Zebrafish IRF1, IRF3, and IRF7 Differentially Regulate IFN Φ1 and IFNΦ3 Expression through Assembly of Homo- or

Heteroprotein Complexes Downloaded from This information is current as of August 16, 2019. Hui Feng, Qi-Min Zhang, Yi-Bing Zhang, Zhi Li, Jun Zhang, Ya-Wei Xiong, Min Wu and Jian-Fang Gui J Immunol 2016; 197:1893-1904; Prepublished online 5

August 2016; http://www.jimmunol.org/ doi: 10.4049/jimmunol.1600159 http://www.jimmunol.org/content/197/5/1893

References This article cites 57 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/197/5/1893.full#ref-list-1 at Univ of NC Chapel Hill Health Sciences Library on August 16, 2019

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Zebrafish IRF1, IRF3, and IRF7 Differentially Regulate IFNF1 and IFNF3 Expression through Assembly of Homo- or Heteroprotein Complexes

Hui Feng,1,2 Qi-Min Zhang,1 Yi-Bing Zhang, Zhi Li, Jun Zhang, Ya-Wei Xiong, Min Wu, and Jian-Fang Gui Downloaded from

In mammals, IFN regulatory factor (IRF)1, IRF3, and IRF7 are three critical transcription factors that are pivotal for cooperative regulation of the type I IFN response. In this study, we explored the relative contribution of zebrafish (Danio rerio) IRF1 (DrIRF1), IRF3 (DrIRF3), and IRF7 (DrIRF7) (DrIRF1/3/7) to zebrafish IFNF1 (DrIFNF1) and IFNF3 (DrIFNF3) (DrIFNF1/3) activa- tion. Following spring viremia of carp virus infection, DrIFNF1/3 and DrIRF1/3/7 transcripts are significantly induced in zebra- http://www.jimmunol.org/ fish tissues, which correlates with the replication of spring viremia of carp virus. DrIRF1/3/7 selectively bind to the IRF-binding element/IFN-stimulated regulatory element sites of DrIFNF1/3 promoters, with the exception that DrIRF3 has no preference for two IRF-binding element/IFN-stimulated regulatory element motifs within the DrIFNF3 promoter. Consistently, DrIRF3 alone activates DrIFNF1, but not DrIFNF3; DrIRF7 predominantly stimulates DrIFNF3; and DrIRF1 has similar potential to DrIFNF1 and DrIFNF3. Strikingly, DrIRF3 facilitates the binding of DrIRF1 and DrIRF7 to both zebrafish IFN promoters, and so does DrIRF7 for the binding of DrIRF1, particularly to the DrIFNF3 promoter. These binding properties correlate with differential responses of DrIFNF1 and DrIFNF3 to the combinatory stimulation of DrIRF1/3/7, depending on their relative amounts. Similar to the dual roles of human IRF3 in regulating IRF7-activated IFNa , DrIRF3 exerts dual effects on DrIRF1-mediated DrIFNF3 expression: an inhibitory effect at lower concentrations and a synergistic effect at higher at Univ of NC Chapel Hill Health Sciences Library on August 16, 2019 concentrations. These data provide evidence that fish and mammals have evolved a similar IRF-dependent regulatory mechanism fine-tuning IFN gene activation. The Journal of Immunology, 2016, 197: 1893–1904.

n mammals, type I IFNs (primarily IFN-a/b) are induced to converge on the activation of a family of transcriptional factors boost the immune response, protecting hosts from viral and called IFN regulatory factors (IRFs) (1). The IRF family includes I nonviral pathogens (1). Transcriptional initiation of type I 9 members in mammals, 10 members in birds, and 11 members in IFN genes depends on timely recognition of pathogenic signals fish (3). All IRFs contain a conserved N-terminal DNA-binding through host pattern recognition receptors within endosomes domain (DBD) characterized by five tryptophan repeat elements. (such as TLR2/3/4/7/8/9) or in the cytosol (such as cytosolic RNA By folding to a helix-turn-helix motif, DBD specifically recog- sensors, RNA helicases, and cytosolic DNA sensors) (2). Such nizes and binds to a similar DNA sequence termed an IRF-binding recognition events trigger distinct signaling pathways that finally element (IRF-E)/IFN-stimulated response element (ISRE) within the promoters of type I IFN genes and IFN-stimulated genes State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of (4–7). In addition to DBD, each family member contains a unique Hydrobiology, Chinese Academy of Sciences, University of Chinese Academy of C-terminal IRF-associated domain that accounts for interactions Sciences, Wuhan 430072, China with other IRFs and transcription factors (1). 1 H.F. and Q.-M.Z. contributed equally to this work. Among the IRF family, IRF1, IRF3, and IRF7 are characterized 2 Current address: Lineberger Comprehensive Cancer Center, University of North early in regulating type I IFN expression (1). In most cell types, the Carolina at Chapel Hill, Chapel Hill, NC. IRF3/7-dependent IFN response is activated through the cytosolic Received for publication January 28, 2016. Accepted for publication July 4, 2016. RNA-mediated mitochondrial antiviral signaling protein path- This work was supported by Grants 31272690 and 31572646 from the National Natural Science Foundation and Grant XDA08010207 from the Strategic Priority way and the cytosolic DNA-mediated mediator of IRF3 activator Research Program of the Chinese Academy of Sciences. pathway (1, 2). IRF3 and IRF7 also contribute to TLR3-dependent Address correspondence and reprint requests to Dr. Yi-Bing Zhang, State Key Lab- IFN expression, and IRF3 is primarily responsible for TLR4-IFN oratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Uni- signaling through the TIR domain-containing adaptor protein versity of Chinese Academy of Sciences, Wuhan 430072, China. E-mail address: [email protected] pathway (8). However, TLR2/7/8/9-mediated induction of type I Abbreviations used in this article: cDC, conventional DC; co-IP, coimmunoprecipita- IFN appears to use distinct IRFs in a cell type–specific fashion but tion; DBD, DNA-binding domain; DC, dendritic cell; DrIFNF1, zebrafish IFNF1; through a common adaptor called MyD88 (9–12). For example, DrIFNF1/3, DrIFNF1andDrIFNF3; DrIFNF3, zebrafish IFNF3; DrIRF1, zebrafish the TLR9-triggered type I IFN response exclusively requires IRF7 (D. rerio) IRF1; DrIRF1/3/7, zebrafish IRF1, IRF3, and IRF7; DrIRF3, zebrafish IRF3; DrIRF7, zebrafish IRF7; DrMyD88, zebrafish myeloid differentiation factor 88; in plasmacytoid dendritic cells (DCs) (9), IRF1 in conventional HA, hemagglutinin; IRF, IFN regulatory factor; IRF-E, IRF-binding element; IRF1-HA, DCs (cDCs) (13, 14), and IRF3 in B lymphocytes (15); IRF1 and HA-tagged DrIRF1; IRF3-HA, HA-tagged DrIRF3; IRF7-HA, HA-tagged DrIRF7; ISRE, IFN-stimulated response element; lambda PPase, lambda protein phosphatase; IRF7 are necessary for the TLR2-driven type I IFN response in ORF, open reading frame; poly(I:C), polyinosinic-polycytidylic acid; qRT-PCR, quan- immune cells, including macrophages and cDCs (12), and for the titative RT-PCR; rIFN, recombinant crucian carp IFN; RLR, retinoic acid–inducible TLR7/9-triggered IFN response in cDCs (11). Therefore, like gene I–like ; SVCV, spring viremia of carp virus; ZFL, zebrafish liver cell. IRF3 and IRF7, IRF1 is another critical regulator of the type I IFN Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 response (16, 17). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600159 1894 DIFFERENTIAL ACTIVATION OF ZEBRAFISH IFNs BY IRF1/3/7

