by a red slash indicate two homologues produced in the genome duplication.
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Fig. 3. The genomic neighborhood surroundings akirin2(1) and akirin2(2) of fish. Details are as in the Fig. 2 legend.
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Fig. 4. Phylogenetic tree of collected akirin genes was constructed using Bayesian method. The numbers in topologies represent Bayesian posterior probability values.
Akirins are present throughout the metazoan, but their gene structures were different in different species. The earliest akirin genes were identified in the Protista taxa Alveolata (in cryptomonas phi), and Heterolobosea (in brain-eating amoeba) only had a single exon. The akirin genes that subsequently undergo the exon gain event and the intron insertion event were organized as five exons of comparable size and four more variable introns in most vertebrates (Fig. 1A) (Macqueen and Johnston, 2009). The akirin1 and akirin2(1) of miiuy croaker, similar to the akirin homologues of the teleost and mammalian, consisted of five exons and four variable introns and possessed the start codon in the first exon itself (Fig. 1A). However, in akirin2(2) of miiuy croaker, six exons and five introns are different from other teleosts. The first exon of the miiuy croaker akirin2(2) consisted of non-coding nucleotides, and the start codon was present in the second exon (Fig. 1A). Given that exons 3, 4, 5, and 6 of
akirin2(2) from miiuy croaker were consistent with other teleost and mammalian species and the untranslated exon formed in the untranslated 5’ region, we can speculate that a second intron insertion event would have occurred between exons 1 and 2 (Kasthuri et al., 2013). This phenomenon may be specific to a particular species but the same phenomenon also appeared in akirin2(2) of rock bream. This phenomenon is a novel evidence for the variation of akirin structure in some species for some unclear reason. To clarify the reason of the phenomenon, more data and further research are necessary. To understand the evolutionary relationship of akirin genes, we analyzed the genomic neighborhood that surrounds the akirin genes of teleost and lamprey. The result showed that akirin1 and akirin2 were both present in all examined teleost genomes (except for green spotted puffer) and were positioned on different chromosomes or scaffolds. However, akirin2 was not found in lamprey (Figs 2 and 3).
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Fig. 5. The expression profiles of akirin genes and rel gene in ten specific tissues of miiuy croaker (A) and expression analysis in liver (B), spleen (C) and head kidney (D) during 6, 12, 24, 36, 48, and 72 h post-inoculation with V. anguillarum. The C on the abscissa stand for the control; the expression level of β-actin was set as the internal control for real-time PCR. Deviation bars represent the standard errors. Values with the same superscript were not significantly different (P > 0.05). Data indicated with asterisk symbol (*) were significantly different (P < 0.05) between injection and control groups.
Macqueen and Johnston (2009) and Macqueen et al. (2010a, 2010b) demonstrated a genome duplication event, which occurred in a common ancestor to the vertebrate lineage, prior to the separation of gnathostome and agnathan lineages, which lead to the generation of akirin1 and akirin2 from an akirin proto-orthologue. Our result further proved the viewpoint. In all examined teleost genomes, two akirin2 homologues (akirin2(1) and akirin2(2)) were found on different chromosomes or scaffolds. Meanwhile, some genomic neighborhood genes of akirin2(1) or akirin2(2) gene in all examined teleosts had two homologues (Fig. 3). If different gene members arose from the duplication of the genome, other genes around them would have been duplicated at the same time (Macqueen and Johnston, 2009). Thus, if teleosts have been subjected to WGD, two pairs of paralogues, namely, akirin1(1) and akirin1(2) (from akirin1) and akirin2(1) and akirin2(2) (from akirin2), exist on different chromosomes in teleosts. In addition, the genomic neighborhood genes of akirin1 and akirin2 should have two homologues. However, the paralogue gene of akirin1 is not present in all teleost genomes that were examined (except for Salmonid), and most genomic neighborhood genes of akirin1 and akirin2 are also only
