Short communicationCloning and expression study of an IRF4a gene and its two transcript variants in turbot, Scophthalmus maximus
Introduction
Interferon regulatory factors (IRFs) are a family of transcription mediators involved in the transcriptional regulation of type Ⅰ interferon (IFN) and IFN-stimulated genes (ISGs) [1]. They play a role in virus-mediated signaling, early immune response to pathogens and hematopoietic cell development [2]. To date, a total of 11 IRF members have been identified in vertebrates, with IRF10 being present in avian and fish and IRF11 only in fish [3]. All IRF proteins share a significant homology in the amino (N) terminus comprising a DNA-binding domain (DBD) characterized by five tryptophan repeats responsible for binding to the AANNGAAA elements in interferon stimulatory response elements (ISRE) that are located in the promoters of a diverse range of immune or immune-related genes [4]. In addition, IRFs, except IRF6 and -10, possess one or two nuclear localization signals (NLS) in the N-terminus, which relate to nuclear translocation and reservation of IRFs [5]. Moreover, in the carboxyl (C) terminus, IRFs (IRF3-10) contain an IRF-associated domain (IAD) which mediates the formation of homo- or heterodimers with other IRFs or transcription factors to target gene promoters, whereas IRF1 and -2, and possibly IRF11, possess an IAD2, another type of association module of IRF proteins [6]. Outside IAD, the C-terminus is not well conserved and contributed to specific functions of each IRF. Functionally, IRFs may be classified into two groups: IRF1, -3, -7, -9 and -10 act as transcriptional activators, while IRF2, -4, -5 and -8 are bi-functional factors that both activate and repress transcription depending on the target gene promoters, nature of the partner transcription factors, cell types and differentiation status [7].
IRF4, also known as LSIRF, LCSAT, MUM1 or PU.1 interaction partner (Pip), is involved in the regulation of both innate and adaptive immunity [8] and is found to play key roles in regulating expression of genes specific to T and B cells, macrophages and dendritic cells by the interaction with Ets family members, PU.I and Sp.I [9], [10]. IRF4 is a multifunctional transcription factor that tends to act positively towards ISG15 transcription when bound to composite motifs, and negatively modulates Toll-like receptor (TLR) signalings by binding to ISRE motifs in their promoters [11], [12]. Consistent with the functional importance in lymphomyeloid cells, the expression of IRF4 is up-regulated in splenocytes, purified B/T cells, macrophages and dendritic cells by the TLR ligands, LPS and CpG-containing DNA [4], [13]. In addition, human T cell leukemia virus type Ⅰ (HTLV-Ⅰ) and Epstein-Bar virus (EBV) was reported to elevate IRF4 expression through activating the NF-κB pathway [14].
To date, the fishes with IRF4 characterized include rainbow trout [10], rock bream [8], Asian swamp eel [15], half-smooth tongue sole [16], Atlantic cod [17] and Japanese flounder [18]. Teleost fish have two copies of IRF4 genes, IRF4a and IRF4b, while tetrapods have a single IRF4 gene [3]. Fish IRF4 was predominantly expressed in lymphomyeloid-rich tissues, and shown to be transcriptionally up-regulated by phytohaemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) treatment [10]. PMA is a potent activator of protein kinase C (PKC) which is a B-cell linker (BLNK) downstream signaling molecule inducing the expression of IRF4 [19]. Further, poly(I:C), DNA virus and Gram-positive and -negative bacteria stimulations elicited a significant up-regulation of expression of fish IRF4 [8], [15], [16], [17], [18], suggesting its role in host's antiviral and antibacterial responses.
Turbot, Scophthalmus maximus, is one of the most important marine species cultured widely in the world, but its farming industry has been plagued by viral diseases. The study of turbot (Sm)IRF4a will undoubtedly enrich the knowledge of turbot immune defense system, which is helpful for the development of effective strategies for infectious disease control of this economically important species. Previously, turbot IRF3, -5, -7 and -8 have been studied by our team, with all shown to be involved in regulating antiviral immune responses [20], [21], [22], [23]. In this study, a SmIRF4a gene, as well as its two transcript variants SmIRF4a1 and -2, was cloned. In order to understand potential roles of SmIRF4a in antiviral response, the tissue specific expressions of the two transcripts and their temporal expression profiles after stimulation with polyinosinic:polycytidylic acid [poly(I:C)] and turbot reddish body iridovirus (TRBIV), one of the most prevalent viral pathogens in farmed turbot in China, were examined.
Section snippets
Fish and challenge experiments
Turbot (S. maximus) juveniles (63.5 ± 3.0 g, n = 120) were purchased from a local mariculture farm and maintained in 50 L tanks with aerated seawater (salinity 34 ± 0.6%, pH 7.6 ± 0.5) at 16 °C. Fish were acclimated for 1 week prior to sampling. Poly(I:C) was purchased from Sigma (San Diego, CA, USA). TRBIV was isolated from cultured turbots with TRBIV disease as previously described [24]. The viral titers were measured by a 50% tissue culture infective dose (TCID50) assay according to the
Molecular characterization of SmIRF4a gene and its two transcript variants
Sequencing and assembly of SmIRF4a genomic region produced an 8367-nt consensus sequence (accession no. KP985235) consisting of 8 exons and 7 introns (Fig. 2). All exon/intron junctions of the SmIRF4a gene follow the consensus rule of the splice acceptor -AG/GT-splice donor for splicing [28]. The analysis of several clones showed the existence of two transcript variants, SmIRF4a1 and -2. The full-length SmIRF4a1 (accession no. KP985233) was 3185 nt, including a 5′-untranslated region (UTR) of
Discussion
In mammals, IRF4 has been extensively studied and known to be essential to the development and functions of lymphomyeloid cell lineages [5], [32]. However, information related to its functions in fish species is very limited. In the present study, we cloned and tested expression patterns of an IRF4a gene and its two transcript variants, SmIRF4a1 and -2, from turbot. The SmIRF4a gene shows a typical exon-intron organization of vertebrate IRF4 (Fig. 2). Phylogenetic analysis showed two copies of
Acknowledgment
This work was supported by grants from the Special Scientific Research Fund of Marine Public Welfare Industry of China (201505001) and Fundamental Research Funds for the Central Universities (201762003).
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