Interestingly, accumulating data showed that other IRF members Luciferase activity assays also participate in retinoic acid–inducible gene I–like receptor Stimulation, transfection, and subsequent luciferase activity assays were (RLR)-IFN or TLR-IFN signaling. IRF8 directs the IFN response, performed as previously described (28, 29, 37). together with IRF3/7, in DCs (18) and with IRF3 in human blood monocytes (19). IRF3, IRF5, and IRF7 coordinately act in mye- DNA pull-down assays loid DCs downstream of mitochondrial antiviral signaling protein DNA pull-down assays were performed as described previously (36, 39), signaling (20). Following Lactobacillus acidophilus infection, with in vitro–translated IRF proteins, prokaryotically expressed proteins, or MyD88 triggers IFN-b responses in DCs through a mechanism cell lysates of HEK293T transfected or not with IRF expression plasmids. Zebrafish IRF proteins were in vitro–translated in the TNT SP6 Quick involving IRF1, IRF3, and IRF7 (21). These results suggest that Coupled Transcription/Translation System supplemented with 40 mM KCl multiple IRF members often work together to coordinately reg- (Promega) or expressed as recombinant proteins by a prokaryotic expression ulate type I IFN gene activation, although the molecular mecha- system, according to previous studies (27, 28), or they were overexpressed nisms involved remain unclear. as tag-fused proteins by transfection of HEK293T cells for 24 h. Briefly, Downloaded from m m Studies during the past decade have led to tremendous ad- 25 l of individual translated products, 4 g of recombinant products, or an adequate amount of IRF-overexpressing HEK293T cell lysates (one fifth of vances in the understanding of fish IFN responses (22–24). Despite a 10 cm-dish, unless otherwise indicated), together with 1 mg of biotinylated functional similarity, fish IFNs are classified into group I and promoter dsDNA, was incubated in HKMG buffer at 4˚C for 24 h. The bead- group II IFNs (25–27), and they signal through distinct receptors bound DNA-protein complex was captured with M-280 streptavidin-coupled that are different from mammalian type I IFNRs (26). Neverthe- Dynabeads (Invitrogen) and washed with incubation buffer five times, fol- lowed by further Western blotting analysis.