single-copy genes (Figs 2 and 3). Macqueen and Johnston (2009) indicated that a paralog may be lost after WGD because one of two paralogs in 85% or higher of pairs was lost after the WGD (Brunet et al., 2006). Although the expression levels of akirin and rel genes in the expression analysis were slightly different in all 10 tissues from healthy miiuy croaker, their highest expression level all appeared in the liver (Fig. 5A). Previous studies demonstrated that the highest expression of HAMP (also named LEAP-1) and LEAP-2 from miiuy croaker was also present in the liver (Liu et al., 2014; Xu et al., 2012). This finding suggests that these genes may have some connection in biological functions. To further explore the possible biological functions of akirins, the temporal expression of akirins was examined at different time points after the challenge with V. anguillarum, which was defended against by akirin in flies and mice (Goto et al., 2008) in three main immune tissues. The temporal expression of akirins revealed that different expression patterns existed in the liver, spleen, and head kidney. In the liver, the expression level of akirins and rel at 6 h after infection was about 45–250 times of the control level (Fig. 5B). This
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result suggests that they may play a role in the early defense against invading pathogens in miiuy croaker. The expression level of akirins and rel was significantly upregulated within 6 h; this finding was also observed in LEAP-2 of miiuy croaker, but was not observed in LEAP-1 of miiuy croaker (Liu et al., 2014; Xu et al., 2012). However, in the spleen and head kidney, akirins and rel transcripts were upregulated at 24 h (P < 0.05) (Fig. 5C,D). Interestingly, the same expression patterns existed in LEAP-1 of miiuy croaker but did not appear in LEAP-2 of miiuy croaker (Liu et al., 2014; Xu et al., 2012). Although the reason behind this difference is unknown, the above results further support that akirins and rel induced the expression of LEAP-1 and LEAP-2, i.e., the downstream elements of NF-kBdependent signaling pathway (Goto et al., 2008; Hou et al., 2013; Xue et al., 2014). Furthermore, we found that the gene expression patterns of akirin paralogues and rel were often highly correlated, which suggests that natural selection has maintained an intricate network of co-regulation among family members (Macqueen et al., 2010b) along with rel gene. Acknowledgements This study was supported by the National Natural Science Foundation of China (31370049), Zhejiang Provincial Natural Science Foundation of Distinguished Young Scientists (LR14C040001) and National Spark Program (2013GA700247). Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.dci.2015.04.010. References Almazán, C., Blas-Machado, U., Kocan, K.M., Yoshioka, J.H., Blouin, E.F., Mangold, A.J., et al., 2005. Characterization of three Ixodes scapularis cDNAs protective against tick infestations. Vaccine 23, 4403–4416. Bonnay, F., Nguyen, X.H., Cohen-Berros, E., Troxler, L., Batsche, E., Camonis, J., et al., 2014. Akirin specifies NF-κB selectivity of Drosophila innate immune response via chromatin remodeling. EMBO J. 33, 2349–2362. Brunet, F.G., Crollius, H.R., Paris, M., Aury, J.M., Gibert, P., Jaillon, O., et al., 2006. Gene loss and evolutionary rates following whole-genome duplication in teleost fishes. Mol. Biol. Evol. 23, 1808–1816. Che, R., Sun, Y., Sun, D., Xu, T., 2014. Characterization of the miiuy croaker (Miichthys miiuy) transcriptome and development of immune-relevant genes and molecular markers. PLoS ONE 9, e94046. Chen, X., Huang, Z., Jia, G., Wu, X., Wu, C., 2012. Molecular cloning, tissue distribution and functional analysis of porcine Akirin2. Anim. Biotechnol. 23, 124–131. de la Fuente, J., Almazán, C., Blas-Machado, U., Naranjo, V., Mangold, A.J., Blouin, E.F., et al., 2006. The tick protective antigen, 4D8, is a conserved protein involved in modulation of tick blood ingestion and reproduction. Vaccine 24, 4082–4095. de la Fuente, J., Maritz-Olivier, C., Naranjo, V., Ayoubi, P., Nijhof, A.M., Almazán, C., et al., 2008. Evidence of the role of tick subolesin in gene expression. BMC Genomics 9, 372. Dai, F., Huang, K., Liu, H., Han, C., Li, L., Wang, J., 2011. Cloning, sequence analysis and specific expression in different tissues of duck Akirin2 gene. Acta Vet. Zootech. Sin. 42, 33–38. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Gonzalez, K., Baylies, M., 2005. Bhringi: a novel Twist co-regulator. A Dros. Res. Conf. 46, 320B. Goto, A., Matsushita, K., Gesellchen, V., Chamy, L.E., Kuttenkeuler, D., Takeuchi, O., et al., 2008. Akirins are highly conserved nuclear proteins required for NF-κBdependent gene expression in drosophila and mice. Nat. Immunol. 9, 97–104. Hou, F., Wang, X., Qian, Z., Liu, Q., Liu, Y., He, S., et al., 2013. Identification and functional studies of Akirin, a potential positive nuclear factor of NF-κB signaling pathways in the Pacific white shrimp, Litopenaeus vannamei. Dev. Comp. Immunol. 41, 703–714. Jaillon, O., Aury, J.M., Brunet, F., Petit, J.L., Stange-Thomann, N., Mauceli, E., et al., 2004. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431, 946–957.
