Full length articleMolecular characterization of the interferon regulatory factor (IRF) family and functional analysis of IRF11 in the large yellow croaker (Larimichthys crocea)
Introduction
Interferon regulatory factors (IRFs) are a family of transcription factors mainly involved in the regulation of interferon (IFN) gene expression [1]. This transcription factor family has been identified in many species and the members function in the regulation of innate and adaptive immune responses, immune cell development, and oncogenesis [2]. The mammalian IRF family consists of nine members, IRF1–9. These IRFs all possess a highly conserved N-terminal helix-turn-helix DNA-binding domain (DBD), which consists of about 120 amino acids containing five regularly spaced tryptophan residues. This domain functionally recognizes the IFN-stimulated response elements (ISREs), which are found in the promoters of type I IFN, type III IFN, and IFN-stimulated genes (ISGs) [3]. All IRFs contain a conserved C-terminal IRF-association domain (IAD) or a similar IAD2, which mainly mediates the homomeric or heteromeric formation of IRFs and the interaction of IRF with non-IRF members, such as the signal transducer and activator of transcription (STAT). Outside the IAD, the C-terminus is not well conserved, which may confer specific functions to each IRF [4]. Mammalian IRF1, IRF3, IRF5, IRF7, and IRF9 mainly function as activators in the regulation of immune response, whereas IRF4 plays a role as a negative regulator [5,6]. IRF2 and IRF8 can both activate and repress the gene transcription depending on the nature of the pathogen and signaling pathways that are activated [7,8]. Additionally, IRF1, IRF2, IRF4, and IRF8 have essential roles in the development of various immune cells [9].
Eleven IRF family members have been identified in fish species, including zebrafish (Danio rerio) [10], mandarin fish (Siniperca chuatsi) [11], and grass carp (Ctenopharyngodon idella) [12], and IRF11 was only found in fish. In some species, there are two paralogues of IRF4 and IRF10, probably due to whole genome duplication (WGD) or locus duplication [13,14]. Based on phylogenetic analysis, these 11 IRF family members are classified into four subfamilies: IRF1 subfamily (IRF1, IRF2, and IRF11), IRF3 subfamily (IRF3 and IRF7), IRF4 subfamily (IRF4a, IRF4b, IRF8, IRF9, IRF10a, and IRF10b), and IRF5 subfamily (IRF5 and IRF6) [15]. Structurally, all the IRFs in fish also contain an N-terminal DBD region with high similarity to mammalian IRFs, which is responsible for binding the ISREs in the target gene promoter [16]. In addition, all IRFs except IRF1 subfamily members contain an IAD at the C-terminus, while IRF1 subfamily members (IRF1, IRF2, and IRF11) share a similar IAD2. Both IAD and IAD2 mediate the formation of homo- or hetero-dimers or the recruitment of other transcription factors to the promoter of target genes [16].
Fish IRFs can be induced by various immune stimulants, such as viral mimic poly (I:C), viruses, bacterial LPS, and bacteria [12,16]. In mandarin fish, all IRFs are induced by both poly (I:C) stimulation and infection of the infectious spleen and kidney necrosis virus (ISKNV) [11]. IRFs in grass carp [12] and IRF1–9 in half-smooth tongue sole (Cynoglossus semilaevis) [16] are also induced by viral infections. In Atlantic cod (Gadus morhua), IRF4a, IRF4b, IRF7, IRF8, and IRF10 were upregulated by poly (I:C) [17], while IRF1, IRF2, IRF3, IRF9, and IRF11 in the miiuy croaker (Miichthys miiuy) were responsive to poly (I:C) stimulation [[18], [19], [20]]. The expression of IRF1–9 in half-smooth tongue sole was induced by different bacterial pathogens [16]. IRF2, IRF7, and IRF9 in blunt snout bream (Megalobrama amblycephala) were upregulated by Aeromonas hydrophila [21], while IRF4b, IRF7, IRF8, and IRF10 in Atlantic cod were responsive to Aeromonas salmonicida [17]. Functional studies confirmed that grass carp IRF2 played an antagonistic role to IRF1 and acted as a negative regulator in transcriptional regulation of IFN and ISGs. This was similar to mammalian counterparts [22]. IRF3 and IRF7 showed distinct features in regulating type I IFN expression depending on different IFN subgroups [23]. Zebrafish IRF6 is involved in both the MyD88 (myeloid differentiation primary response protein 88) and TBK1 (TANK-binding kinase 1) pathways to induce IFN expression [24]. IRF3 in teleost can negatively regulate the TRIF-mediated NF-κB signaling pathway by targeting adaptor TRIF for degradation, which mainly depended on the IAD domain [25]. In contrast, fish IRF9 also acted as an inhibitor of TRIF-mediated NF-κB pathway through the DBD domain [26]. The fish IRF10 was shown to repress the IFN transcription that was activated by retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) signaling pathway [27]. IRF11, as a fish specific IRF, has been found to positively regulate antiviral response in zebrafish [28] and mandarin fish [29]. IRF11 exerts its antiviral effect by upregulating the expression of type I IFNs through binding to their promoters [28,29].