less, fish IRF family members exhibit an orthologous relationship http://www.jimmunol.org/ with mammalian homologs (3, 28) and play conserved roles in the Quantitative RT-PCR host antiviral response (28–34). Notably, IRF3 and IRF7 are Tissues were sampled from healthy zebrafish or ones that had been infected necessary for the RLR-mediated IFN response in zebrafish (Danio for 48 h with SVCV, and total RNA was extracted for further expression rerio) (29, 32), as well as for the MyD88-mediated IFN response analysis of zebrafish IRFs, zebrafish IFNs, and SVCV genes, including L, N, in Atlantic salmon (Salmo salar) (35); zebrafish (D. rerio) IRF1 and G, by quantitative RT-PCR (qRT-PCR). qRT-PCR was also used to (DrIRF1) synergizes with MyD88 to regulate the expression of detect zebrafish IRF and IFN expression in ZFLs, which were seeded in six- well plates and transfected with polyinosinic-polycytidylic acid [poly(I:C); zebrafish IFNF3 (DrIFNF3) (36). Furthermore, fish IRF3 is an 2 mg/ml] or treated with SVCV (1000 TCID50/ml) and recombinant crucian IFN-inducible protein and is translocated to the nucleus following carp IFN (rIFN) protein (5 ng/ml). rIFN protein was generated by a pro- virus infection or IFN treatment (28). These unique findings karyotic expression system (27). qRT-PCR was performed in a DNA En- at Univ of NC Chapel Hill Health Sciences Library on August 16, 2019 prompted us to explore the coordination of fish IRF3 with other gine Chromo4 Real-Time PCR System with SYBR Green Real-Time PCR IRF members, such as IRF1 and IRF7, on the IFN antiviral re- Master Mix (both from Bio-Rad) (27). All samples were analyzed in trip- licates, and the expression values were normalized to b-actin. The following sponse. primer sequences were used: DrIRF1 (F: 59-CCGGCGGATGAAGGGTC- In the current study, we determined the relative contribution TGT-39,R:59-CGTTGCATGTGCTGTCAGGG-39); DrIRF3 (F: 59-CAA- of zebrafish IRF1, IRF3, and IRF7 (DrIRF1/3/7) to distinct zebra- AACCGCTGTTCGTGCC-39,R:59-CATCGTCGCTGTTGGAGTCCT-39); fish IFN activation. RT-PCR revealed the expression of zebrafish DrIRF7 (F: 59-AGGCAGTTCAACGTCAGCTACCAT-39,R:59-TTCCAC- F F F F CAAGTTGAGCAATTCCAG-39); DrIFNF1 (universal primers F: 59-ACG- IFN 1(DrIFN 1) and DrIFN 3(DrIFN 1/3), together with ACAGAATCTCTGAACCT-39,R:59-GTCAGGACTAAAAACTTCAC-39); DrIRF1/3/7, in some zebrafish tissues, and coimmunoprecipitation the secreted DrIFNF1 isoform (F: 59-TGAGAACTCAAATGTGGACCT-39, (co-IP) showed the interaction of DrIRF1/3/7 and MyD88, implying R: 59-GTCCTCCACCTTTGACTTGT-39); the intracellular DrIFNF1iso- that DrIRF1/3/7 might coregulate DrIFNF1 and DrIFNF3gene form (F: 59-ACGGCAGCCTGAAATACGTT-39,R:59-GTCCTCCACCT- 9 F 9 9 activation. Based on in vitro pull-down assays and promoter acti- TTGACTTGT-3 ); DrIFN 3(F:5-TTCTGCTTTGTGCAGGTTTG-3 ,R: 59-GGTATAGAAACGCGGTCGTC-39); SVCV N gene (F: 59-GGTGCGA- vation analyses, we found that DrIRF1/3/7 coordinately bound to GTAGAAGACATCCCCG-39,R:59-GTAATTCCCATCATTGCCCCAGA- IRF-E/ISRE motifs within DrIFN promoters as homo- or hetero- C-39); SVCV L gene (F: 59-CAAGTTCACAATCGGGAAGACGC-39,R: protein complexes to synergistically stimulate or downregulate the 59-CCAGTTGCTTGTTGGCTTATCCG-39); and SVCV G gene (F: 59-CC- expression of DrIFNF1 and DrIFNF3, depending on their combi- ATTCTGTTCATTTGGAGCCGTA-39,R:59-AATTTCATTCGACAAGA- CCCCC-39). natory binding and the relative amounts. Co-IP and Western blots Materials and Methods Co-IP assays and Western blots were performed, as previously described (28, Cells, virus, and zebrafish 29, 37). The following commercial Abs were used: anti-, anti-HA, anti- Flag ( Technology, Danvers, MA), and anti-GST (Merck Crucian carp (Carassius auratus L.) blastula embryonic cells, epithe- Millipore, Darmstadt, Germany). Anti-crucian carp IRF3 and IRF7 Abs were lioma papulosum cyprini cells, and zebrafish liver cells (ZFLs) were raised by immunization of rabbit with prokaryotically expressed crucian carp cultured as described previously (37, 38). Spring viremia of carp virus IRF3-DBD or IRF7-DBD, as described previously (28). We confirmed the (SVCV) was propagated and titered in epithelioma papulosum cyprini binding specificity of both fish IRF Abs to zebrafish IRF3 and IRF7. cells. Zebrafish (D. rerio) was maintained and infected i.p. with 50 mlof SVCV (108 TCID /ml)/fish, as described (36). 50 Results Plasmids Expression patterns of DrIRF1/3/7 with DrIFNF1 and F For in vitro translation and co-IP assays, the expression plasmids were DrIFN 3 in stimulated cells and tissues generated by inserting the open reading frames (ORFs) of DrIRF1, Previous results showed that DrIRF1/3/7 upregulate the expres- + zebrafish IRF3 (DrIRF3), and zebrafish IRF7 (DrIRF7) into a pCS2( ) sion of DrIFNF1 and DrIFNF3 (29, 36). To further delineate the vector that had precloned the HA/Myc coding sequence into the XbaI restriction site. For luciferase assays, the ORFs of DrIRF1, DrIRF3, and functional link among them, we determined their expression pat- DrIRF7 were cloned into the EcoRI and NotI sites of the pcDNA3.1(+) terns in ZFLs and selected zebrafish tissues. As shown in Fig. 1, vector. For prokaryotic expression, the ORF of DrIRF3 was cloned into all five molecules were constitutively transcribed in nonstimulated EcoRI and NotI sites of pGEX4T-1. All constructs were confirmed by ZFLs, albeit at varied expression levels; three stimuli, including sequencing. Other plasmids, including zebrafish IFN promoter-driven luciferase plasmids DrIFNF1pro-luc and DrIFNF3pro-luc, zebrafish poly(I:C), SVCV, and rIFN protein, induced their expression myeloid differentiation factor 88 (DrMyD88)-Flag were described pre- quickly over a 24-h time course. DrIRF1 and DrIRF7 were in- viously (29, 36). duced continuously, displaying similar levels during early stimu- The Journal of Immunology 1895 Downloaded from http://www.jimmunol.org/