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Contents lists available at ScienceDirect
Developmental and Comparative Immunology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d c i
Evolution of akirin family in gene and genome levels and coexpressed patterns among family members and rel gene in croaker Tianxing Liu a, Yunhang Gao b, Tianjun Xu a,* a b
Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan 316022, China College of Animal Science and Veterinary Medicine, Jilin Agriculture University, Changchun 130118, China
A R T I C L E
I N F O
Article history: Received 16 January 2015 Revised 17 April 2015 Accepted 19 April 2015 Available online 23 April 2015 Keywords: Akirin NLS Gene structure Evolution Expression patterns
A B S T R A C T
Akirins, which are highly conserved nuclear proteins, are present throughout the metazoan and regulate innate immunity, embryogenesis, myogenesis, and carcinogenesis. This study reports all akirin genes from miiuy croaker and analyzes comprehensively the akirin gene family combined with akirin genes from other species. A second nuclear localization signal (NLS) is observed in akirin2 homologues, which is not in akirin1 homologues in all teleosts and most other vertebrates. Thus, we deduced that the loss of second NLS in akirin1 homologues in teleosts likely occurred in an ancestor to all Osteichthyes after splitting with cartilaginous fish. Significantly, the akirin2(2) gene included six exons interrupted by five introns in the miiuy croaker, which may be caused by the intron insertion event as a novel evidence for the variation of akirin gene structure in some species. In addition, comparison of the genomic neighborhood genes of akirin1, akirin2(1), and akirin2(2) demonstrates a strong level of conserved synteny across the teleost classes, which further proved the deduction of Macqueen and Johnston 2009 that the produce of akirin paralogues can be attributed to whole-genome duplications and the loss of some akirin paralogues after genome duplications. Furthermore, akirin gene family members and relish gene are ubiquitously expressed across all tissues, and their expression levels are increased in three immune tissues after infection with Vibrio anguillarum. Combined with the expression patterns of LEAP-1 and LEAP-2 from miiuy croaker, an intricate network of co-regulation among family members is established. Thus, it is further proved that akirins acted in concert with the relish protein to induce the expression of a subset of downstream pathway elements in the NF-kB dependent signaling pathway. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Akirins, conserved nuclear resident NF-kB signaling pathway molecules, are present throughout the metazoan. To date, akirin genes are identified from eukaryotes, including coelenterates, arthropods, fish, amphibians, birds, reptiles, and mammals (Chen et al., 2012; Dai et al., 2011; Goto et al., 2008; Macqueen et al., 2010a, 2010b; Man et al., 2011; Yang et al., 2011). Furthermore, the akirin gene family consists of two members in amphibians and mammals (akirin1 and akirin2), a single member in birds and reptiles (akirin2), and two to three members in teleosts (akirin1(1) and akirin2(1) and/ or akirin2(2)) (Macqueen et al., 2010a, 2010b). However, teleost species of the Salmonidae family include eight akirin family members (akirin1(1a), 1(1b), 1(2a), 1(2b), 2(1a), 2(1b), 2(2a), and 2(2b)) (Macqueen et al., 2010a). Meanwhile, Macqueen and Johnston (2009)
* Corresponding author. Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan 316022, China. Tel.: +580 2550826; fax: 0086-580-2550826. E-mail address: [email protected] (T. Xu). http://dx.doi.org/10.1016/j.dci.2015.04.010 0145-305X/© 2015 Elsevier Ltd. All rights reserved.
and Macqueen et al. (2010a) proposed that the number of akirin gene family members is closely related with whole genome duplications (WGDs). Akirin1 and akirin2 are derived from basal vertebrate WGD (2R) (Putnam et al., 2008). Akirin1(1) and 1(2) along with akirin2(1) and 2(2) are retained from the teleost WGD (3R) (Jaillon et al., 2004). However, akirin1(2) is lost in most species. Eight members of the akirin gene family in the Salmonidae family are paralogues retained from the salmonid WGD (4R) (Macqueen and Johnston, 2014). The innate immune system, which serves as the first line of defense, shields all metazoans against invasion of pathogens and deals with any foreign material until the adaptive immune system can sufficiently take over (Medzhitov and Janeway, 1997; Sinyakov et al., 2002). As an indispensable component of the innate immune responses, akirins can coordinate with “14-3-3” proteins to promote or to inhibit mRNA transcription (Gonzalez and Baylies, 2005; Komiya et al., 2008) and interact genetically and physically with the basic helix–loop–helix transcription factor (Twist) to facilitate the expression of a number of Twist-regulated genes during embryonic myogenesis (Nowak et al., 2012). Akirins are essential in animal development, owing to the lethal embryonic phenotype of mice
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knockouts as well as the lethal or reduced growth phenotypes demonstrated by targeted knockdown in Drosophila, ticks, and nematodes (de la Fuente et al., 2006; Goto et al., 2008; Maeda et al., 2001). Meanwhile, akirins participate in regulating gene expressions in numerous physiological processes, including the innate immune responses of mammalians and insects (Goto et al., 2008; Tartey et al., 2014), metazoan myogenesis (Marshall et al., 2008; Salerno et al., 2009), mammalian carcinogenesis (Komiya et al., 2008), insect reproduction, and arthropod growth (Almazán et al., 2005; de la Fuente et al., 2006, 2008). Furthermore, the study demonstrated that akirins are required to activate a subset of Relish-dependent genes (Bonnay et al., 2014). Akirins acted in concert with relish (rel) protein in the NFkB dependent signaling pathway, which induces the expression of a subset of downstream pathway elements (e.g., LEAP-1 and LEAP2) (Goto et al., 2008; Hou et al., 2013; Xue et al., 2014). In this study, we extended the comparative genomics and phylogenetics work by Macqueen and Johnston (2009) and analyzed the akirin gene structure and the genomic neighborhood surrounding of akirin genes to better understand akirin genes in miiuy croaker (Miichthys miiuy) and to establish the chromosomal locations of akirin genes in teleosts. Furthermore, we conducted realtime quantitative PCR (qRT-PCR) assays by using akirin genes and relish (rel) gene of the miiuy croaker to investigate the regulation relationship among akirin gene family members, along with rel gene.
release Ensembl genome assemblies for teleost and lamprey (Petromyzon marinus). The miiuy croaker corresponding data were obtained from the whole-genome sequences of miiuy croaker (unpublished).