Large yellow croaker (Larimichthys crocea), a perciform fish, is an economically important mariculture species in China. With the rapid development of large yellow croaker farming, diseases caused by various pathogens are becoming more severe, and causing economic losses. To reveal the molecular basis of the large yellow croaker immunity, we have sequenced its whole genome [30]. Genome analysis revealed a complete repertoire of 11 IRF genes, of which LcIRF1, LcIRF3, LcIRF4, LcIRF7, LcIRF8, and LcIRF9 had been previously characterized [[31], [32], [33]]. To delineate the characterization of the whole IRF family in this species, we cloned and identified the other members, LcIRF2, LcIRF5, LcIRF6, LcIRF10, and LcIRF11, and analyzed the expression profiles of all 11 LcIRFs under normal and induced conditions. We also explored the functions of LcIRF11 on the regulation of type I IFN expression and antiviral response.
Section snippets
Fish and challenge experiments
Large yellow croakers (weight: 102 ± 17.3 g; length: 21 ± 1.3 cm) were purchased from Ningde Fufa Aquaculture Co. Ltd in Ningde, Fujian province, China. Healthy fish were acclimatized in a flow-through seawater tank at 25 °C for one week and fed with the commercialized formula feed once daily prior to the experiments. After the fish were anesthetized with eugenol, tissue samples of spleen, brain, head kidney, heart, skin, intestine, stomach, muscle, gills, liver, and blood were immediately
Identification and characterization of LcIRF2, LcIRF5, LcIRF6, LcIRF10, and LcIRF11
LcIRF1, LcIRF3, LcIRF4, LcIRF7, LcIRF8, and LcIRF9 were identified in previous studies [[31], [32], [33]]. Here, we cloned the other five members, LcIRF2 (MT767748), LcIRF5 (MT767749), LcIRF6 (MT767750), LcIRF10 (MT767751), and LcIRF11 (MT767752). The complete ORF sequences of these five LcIRFs are shown in Supplementary Fig. 1A–E. The characteristics of LcIRFs including ORF length, number of amino acids, predicted molecular weight, isoelectric point (pI), number of exons, DBD, IAD, and
Discussion
In the present study, we identified five new members of the large yellow croaker IRF family, LcIRF2, LcIRF5, LcIRF6, LcIRF10, and LcIRF11. Each member had high identity with its counterpart in other fish species in the DBD and IAD. This suggests that the function of fish IRFs has been evolutionarily conserved. In the DBD, there are six spaced tryptophan repeats in the LcIRF2 and LcIRF11, whereas five conserved tryptophans exist in the LcIRF5, LcIRF6, and LcIRF10 (Supplementary Fig. 1A–E), which
CRediT authorship contribution statement
Yanyun Guan: Conceptualization, Methodology, Validation, Formal analysis, Writing - original draft. Xiaojuan Chen: Investigation, Validation, Formal analysis. Tian Luo: Investigation, Validation, Formal analysis. Jingqun Ao: Supervision. Chunxiang Ai: Supervision. Xinhua Chen: Supervision, Resources, Writing - review & editing.
Acknowledgments
The work was supported by grants from the National Natural Science Foundation of China (31530080, U1905204 and 31772874), China Agriculture Research System (CARS-47), and Marine Economic Development Subsidy Fund of Fujian Province(FJHJF-L-2019-2).
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