FIGURE 1. RT-PCR analysis of zebrafish IRF1, IRF3, and IRF7 together with zebrafish IFNF1 and IFNF3 in ZFLs. ZFLs seeded in six-well plates were transfected with poly(I:C) (2 mg/ml) or treated with SVCV (1000 TCID50/ml) or recombinant IFN protein (5 ng/ml). At the indicated time points, the cells were sampled for real-time PCR analysis of DrIRF1/3/7 (A) and DrIFNF1/3 (B) expression by different stimuli. Amplification of DrIFNF1 was conducted by universal primers. The expression level is relative to the corresponding expression level of b-actin. Error bars represent SDs obtained by measuring each sample in triplicate. at Univ of NC Chapel Hill Health Sciences Library on August 16, 2019 lation and obviously different expression values afterward (6 h FLAG-tagged DrMyD88 with hemagglutinin (HA)-tagged DrIRF1 posttreatment), whereas DrIRF3 transcripts reached a peak at 12 h (IRF1-HA), HA-tagged DrIRF3 (IRF3-HA), or HA-tagged DrIRF7 posttreatment and decreased slightly thereafter (Fig. 1A). Notably, (IRF7-HA). Consistent with our previous results (36), DrIRF1 was DrIRF1 and DrIRF7 were transcriptionally expressed much more physically associated with DrMyD88, because the protein complex than IRF3 in nonstimulated and stimulated cells (Fig. 1A). DrIFNF1 immunoprecipitated by anti-FLAG Ab was also recognized by anti- and DrIFNF3 were also induced under the same conditions, with HA Ab (Fig. 3A, upper panels); however, an unrelated protein a higher constitutive expression and a more rapid induction for (EGFP) was not under the same condition (Fig. 3A, lower panels). DrIFNF1(Fig.1B). Similar results were observed for transfection of DrMyD88 with Detection of these five transcripts in 13 tissues of healthy either DrIRF3 or DrIRF7 (Fig. 3B, 3C). Combined with the ex- zebrafish showed that DrIRF1 had the highest level of constitutive pression analysis of DrIFN1/3/7 and DrIFNF1/3(Figs.1,2),these expression among the three IRFs (Fig. 2A). DrIFNF1 has two results suggest that zebrafish IRF1, IRF3, and IRF7 might simulta- isoforms (40, 41). RT-PCR analysis showed that the intracellular neously regulate IFN in certain tissues or cell types DrIFNF1 isoform (without a secretion peptide) was more sig- through similar signaling pathways, such as the MyD88 pathway. nificantly expressed than the secreted one (with a secretion pep- Binding of DrIRF1/3/7 to IRF-E/ISRE motifs within promoters tide) in healthy tissues, and DrIFNF3 transcripts were richer than of DrIFNF1/3 secreted DrIFNF1. Despite the varied expression levels, IRF and IFN molecules were generally abundant in thymus, gill, heart, and It is well-known that mammalian IRF-activated type I IFN ex- muscle, whereas their abundance was moderate or poor in the rest pression depends on direct binding to IFN promoter regions (1). We of the tissues (Fig. 2A). After SVCV infection, these molecules confirmed that DrIRF1 binds to DrIFNF1 (corresponding to the were transcriptionally upregulated to different extents in all tis- secreted isoform hereafter, unless indicated) and DrIFNF3 pro- sues, predominantly liver, intestine, and skin (Fig. 2B). With moters (36). Thus, we asked whether this might be the case for regard to the two DrIFNF1 isoforms, although the secreted one DrIRF3 and DrIRF7 as well. To this end, DNA pull-down assays had low basal expression in healthy tissues (Fig. 2A), it was more were performed by incubation of biotin-labeled promoter DNAs of strongly induced by viral infection than the intracellular one and DrIFNF1(2596 to +38) or DrIFNF3(21455 to 21102), which DrIFNF3 (Fig. 2B). Subsequent analysis of three SVCV gene bear three or two putative ISRE/IRF-E–like motifs (36), with transcripts, including L, N, and G, showed relatively high ex- in vitro–translated DrIRF1/3/7 proteins to determine whether each pression in hypophysis, thymus, gill, liver, and muscle (Fig. 2C), of them was able to bind to IFN promoters. As shown in Fig. 4A, in which zebrafish IFN transcription was also induced (Fig. 2B). in vitro translation of an HA-tagged DrIRF1 plasmid (IRF1-HA) These results suggested that SVCV infection induces simulta- generated a theoretically sized protein and a large-sized one; neous expression of DrIFNF1/3 and DrIRF1/3/7 in zebrafish cells Western blot analysis of promoter-bound products showed that and tissues. both forms of DrIRF1 effectively bound to IFNF1/3 promoters. Two protein forms were also observed for in vitro–translated Interaction of zebrafish IRF1/3/7 with MyD88 DrIRF7 or DrIRF3, with a main one corresponding to the expected In mammals, IRF1/7 interact with MyD88 (9, 13, 14), and so does m.w. and a weak one that is likely to be a degraded form. Only the mammalian IRF3 (42). To determine whether DrIRF3 and theoretically sized DrIRF7 bound to IFN promoters and, unex- DrIRF7, like salmon homologs (35), interact with DrMyD88, we pectedly, none of the in vitro–translated DrIRF3 do so under the performed co-IP assays in HEK293T cells co-overexpressing same conditions (Fig. 4A). 1896 DIFFERENTIAL ACTIVATION OF ZEBRAFISH IFNs BY IRF1/3/7 Downloaded from http://www.jimmunol.org/ at Univ of NC Chapel Hill Health Sciences Library on August 16, 2019

FIGURE 2. RT-PCR analysis of DrIRF1/3/7 and DrIFNF1/3 in healthy or infected zebrafish. The indicated tissues were collected from wild-type 6 zebrafish (A) or from zebrafish injected i.p. with PBS or SVCV (5 3 10 TCID50)(B) for qRT-PCR detection of DrIFNF1/3 and DrIRF1/3/7. Universal primers were used for analysis of total DrIFNF1, and specific primers were used for the secreted or intracellular isoforms. (C) At the same time, the expression of SVCV genes G, L, and N was determined in virally infected tissues. The expression level is shown relative to the corresponding expression level of b-actin (A) or as fold induction relative to that in corresponding tissues injected with PBS i.p., which was set to 1 (B and C). Error bars represent SDs obtained by measuring each sample in triplicate.

To further characterize the binding nature of DrIRF3, DrIRF3 motif. Deleting the proximal IRF-E3 motif caused a moderate re- (IRF3-HA) or DrIRF7 (IRF7-HA) was overexpressed in HEK293T duction in the band intensities of three DrIRFs, whereas deleting the cells, from which the cell lysates were used to repeat DNA pull- middle IRF-E2 motif only led to a slight decrease in DrIRF3 down assays. Western blot detection of the transfected cell lysates binding (Fig. 5, upper panels). With regard to the DrIFNF3 pro- showed a theoretically sized form and at least one large-sized form of moter, deleting the distant IRF-E1 motif led to a significant re- DrIRF3 (Fig. 4B). As a positive control, overexpression of DrIRF7 duction in DrIRF7 binding, a moderate reduction in DrIRF1 resulted in an intense theoretically sized protein that bound to binding, and a slight decrease in DrIRF3 binding. Deleting the DrIFNF1/3 promoters and sometimes a faint slow-migrating band proximal IRF-E2 motif caused a moderate reduction in DrIRF7 that was undetectable in the promoter-bound products. In contrast to binding, a slight decrease in DrIRF3 binding, and no obvious effect in vitro–translated DrIRF3, all overexpressed DrIRF3 forms bound on DrIRF1 binding (Fig. 5, lower panels). Collectively, these data to both zebrafish IFN promoters (Fig. 4B). Further, a recombinant suggest that DrIRF1, DrIRF3, and DrIRF7 differentially bind to the fusion protein (GST-IRF3) was made by a prokaryotic expression IRF-E/ISRE motifs within DrIFNF1 and DrIFNF3 promoters. system (Fig. 4C, left panels), and, expectedly, GST-IRF3 exhibited a strong binding affinity to DrIFN promoters (Fig. 4C, right panel). Physical binding of DrIRF1/3 to IFN promoters independently This prokaryotic expression system simultaneously produced a of phosphorylation degraded form of GST-IRF3 that retained the IFN promoter binding It was possible that the slow-migrating protein of DrIRF3 in the affinity due to the existence of N-terminal DBD (Fig. 4C). Thus, overexpressed HEK293T cells represented a phosphorylated form. DrIRF3 physically binds to zebrafish IFN promoters. To address this and determine whether such phosphorylation was In subsequent assays, we constructed a set of IFN promoter essential for the binding affinity of IRF proteins to IFN promoters, mutants lacking the individual IRF-E/ISRE motif to examine the lambda protein phosphatase (lambda PPase) was used to treat exact binding affinity of DrIRF1, DrIRF3, and DrIRF7 (Fig. 5). DrIRF1 and DrIRF3 before or after DNA pull-down assays. As Compared with the amounts of three IRF proteins bound to the shown in Fig. 6, in vitro translation gave two forms of DrIRF1; wild-type DrIFNF1 promoter, significantly less IRF protein was transfection of HEK293T cells with DrIRF3 plasmid generated pulled down by the DrIFNF1 promoter lacking the distant IRF-E1 three migrating protein forms (i.e., one main band at the expected The Journal of Immunology 1897