2. Materials and methods
2.5. RNA isolation and cDNA synthesis
2.1. Sequence analysis
Total RNA was extracted from diverse tissues from individuals following the manufacturer’s instructions by using RNAiso Reagent (Takara). The cDNA templates were synthesized through reverse transcriptase M-MLV (Takara) according to the manufacturer’s protocol. Extracted RNA samples were stored at −80 °C until its use, whereas cDNA samples were diluted 10-fold and stored at −20 °C until further analysis.
To identify the akirin1, akirin2(1), and akirin2(2) genes from the miiuy croaker, we used the available akirin homologues reported in other organisms as queries to seek for the transcriptome (Che et al., 2014) and whole-genome sequences of the miiuy croaker (unpublished) by using local BLASTN program. Three corresponding scaffolds were identified. The cDNA sequences were aligned with the corresponding scaffolds by using MAFFT (http://mafft.cbrc .jp/alignment/software/) to determine the exon–intron structure of akirin genes from the miiuy croaker. Open Reading Frame Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to predict the open reading frame (ORF) and to translate nucleotide into protein. Nuclear localization signal (NLS) was forecasted using PSORT II server (http://psort.hgc.jp/form2.html), and SignalP 4.1 server was used to predict the presence and location of signal peptide cleavage sites (Nielsen et al., 1997). 2.2. Sequence alignment and phylogenetic analyses The akirin genes used in this study were derived from GenBank (http://www.ncbi.nlm.nih.gov) and Ensemble database (http:// www.ensembl.org) (Supporting information, Table S1). Multiple alignments of the akirin sequences from different species were performed under codon model by using MUSCLE software (Edgar, 2004). Before constructing the phylogenetic tree, jModeltest software was used to select an optimal substitution model (Posada, 2008), through which GTR + I + G was considered the best-fit model based on Bayesian information criterion (Posada and Buckley, 2004). Phylogenetic analysis was performed using Bayesian approach in MrBayes v3.2 (Ronquist and Huelsenbeck, 2003), which was ran for 5,000,000 generation with the first 25% of trees burned.
2.4. Fish sampling and challenge experiment Healthy miiuy croakers (mean weight: 800g) were collected from Zhoushan Fisheries Research Institute (Zhejiang, China). Fish were acclimatized in a recirculating seawater system at an ambient temperature (25 °C) for one week. Challenge experiments, as previously described by Zhu et al. (2013), were performed on the miiuy croaker. Fish were randomly divided into two groups, namely, injection and control groups. In the injection group, fish were challenged with 1 mL of Vibrio anguillarum suspension (approximately 3.0 × 107 CFU/mL) by intraperitoneal injection as the control fish were injected with 1 mL of phosphate-buffered saline. The infected and control fish samples were killed at 6, 12, 24, 36, 48, and 72 h after the challenge. Three immune tissues (head kidney, liver, and spleen) from three individuals were sampled. Furthermore, 10 tissues (head kidney, liver, spleen, intestines, heart, muscle, gill, brain, eye, and skin) from healthy miiuy croaker were removed. All tissues were immediately frozen in liquid nitrogen after dissection and then separately stored at −80 °C until RNA extraction.