FIGURE 3. Immunoprecipitation anal- ysis of the association between zebrafish IRF1 and MyD88. HEK293T cells, seeded Downloaded from in 10-cm dishes, were cotransfected with 5 mg of FLAG-tagged DrMyD88 and 5 mgof IRF1-HA (A), IRF3-HA (B), or IRF7-HA (C) or empty vector pCS2+ (A–C,upper panels) and EGFP-HA (A–C, lower panels) as control. Twenty-four hours later, the trans- fected cells were immunoprecipitated with http://www.jimmunol.org/ anti-FLAG Ab, followed by analysis of the immunoprecipitates by Western blotting. at Univ of NC Chapel Hill Health Sciences Library on August 16, 2019

m.w. was accompanied by two weak, slow-migrating ones). promoters, DNA pull-down assays showed that neither IRF1 Incubation of lambda PPase did not affect the mobility of any nor IRF3 lost their binding affinities. As control, pretreatment rapid-migrating protein, but it resulted in the disappearance of of DrIRF7 with the protein phosphatase did not influence its the more slowly migrating form of DrIRF1 and only the most binding to IFN promoters. These results suggest that physical slowly migrating one of DrIRF3. Regardless of whether lambda binding of DrIRF1/3 to IFN promoters is a phosphorylation- PPase treatment occurred before or after incubation with IFN independent action.

FIGURE 4. The binding affinity of zebrafish IRF1, IRF3, and IRF7 to zebrafish IFNF1 and IFNF3 promoters. (A) In vitro-translated DrIRF1 and DrIRF7, but not DrIRF3, bind to both zebrafish IFN promoters by pull-down assays. A total of 1 mg of biotin-labeled DrIFNF1(2596 to +38) or DrIFNF3 (21455 ∼ 21102) promoter DNAs was incubated with the indicated in vitro–translated DrIRF1, DrIRF3, or DrIRF7 at 10-fold amounts of input. The bead- bound DNA-protein complex was detected by Ab specific to HA tag. (B) DrIRF3 and DrIERF7 overexpressed in HEK293T cells are capable of binding to zebrafish IFNF1 or IFNF3 promoters by pull-down assays. HEK293T cells seeded in 10-cm dishes were transfected with 10 mg of DrIRF3 or DrIRF7 or empty vector pCS2 as control. At 24 h posttransfection, cells were harvested for sonication in HKMG buffer. A total of 2% of DrIRF3- or DrIRF7- containing cell lysates was loaded as input, and 45% was used for DrIFNF1 or DrIFNF3 promoter binding analysis, as described in (A). (C) Prokaryotically expressed DrIRF3 is capable of binding to zebrafish IFNF1 or IFNF3 promoters by pull-down assays. Zebrafish GST-IRF3 was expressed by a prokaryotic expression system and purified (left panels). A total of 4 mg of the purified GST-IRF3 protein was incubated with 1 mg of biotin-labeled DrIFNF1or DrIFNF3 promoter DNA, and the bead-bound DNA-protein complexes were detected by Ab specific to GST tag. 1898 DIFFERENTIAL ACTIVATION OF ZEBRAFISH IFNs BY IRF1/3/7 Downloaded from http://www.jimmunol.org/

FIGURE 6. Physical binding of DrIRF1 and DrIRF3 to DrIFNF1 and DrIFNF3 promoters without phosphorylation. DNA pull-down assays were performed as in Fig. 5. For in vitro–translated DrIRF7, the protein was pretreated or not with lamda PPase before pull-down assays. For

in vitro–translated DrIRF1 and DrIRF3-overexpressing HEK293T cell at Univ of NC Chapel Hill Health Sciences Library on August 16, 2019 FIGURE 5. The binding of zebrafish IRF1, IRF3, and IRF7 to zebrafish lysates, the indicated proteins were treated with lambda PPase before or F F IFN 1- and IFN 3-derived IRF-E/ISRE binding sites. DNA pull-down after incubation with DrIFNF1 or DrIFNF3 promoters or were left m assays were performed by incubating 1 g of biotin-labeled mutant pro- treated. The bead-bound DNA-protein complexes were detected by F moter DNA, which lacked the individual IRF-E/ISRE motif of DrIFN 1 Western blotting. * and **, phosphorylation form of DrIRF1 and DrIRF3. or DrIFNF3, with in vitro–translated DrIRF1, in vitro–translated DrIRF7, and DrIRF3-overexpressing HEK293T cell lysates. The bead-bound DNA- protein complexes were detected by Ab specific to HA tag. translated DrIRF3 was still undetectable in the combined reaction setups in which DrIRF1 or DrIRF7 or both were included. Inter- estingly, compared with the reaction setup that included only DrIRF1, Binding of DrIRF1/3/7 to IFN promoters as homo- or two combined reaction setups (DrIRF1+DrIRF7 or DrIRF1+ heteroprotein complexes DrIRF3+DrIRF7) displayed an obviously enhanced binding of To determine whether zebrafish IRF1, IRF3, and IRF7 could coop- DrIRF1, particularly to the DrIFNF3 promoter (Fig. 7G), implying eratively regulate IFN responses, co-IP experiments were first per- abenefitofDrIRF7onDrIRF1binding to fish IFN promoters. formed to examine their potential to form homo- or heteroprotein To determine whether DrIRF3 would bind to IFN promoters complexes. Two sets of IRF constructs carrying the myc tag or the through the formation of homoprotein complexes, we set up HA tag were used to transfect HEK293T cells. In IRF1-myc–over- reconstitution reaction-based DNA pull-down assays by mixing expressing cells and IRF1-HA–overexpressing cells, anti-myc Ab– in vitro–translated DrIRF3 (IRF3-HA) with the lysates of immunoprecipitated protein complex was recognized by anti-HA Ab HEK293T cells transfected or not with IRF3-HA. As shown in (Fig. 7A, upper panels). However, an unrelated protein (EGFP-HA) Fig. 7H, Western blot with anti-HA Ab failed to detect in vitro– was not immunoprecipitated by IRF1-myc (Fig. 7A, lower panels). translated DrIRF3 in IRF3-alone–included pull-down assays; These results indicated that DrIRF1 was able to form homodimers. however, the combination of the same amount of in vitro– The same was true for DrIRF3 and DrIRF7 (Fig. 7B, 7C). In another translated DrIRF3 and regular HEK293T cell lysates led to experiment in which HEK293T cells were cotransfected with detectable binding (Fig. 7H), probably because human IRF3 IRF1-HA and IRF3-myc, anti-HA Ab–immunoprecipitated protein proteins that were expressed constitutively in HEK293T cells complex was detected by anti-IRF3 Ab (Fig. 7D, upper panels) or helped in vitro–translated DrIRF3 bind to IFN promoters by anti-myc Ab–immunoprecipitated protein complex was detected by forming a homoprotein complex. No binding was detected in anti-HA Ab (Fig. 7D, lower panels), suggesting that DrIRF1 was the solely HEK293T cell lysate–included reaction because hu- associated with DrIRF3. Similar results showed that DrIRF1 also man IRF3 was not recognized by anti-HA Ab; however, a strong interacted with DrIRF7 and so did DrIRF3 with DrIRF7 (Fig. 7E, 7F). binding was seen when HEK293T cells were transfected with Considering that in vitro–translated DrIRF3 protein alone IRF3-HA (Fig. 7H). Further assays used a reconstituted DrIRF3 failed to bind to IFN promoters, we wondered whether it would protein mixture consisting of in vitro–translated IRF3 and do so by virtue of in vitro–translated DrIRF1 or DrIRF7. DNA HEK293T cell lysates in which free-tagged or HA-tagged DrIRF3 pull-down assays were performed with DrIRF1/3/7 individually had been overexpressed; anti-HA Ab detection of the pull-down or collectively at a constant amount for each IRF protein proteins showed an obvious binding for the former mixture (over- (Fig. 7G). Western blot analysis of DrIFNF1orDrIFNF3promoter- expression of free-tagged IRF3) and an enhanced binding for the bound IRF proteins showed that in vitro–translated DrIRF1 and latter (overexpression of IRF3-HA) (Fig. 7I). Collectively, these DrIRF7, but not in vitro–translated DrIRF3, were easily detected data suggested that zebrafish IRF1/3/7 bind to fish IFN promoters as in single IRF-included reaction setups. Unexpectedly, in vitro– homo- and heteroprotein complexes. The Journal of Immunology 1899 Downloaded from http://www.jimmunol.org/ at Univ of NC Chapel Hill Health Sciences Library on August 16, 2019

FIGURE 7. The binding of zebrafish IRF1, IRF3, and DrIRF7 to zebrafish IFN promoters through the assembly of homo- or heteroprotein complexes. (A–F) Immunoprecipitation analysis of the association among zebrafish IRF1, IRF3, and IRF7. HEK293T cells seeded in 10-cm dishes were cotransfected with IRF1-myc and IRF1-HA (A), IRF3-myc and IRF3-HA (B), IRF7-myc and IRF7-HA (C), IRF1-HA and IRF3 (D), IRF1-HA and IRF7 (E), or IRF3 and IRF7-HA (F), each with 5 mg (upper panels). Cells were transfected with the indicated IRF expression plasmids and an unrelated vector EGFP-HA as control (lower panels). Twenty-four hours later, the transfected cells were immunoprecipitated with anti-myc Ab (A–C) or anti-HA Ab (D–F), followed by Western blotting with the indicated Abs. Arrows indicate nonspecific binding. (G–I). Cooperative binding of zebrafish IRF1, IRF3, and IRF7 to zebrafish IFN promoters. DNA pull-down assays were performed as in Fig. 4, by incubation of DrIFNF1 and DrIFNF3 promoter DNA with in vitro–translated DrIRF3, in vitro–translated DrIRF1, and in vitro–translated DrIRF7, individually and collectively (G), or with in vitro–translated DrIRF3 and regular HEK293T cell lysates, individually and collectively, and DrIRF3-transfected HEK293T cell lysates alone as control (H), or with in vitro–translated DrIRF3 and free-tagged DrIRF3-transfected HEK293T cell lysates collectively and HA-tagged DrIRF3-transfected HEK293T cell lysates as control (I). A constant amount of in vitro–translated DrIRF3 was included, and the bead-bound DNA-protein complexes were detected by Ab specific to HA tag.

Recruitment of DrIRF1/7 to bind to IFN promoters by DrIRF3 DrIRF3 binding. Notably, all reactions contained equal amounts of We further reconstituted DrIRF3-overexpressed HEK293T cell ly- the same DrIRF proteins and DrIFN promoter DNAs; however, the sates together with in vitro–translated DrIRF7 and/or DrIRF1 proteins amounts of IRF proteins that were pulled down by IFN promoter DNAs varied among different experiment combinations, and the to perform DNA pull-down assays. As shown in Fig. 8, each fish IRF amounts of promoter DNA-bound multiple IRF proteins in a given protein could bind to zebrafish IFN promoters, with a strong binding reconstituted reaction setup are not equal to the sum of those from for the overexpressed DrIRF3 and a weak one for DrIRF1 or DrIRF7 single component–included reaction. Therefore, DrIRF3 might fa- alone. In comparison with the relatively weak binding by DrIRF1 or cilitate the binding of DrIRF1 and/or DrIRF7 to fish IFN promoters. DrIRF7 alone, significantly enhanced binding of DrIRF1 or DrIRF7 was observed for the reconstituted fish IRF protein mixtures that Differential response of DrIFNF1/3 promoters to DrIRF/3/7 were made up of overexpressed DrIRF3 plus DrIRF7, DrIRF1, or individually and collectively both (Fig. 8). Interestingly, this effect was not reciprocal, because this Luciferase assays were used to determine the expression regu- supplementation resulted in an obvious reduction, not an increase, in lation of DrIFNF1 and DrIFNF3 by DrIRF1, DrIRF3, and 1900 DIFFERENTIAL ACTIVATION OF ZEBRAFISH IFNs BY IRF1/3/7 Downloaded from