2.6. Expression analysis of akirin genes and rel gene To examine the tissue distribution of akirin mRNAs in different tissues of healthy miiuy croaker and to investigate the regulation relationship among akirin and rel genes after immune challenges, real-time qPCR was conducted on a 7300 Real-time PCR system (Applied Biosystems, USA) by using a RealMaster Mix kit (TIANGEN) following the manufacturer’s instructions. Reaction was carried out without the template used as blank control. Five pairs of qPCR primers were used to amplify akirin, rel, and β-actin genes as normalizer gene fragments (Supporting information, Table S2). Realtime qPCR reactions were carried out as described by Liu et al. (2014). The reaction for each sample was conducted in triplicate, and the cycling conditions were as follows: 15 min at 95 °C, followed by 45 cycles of 15 s at 95 °C, 60 s at 60 °C, and 31 s at 72 °C. Dissociation curve analysis was performed after each assay to determine target specificity. The PCR efficiency was determined using LinRegPCR (Karlen et al., 2007; Ruijter et al., 2014). Pfaffl (2001) method was selected as the relative quantification calculation method. Statistical analysis was performed using one-way analysis of variance statistical test followed by Duncan’s multiple comparison tests. P-values of less than 0.05 were considered statistically significant. All data were expressed as mean ± standard error. 3. Results
2.3. Comparative analysis of gene synteny To obtain insight on whether the genomic neighborhoods surrounding akirin genes are evolutionary conserved, synteny maps for the genomic neighborhoods surrounding akirin1, akirin2(1), and akirin2(2) were constructed. Data were manually obtained from
3.1. Characterization and structure of akirin genes from miiuy croaker The molecular characteristics of akirin1, akirin2(1), and akirin2(2) from the miiuy croaker are compiled in Table 1. The complete cDNA
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Table 1 Summary of characteristic features of akirin gene family members from miiuy croaker. Features
akirin1
akirin2(1)
akirin2(2)
GenBank accession number Gene size Length of cDNA Open reading frame 5’-UTR 3’-UTR Polyadenylation signals RNA instability motif Amino acids Molecular mass of protein Isoelectric point of protein Signal peptide Nuclear localization signal (NLS) Second NLS Number of exons Number of introns
KP340435 12267 1337 564 56 717 AATAAA ATTTA 187 21.2KDa 9.2 Nil 19PQSPKRRRCN28 Nil 5 4
KP340436 3805 1515 540 204 771 AATAAA ATTTA 179 20.4 9.01 Nil 19PASPKRRRCA28 73KRRH76 5 4
KP340437 2736 1498 555 138 805 AATAAA ATTTA 184 20.8 8.79 Nil 19PTSPKRRRCI28 75KRKH78 6 5
sequence of akirin1 consisted of 1337 bp, which contained a 5’terminal untranslated region (UTR) of 56 bp; an ORF of 564 bp; a 3’-UTR of 717 bp; and encoded a protein with 187 amino acids (Supporting information, Fig. S1). The full length of akirin2(1) cDNA was 1515 bp, with an ORF of 540 bp encoding for 179 amino acids. The 5’-UTR and 3’-UTR were 204 and 771 bp, respectively (Supporting information, Fig. S2). The complete cDNA sequence of akirin2(2) was 1498 bp, consisting of a 5’-UTR of 138 bp, an ORF of 555 bp encoding for 184 amino acids, and a 3’-UTR of 805 bp (Supporting information, Fig. S3). The instability index (II) of akirin1, akirin2(1), and akirin2(2) was computed as 90.61, 94.98, and 92.21, respectively, which indicated that akirins are unstable. The protein sequence analysis for the motifs, domains, and signal peptide revealed that no signal peptide was present in akirin1, akirin2(1), and akirin2(2), which indicated that they were non-secretory proteins. A putative 10-peptide nuclear localization signal (NLS) was found in the N-terminal region in akirin1 (19PQSPKRRRCN28), akirin2(1) (19PASPKRRRCA28), and akirin2(2) (19PTSPKRRRCI28), and their presence was approved in the nucleus. Furthermore, a putative second NLS was presented in akirin2(1) (73KRRH76) and akirin2(2) (75KRKH78), but was absent in akirin1 in miiuy croaker. One NLS was observed in akirin1 homologues, whereas two NLSs were present in akirin2 homologues. This phenomenon was similarly found in all teleosts. However, two NLSs in akirin1 of elephant shark (Callorhinchus milii) were derived. The genomic DNA sequences of akirin1, akirin2(1), and akirin2(2) from the miiuy croaker contained 12267, 3805, and 2736 bp, respectively (GenBank accession no. KP340435, KP340436, and KP340437). The alignment results of the cDNA sequences and the genomic DNA sequences of akirin1, akirin2(1), and akirin2(2) genes revealed that akirin1 and akirin2(1) contained five exons and four introns (Fig. 1A), which is consistent with the akirin homologues from other teleost species. The exon–intron structure of akirin2(2) from the miiuy croaker indicated the presence of six exons interrupted by five introns (Fig. 1A), which is different from the akirin homologues from other teleost species (except for akirin2(1) of rock bream (Oplegnathus fasciatus). It should be named akirin2(2) according to the analyses of phylogenies and conserved synteny. Thus, it was named akirin2(2) in this paper.). All introns of akirin genes in the miiuy croaker completely conformed with the GT-AG rule (Supporting information, Figs. S1, S2, and S3). 3.2. Evolution of akirin gene structure Akirin1 and akirin2(1) genes of the miiuy croaker consisted of five exons and four variable introns, whereas the akirin2(2) gene in the miiuy croaker comprised six exons, and its exon 1 failed to