FIGURE 8. Enhanced binding of zebrafish IRF1 and IRF7 to zebrafish IFN promoters by zebrafish IRF3. DNA pull-down assays were performed with the indicated pro- teinsasinFig.4.Atotalof1mg of biotin-labeled pro- moter DNA of DrIFNF1 or DrIFNF3 was incubated with

in vitro–translated DrIRF1, in vitro–translated IRF7-HA– http://www.jimmunol.org/ and IRF3-HA–transfected HEK293T cell lysates, indi- vidually or collectively. Each IRF was included at a constant amount, and the bead-bound DNA-protein complexes were detected by anti-HA tag Ab. at Univ of NC Chapel Hill Health Sciences Library on August 16, 2019

DrIRF7. Consistent with our previous results (29, 36), over- activities were quantified as 5.5-, 1.7-, and 1.2-fold, respectively expression of each fish IRF resulted in activation of the DrIFNF1 (Fig. 9A, top panel). promoter in a dose-dependent fashion (Fig. 9A). Similar acti- To exclude the interference of additional effects, the coopera- vation of the DrIFNF3 promoter was seen in response to DrIRF1 tivity values were also calculated as the ratio of luciferase activity or DrIRF7; however, DrIRF3 failed to activate the DrIFNF3 values obtained in the presence of two IRFs/sum of luciferase promoter (Fig. 9C). Among the three IRFs, the best stimulator of activity values obtained with each alone (Fig. 9B, 9D). We set the the DrIFNF1 promoter was DrIRF3, followed by DrIRF1 and criteria that values around or equal to 1 indicated no cooperative then DrIRF7. For instance, a 19-fold increase in DrIFNF1pro- effect, values , 1 indicated an inhibitory effect, and values . 1 moter–driven luciferase activities was detected when 200 ng of indicated a synergistic activation. Using this method to reassess DrIRF1 was used, whereas a 160-fold increase was seen with the activation of the DrIFNF1 promoter by DrIRF1 plus DrIRF3, 200 ng of DrIRF3 (Fig. 9A, top panel). In another experiment, a a gradually increasing synergy was observed along with the in- 35-fold increase was observed with DrIRF1 compared with a crease in the relative amounts of DrIRF3, with 1.5–2.2-fold higher 18-fold increase with DrIRF7 (Fig. 9A, middle panel). However, level of luciferase activities at DrIRF1/DrIRF3 ratios of 1:4–1:20 the DrIFNF3 promoter did not show such a preference for (Fig. 9B, top panel). DrIRF1 and DrIRF7 (Fig. 9C, middle panel). Interestingly, although DrIRF3 alone could not stimulate the Considering the varied expression levels of three zebrafish IRF DrIFNF3 promoter, cotransfection of low amounts of DrIRF3 (10 genes in stimulated cells and tissues (Figs. 1, 2), different ratios and 50 ng) and 200 ng of DrIRF1 (DrIRF1/DrIRF3 ratio . 1:1) of any two IRFs were used to evaluate the possible synergistic resulted in significantly decreased luciferase activities relative to effects of IRF expression levels on regulating zebrafish IFN transfection of 200 ng of DrIRF1 alone; conversely, significantly expression. The results showed that IFN promoter activities increased luciferase activities were observed when the DrIRF1/ obtained with two IRFs were often higher than those obtained DrIRF3 ratio was #1:1 (Fig. 9C, top panel). These results indi- with one (Fig. 9A, 9C). For example, compared with transfection cated a dual effect of DrIRF3 on DrIRF1-mediated DrIFNF3 of DrIRF1 alone (200 ng), cotransfection of DrIRF1 (200 ng) expression: a synergistic induction (.1.3–3-fold higher than and increasing amounts (10, 50, 200 ng) of DrIRF3 resulted in a control) at high DrIRF3 concentrations (DrIRF1/DrIRF3 ratio of significant and continuous increase in DrIFNF1promoterac- 1:4 or 1:20) and an inhibitory effect (4–5-fold lower than control) tivities (1.5-, 3.2-, and 5.5-fold higher than control, respec- at low DrIRF3 concentrations (DrIRF1/DrIRF3 ratio of 20:1 or tively); when compared with transfection of DrIRF3 alone at the 4:1) (Fig. 9D, top panel). No obvious effect of DrIRF1/7 on corresponding amounts (10, 50, 200 ng), DrIFNF1promoter DrIFNF1 promoter activation was observed when the DrIRF1/7 The Journal of Immunology 1901 Downloaded from http://www.jimmunol.org/ at Univ of NC Chapel Hill Health Sciences Library on August 16, 2019