translate nucleotides (Figs. 1A, B). However, Macqueen and Johnston (2009) demonstrated that the akirin gene structure was from a single exon into the present-day gene structure of five exons and four introns (Fig. 1A,B). 3.3. Comparison of gene synteny with other vertebrates Synteny analysis results revealed that the miiuy croaker akirin1 gene located between rhbdl2 and cnr2 genes was consistent with most teleosts although different from lamprey, elephant shark, coelacanth (Latimeria chalumnae), and platyfish (Xiphophorus maculatus), in which rhbdl2 or cnr2 was missing. The genomic surrounding genes of akirin1 only had one homologous gene in the lamprey, elephant shark, and coelacanth, whereas a number of genes surrounding akirin1 included two homologous genes on different chromosomes or scaffolds in the other teleosts. Furthermore, the akirin1 gene was not found in the atlantic cod (Gadus morhua) and the green spotted puffer (Tetraodon nigroviridis). However, a single akirin1 gene was identified in the others examined (Fig. 2). However, akirin2(1) and akirin2(2) were present in the Acanthopterygian taxa (i.e., atlantic cod and green spotted puffer), which were located on different chromosomes or scaffolds, as well as in most other teleosts (Fig. 3). Furthermore, the akirin2(1) located between rars2 and cnr1(1) genes, as well as the akirin2(2) located between znf292(2) and cnr1(2) in the miiuy croaker, were consonant with other teleosts (Fig. 3). Most genomic neighborhood genes of akirin1 and akirin2 in the teleosts included two homologues (e.g., cnr2, fhl3, fabp10, rragca, znf292, gabrr1, gabrr2, syncrip, begain, nt5e, cnr1, and pm20d2), which were positioned on different chromosomes or scaffolds. In addition, comparing the genes in the genomic neighborhood of akirin1, akirin2(1), and akirin2(2) indicated a strong level of conserved synteny across teleost classes, with limited intra- or inter-chromosomal rearrangements, which is consistent with the results obtained by Macqueen and Johnston (2009). 3.4. Phylogenetic analysis of akirin genes Phylogenetic analysis of akirin gene family members from different species indicated sequences as they completely clustered in two groups, namely, akirin1 and akirin2 (Fig. 4). In the akirin2 clade, teleost samples split into two subclades, and akirin2(1) and akirin2(2) of the miiuy croaker were divided into two subclades, respectively. In the subclades, salmonid sequences were further split into two groups, each containing four sequence homologues. A similar phenomenon in the akirin2 group was observed in the akirin1 clade, whereas akirin1 included only a single sequence in teleosts (except for Salmonid).
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Fig. 1. The genomic organization of akirin proto-orthologues across eukaryotic lineages. A. Comparison of the miiuy croaker akirin gene structure with the previously published gene structures of other species. The lines stand for introns and exons are shown as boxes. The boxes with slashes represent the untranslated exons. The genes covered by the shadow represent that this gene had been lost after genome duplication. B. Evolution of gene structure and gene duplication of akirin gene family members. The question mark (?) symbol represents the exon gain events or the intron insertion events. The genes covered by the shadow represent that this gene had been lost after genome duplication. (This figure is an extension to Macqueen and Johnston, 2009.)
3.5. Comparative transcript of akirin gene family members and rel gene We examined the tissue expression patterns of akirin1, akirin2(1), akirin2(2), and rel in the miiuy croaker through real-time qPCR. Expression results showed that the akirin genes and the rel gene from the miiuy croaker, as seen in most akirin genes in other teleost species, were ubiquitously expressed across all 10 tissues (head kidney, liver, spleen, intestines, heart, muscle, gill, brain, eye, and skin). However, the expression levels were obviously different in each tissue (Fig. 5A). Within the examined tissues, the high expression level of akirin genes appeared in the liver, spleen, head kidney, and muscle, and the high-level expressions of rel were detected in the liver and spleen. In addition, the lowest expression levels of akirin1, akirin2(2), and rel appeared in the intestine, whereas the heart indicated the lowest expression level for akirin2(1) (Fig. 5A). To determine the potential roles of akirin genes (akirin1, akirin2(1), and akirin2(2)) from the miiuy croaker after injection with the pathogenic bacteria V. anguillarum and to understand the regulating relationship among akirin genes, along with the rel gene, the relative quantity changes of the miiuy croaker in the liver, spleen, and head kidney during 72 h of induction were assessed. In the liver, the relative expressions of akirin and rel genes increased sharply at 6 h, and maximum induction after challenge also occurred at this time point. Subsequently, the expression level gradually decreased at 12, 24, 36, and 48 h but moderately increased at 72 h (Fig. 5B). In the spleen and head kidney, the expressions of akirin and rel genes were gradually increased after infection where the highest expression level was recorded at 24 h and then gradually decreased (Fig. 5C,D). However, the expression of rel gene in the spleen and akirin2(2) gene in the head kidney moderately increased
at 72 h (Fig. 5C,D). To sum up, the expression levels of akirin and rel genes in the liver, spleen, and head kidney were found first upregulated and then downregulated, and finally recovered closer to a normal level (ignoring 72 h) after infection with V. anguillarum.
4. Discussion In this study, we identified first the full length of akirin1, akirin2(1), and akirin2(2) cDNA sequences and genomic DNA sequences from the miiuy croaker and further investigated the evolution of the akirin gene family in gene structure and the duplication of akirin at the genome level. Meanwhile, the transcript relationships of each member from the akirin gene family and rel gene were analyzed using the corresponding genes from miiuy croaker, and the co-expressed patterns among akirin and rel genes were established. The analysis suggested that a typical NLS motif was present in the N-terminal of miiuy croaker akirin1, akirin2(1), and akirin2(2). A putative second NLS was observed in akirin2(1) and akirin2(2). However, this NLS was not identified in akirin1 of miiuy croaker. Furthermore, Macqueen and Johnston (2009) demonstrated that a second NLS was present in akirin and akirin2 but not in akirin1, and the earliest second NLS in akirin could date back to Placozoans. Confusingly, we found that two NLSs exist in akirin1 and akirin2 from other vertebrates (e.g., elephant shark). In all teleosts, second NLS was present only in akirin2 homologues but not in akirin1 homologues. Thus, we deduced that the loss of the second NLS of akirin1 homologues in all teleosts likely occurred in an ancestor to all Osteichthyes after the split with cartilaginous fish for some unclear reason. However, this inference needs further research in the future.
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Fig. 2. The genomic neighborhood surroundings akirin1 of fish and lamprey. The arrows indicate the transcriptional direction. Single, double and triple diagonal lines indicate a genomic distance respectively spanning 1, 2 and >3 genes. Two genes with red shades connected by a red slash indicate two homologues produced in the genome duplication.
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Fig. 3. The genomic neighborhood surroundings akirin2(1) and akirin2(2) of fish. Details are as in the Fig. 2 legend.
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Fig. 4. Phylogenetic tree of collected akirin genes was constructed using Bayesian method. The numbers in topologies represent Bayesian posterior probability values.
Akirins are present throughout the metazoan, but their gene structures were different in different species. The earliest akirin genes were identified in the Protista taxa Alveolata (in cryptomonas phi), and Heterolobosea (in brain-eating amoeba) only had a single exon. The akirin genes that subsequently undergo the exon gain event and the intron insertion event were organized as five exons of comparable size and four more variable introns in most vertebrates (Fig. 1A) (Macqueen and Johnston, 2009). The akirin1 and akirin2(1) of miiuy croaker, similar to the akirin homologues of the teleost and mammalian, consisted of five exons and four variable introns and possessed the start codon in the first exon itself (Fig. 1A). However, in akirin2(2) of miiuy croaker, six exons and five introns are different from other teleosts. The first exon of the miiuy croaker akirin2(2) consisted of non-coding nucleotides, and the start codon was present in the second exon (Fig. 1A). Given that exons 3, 4, 5, and 6 of
akirin2(2) from miiuy croaker were consistent with other teleost and mammalian species and the untranslated exon formed in the untranslated 5’ region, we can speculate that a second intron insertion event would have occurred between exons 1 and 2 (Kasthuri et al., 2013). This phenomenon may be specific to a particular species but the same phenomenon also appeared in akirin2(2) of rock bream. This phenomenon is a novel evidence for the variation of akirin structure in some species for some unclear reason. To clarify the reason of the phenomenon, more data and further research are necessary. To understand the evolutionary relationship of akirin genes, we analyzed the genomic neighborhood that surrounds the akirin genes of teleost and lamprey. The result showed that akirin1 and akirin2 were both present in all examined teleost genomes (except for green spotted puffer) and were positioned on different chromosomes or scaffolds. However, akirin2 was not found in lamprey (Figs 2 and 3).
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Fig. 5. The expression profiles of akirin genes and rel gene in ten specific tissues of miiuy croaker (A) and expression analysis in liver (B), spleen (C) and head kidney (D) during 6, 12, 24, 36, 48, and 72 h post-inoculation with V. anguillarum. The C on the abscissa stand for the control; the expression level of β-actin was set as the internal control for real-time PCR. Deviation bars represent the standard errors. Values with the same superscript were not significantly different (P > 0.05). Data indicated with asterisk symbol (*) were significantly different (P < 0.05) between injection and control groups.
Macqueen and Johnston (2009) and Macqueen et al. (2010a, 2010b) demonstrated a genome duplication event, which occurred in a common ancestor to the vertebrate lineage, prior to the separation of gnathostome and agnathan lineages, which lead to the generation of akirin1 and akirin2 from an akirin proto-orthologue. Our result further proved the viewpoint. In all examined teleost genomes, two akirin2 homologues (akirin2(1) and akirin2(2)) were found on different chromosomes or scaffolds. Meanwhile, some genomic neighborhood genes of akirin2(1) or akirin2(2) gene in all examined teleosts had two homologues (Fig. 3). If different gene members arose from the duplication of the genome, other genes around them would have been duplicated at the same time (Macqueen and Johnston, 2009). Thus, if teleosts have been subjected to WGD, two pairs of paralogues, namely, akirin1(1) and akirin1(2) (from akirin1) and akirin2(1) and akirin2(2) (from akirin2), exist on different chromosomes in teleosts. In addition, the genomic neighborhood genes of akirin1 and akirin2 should have two homologues. However, the paralogue gene of akirin1 is not present in all teleost genomes that were examined (except for Salmonid), and most genomic neighborhood genes of akirin1 and akirin2 are also only
single-copy genes (Figs 2 and 3). Macqueen and Johnston (2009) indicated that a paralog may be lost after WGD because one of two paralogs in 85% or higher of pairs was lost after the WGD (Brunet et al., 2006). Although the expression levels of akirin and rel genes in the expression analysis were slightly different in all 10 tissues from healthy miiuy croaker, their highest expression level all appeared in the liver (Fig. 5A). Previous studies demonstrated that the highest expression of HAMP (also named LEAP-1) and LEAP-2 from miiuy croaker was also present in the liver (Liu et al., 2014; Xu et al., 2012). This finding suggests that these genes may have some connection in biological functions. To further explore the possible biological functions of akirins, the temporal expression of akirins was examined at different time points after the challenge with V. anguillarum, which was defended against by akirin in flies and mice (Goto et al., 2008) in three main immune tissues. The temporal expression of akirins revealed that different expression patterns existed in the liver, spleen, and head kidney. In the liver, the expression level of akirins and rel at 6 h after infection was about 45–250 times of the control level (Fig. 5B). This
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result suggests that they may play a role in the early defense against invading pathogens in miiuy croaker. The expression level of akirins and rel was significantly upregulated within 6 h; this finding was also observed in LEAP-2 of miiuy croaker, but was not observed in LEAP-1 of miiuy croaker (Liu et al., 2014; Xu et al., 2012). However, in the spleen and head kidney, akirins and rel transcripts were upregulated at 24 h (P < 0.05) (Fig. 5C,D). Interestingly, the same expression patterns existed in LEAP-1 of miiuy croaker but did not appear in LEAP-2 of miiuy croaker (Liu et al., 2014; Xu et al., 2012). Although the reason behind this difference is unknown, the above results further support that akirins and rel induced the expression of LEAP-1 and LEAP-2, i.e., the downstream elements of NF-kBdependent signaling pathway (Goto et al., 2008; Hou et al., 2013; Xue et al., 2014). Furthermore, we found that the gene expression patterns of akirin paralogues and rel were often highly correlated, which suggests that natural selection has maintained an intricate network of co-regulation among family members (Macqueen et al., 2010b) along with rel gene. Acknowledgements This study was supported by the National Natural Science Foundation of China (31370049), Zhejiang Provincial Natural Science Foundation of Distinguished Young Scientists (LR14C040001) and National Spark Program (2013GA700247). Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.dci.2015.04.010. References Almazán, C., Blas-Machado, U., Kocan, K.M., Yoshioka, J.H., Blouin, E.F., Mangold, A.J., et al., 2005. Characterization of three Ixodes scapularis cDNAs protective against tick infestations. Vaccine 23, 4403–4416. Bonnay, F., Nguyen, X.H., Cohen-Berros, E., Troxler, L., Batsche, E., Camonis, J., et al., 2014. Akirin specifies NF-κB selectivity of Drosophila innate immune response via chromatin remodeling. EMBO J. 33, 2349–2362. Brunet, F.G., Crollius, H.R., Paris, M., Aury, J.M., Gibert, P., Jaillon, O., et al., 2006. Gene loss and evolutionary rates following whole-genome duplication in teleost fishes. Mol. Biol. Evol. 23, 1808–1816. Che, R., Sun, Y., Sun, D., Xu, T., 2014. Characterization of the miiuy croaker (Miichthys miiuy) transcriptome and development of immune-relevant genes and molecular markers. PLoS ONE 9, e94046. Chen, X., Huang, Z., Jia, G., Wu, X., Wu, C., 2012. Molecular cloning, tissue distribution and functional analysis of porcine Akirin2. Anim. Biotechnol. 23, 124–131. de la Fuente, J., Almazán, C., Blas-Machado, U., Naranjo, V., Mangold, A.J., Blouin, E.F., et al., 2006. The tick protective antigen, 4D8, is a conserved protein involved in modulation of tick blood ingestion and reproduction. Vaccine 24, 4082–4095. de la Fuente, J., Maritz-Olivier, C., Naranjo, V., Ayoubi, P., Nijhof, A.M., Almazán, C., et al., 2008. Evidence of the role of tick subolesin in gene expression. BMC Genomics 9, 372. Dai, F., Huang, K., Liu, H., Han, C., Li, L., Wang, J., 2011. Cloning, sequence analysis and specific expression in different tissues of duck Akirin2 gene. Acta Vet. Zootech. Sin. 42, 33–38. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Gonzalez, K., Baylies, M., 2005. Bhringi: a novel Twist co-regulator. A Dros. Res. Conf. 46, 320B. Goto, A., Matsushita, K., Gesellchen, V., Chamy, L.E., Kuttenkeuler, D., Takeuchi, O., et al., 2008. Akirins are highly conserved nuclear proteins required for NF-κBdependent gene expression in drosophila and mice. Nat. Immunol. 9, 97–104. Hou, F., Wang, X., Qian, Z., Liu, Q., Liu, Y., He, S., et al., 2013. Identification and functional studies of Akirin, a potential positive nuclear factor of NF-κB signaling pathways in the Pacific white shrimp, Litopenaeus vannamei. Dev. Comp. Immunol. 41, 703–714. Jaillon, O., Aury, J.M., Brunet, F., Petit, J.L., Stange-Thomann, N., Mauceli, E., et al., 2004. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431, 946–957.
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