FIGURE 9. Differential effects of zebrafish IRF1, IRF3, and IRF7 on zebrafish IFN promoter activation. (A) Differential response of DrIFNF1 promoter to DrIRF1, DrIRF3, and DrIRF7 by luciferase assays. Crucian carp (C. auratus L.) blastula embryonic cells seeded in 24-well plates were cotransfected with DrIFNF1 promoter–driven luciferase plasmid (0.25 mg) in the presence of two zebrafish IRF plasmids at the indicated amounts; 0.025 mg pRL-TK was introduced as an internal control. At 48 h posttransfection, cells were harvested for detection of luciferase activity. The data shown are representative of three independent experiments, each performed in triplicate. (B) Cooperativity analysis of zebrafish IRF1/3/7-mediated DrIFNF1 activation based on more than three independent luciferase assays, as in (A). Cooperativity values between any two of zebrafish IRF1/3/7 on DrIFNF1 promoter activation were calculated as the ratio of luciferase activities obtained in the presence of both IRF proteins/sum of luciferase activities obtained when each factor was expressed. The values around or equal to 1 indicated no cooperative effects, values , 1 indicated an inhibitory effect, and values . 1 indicated a synergistic activation. (C) Differential response of DrIFNF3 promoter to DrIRF1, DrIRF3, and DrIRF7 by luciferase assays. Luciferase assays were performed, as in (A), using a DrIFNF3 promoter-driven luciferase plasmid instead of a DrIFNF1 promoter–driven luciferase plasmid. (D) Cooperativity analysis of zebrafish IRF1/3/7-mediated DrIFNF3 activation, based on more than three independent luciferase assays, as in (C). ratio was #1:1 because the cooperative value was close to 1, but a most cell types (8), an IRF7-dependent pathway, and/or an IRF1- slight synergistic induction was detected at higher amounts of dependent pathway from endosomes of plasmacytoid DCs, cDCs, DrIRF7 (.1.3-fold higher than control) (Fig. 9A, 9B, middle or other immune cells (9, 11–14). In other instances, invading panels). Similar analyses indicated an obvious synergism on pathogens trigger multiple signaling pathways to activate an IFN DrIFNF3 at DrIRF1/DrIRF7 ratios of 4:1–1:4, with the highest response, such as a Listeria monocytogenes mutant that is pri- level (nearly 4-fold higher than control) at a 1:1 ratio (Fig. 9C, 9D, marily recognized in phagosomes and also in the cytosol of cDCs, middle panels). DrIRF3 and DrIRF7 displayed robust synergism on thus stimulating IFNb dependently of IRF1 in phagosomes and stimulating the DrIFNF1 promoter with increasing the relative IRF3/7 in cytosol (11), and L. acidophilus, which upregulates amounts of DrIRF7 (1.8–3-fold higher than control at DrIRF3/ IFN-b in mouse DCs involving IRF1, IRF3, and IRF7 through the DrIRF7 ratios of 1:1–1:20) (Fig. 9A, 9B, bottom panels) and dis- MyD88 pathway (21). Therefore, IRF1, IRF3, and IRF7 are three play modest synergism on the DrIFNF3 promoter (up to 1.8-fold crucial transcription factors and might be simultaneously assem- higher than control) only at low DrIRF3/DrIRF7 ratios (1:4 and bled to IFN gene promoters, ensuring full release of IFN response. 1:20) (Fig. 9C, 9D, bottom panels). Like mammalian IRF1, DrIRF1 is constitutively expressed in various tissues and is inducible by IFN and IFN stimuli (Fig. 2). Discussion Unlike mammalian IRF3 that is not inducible by IFN stimulation In mammals, accumulating data suggested that the type I IFN and viral infection (43), and mammalian IRF7, whose expression response is contingent on many issues, including intracellular is limited in lymphoid cell types but is highly induced in various locations of pathogen, sensing receptors, and IRF members; these cell types (1), DrIRF3 and DrIRF7 are transcriptionally expressed issues create a functional link directing a specific signaling in all normal tissues tested and significantly induced by SVCV pathway in a given cell type (1, 2). Generally, such a link triggers infection (Fig. 2). This disparate property of DrIRF3 and DrIRF7 an IRF3/7-dependent IFN response from cytosolic location in is also seen in other fish species (28, 44–47), implying the pos- 1902 DIFFERENTIAL ACTIVATION OF ZEBRAFISH IFNs BY IRF1/3/7 sibility that fish IRF3 and IRF7 are required for IFN responses in two coactivators, b-catenin and CBP, by HDAC6 and PKC-b (50). various cell types. Interestingly, very weak transcription of two Therefore, it is important to analyze in vivo binding of fish IRF3 to zebrafish IFN genes, DrIFNF1 and DrIFNF3, is detected in IFN promoters in the future. healthy zebrafish tissues in which DrIRF1/3/7 are also constitu- The binding features of DrIRF1, DrIRF3, and DrIRF7 seem to tively abundant (Fig. 2A). Following SVCV infection, DrIFNF1/3 determine the differential response of both fish IFN gene pro- and DrIRF1/3/7 are significantly induced in another set of zebra- moters. For example, DrIRF3 alone activates the DrIFNF1, but not fish tissues (Fig. 2B), which correlates with the replication of the DrIFNF3, promoter, whereas DrIRF1 and DrIRF7 activate SVCV (Fig. 2C). SVCV is a negative ssRNA virus (48, 49). Al- both; they differ in that DrIRF1 has no preference, and DrIRF7 though it is uncertain how SVCV infection is recognized in fish prefers to activate the DrIFNF3 promoter (Fig. 9). Consistently, cells, the fact that these fish molecules highly respond to SVCV in pull-down assays reveal an unusual binding feature of DrIRF3: it some common tissues prompted us to hypothesize that DrIRF1/3/7, does not have a preference for either of the IRF-E/ISRE motifs like their homologs in mammals (11, 21), might cooperatively within the DrIFNF3 promoters, which is very different from Downloaded from regulate zebrafish IFN gene expression on unknown occasions. In DrIRF3 binding to the DrIFNF1 promoter and DrIRF1/7 binding support of this hypothesis, each of DrIRF1/3/7 interacts with to DrIFNF1/3 promoters, where a preferred motif is selected MyD88 (Fig. 3); DrIRF1, together with MyD88, upregulates (Fig. 5). In mammals, IRF3 possesses a restricted recognizing DrIFNF3 (36), and in Atlantic salmon, IRF3 or IRF7 synergizes DNA sequence, but IRF7 has a broader one (51); this selective with MyD88 to activate the salmon IFN response (35). Notably, binding contributes to differential regulation of early-phase IFN F b zebrafish IFN 1 has two isoforms (40, 41). In the current study, genes, including IFN , and later-phase IFN genes, including most http://www.jimmunol.org/ the secreted isoform of DrIFNF1 is induced more significantly IFNas: the former is primarily controlled by IRF3, and the latter than the intracellular one by SVCV infection (Fig. 2B). The dif- are controlled by IRF7 (6–8, 51–53). Considering that the IRF3/7- ferential induction of both DrIFNF1 isoforms is likely ascribed to dependent IFN response is activated from cytosolic locations in their promoter composition. The secreted DrIFNF1 isoform pro- most cell types (8), the similar binding and regulatory properties moter has three IRF-E motifs (IRF-E1, IRF-E2, and IRF-E3 in of DrIRF3 and DrIRF7 support our previous notion that RLR Fig. 5), and the intracellular isoform promoter has only the distant pathway–triggered DrIFNF1 expression is primarily regulated by IRF-E1. Our previous results showed that the proximal IRF-E DrIRF3, thereby resembling mammalian IFNb, and DrIFNF3is motif primarily contributes to induction of fish IFN gene expres- primarily regulated by DrIRF7, thereby resembling mammalian sion (28), supporting the idea that the secreted one is highly in- IFNas (29). The binding property of DrIRF1 also correlates with at Univ of NC Chapel Hill Health Sciences Library on August 16, 2019 ducible by SVCV. Therefore, the secreted DrIFNF1 isoform its regulatory potential to activate DrIFNF1 and DrIFNF3 (36). promoter was used in the subsequent assays. Interestingly, the magnitude of zebrafish IFN gene expression is The data in the current study indicate that DrIRF1, DrIRF3, and dependent on the combinatory binding of DrIRF1/3/7, as well as their DrIRF7 differentially regulate zebrafish IFN expression, likely relative amounts. In the current study, the cooperativity values were through assembly of homo- and heteroprotein complexes binding determined as the ratio of luciferase activity values obtained in the to IRF-E/ISRE sites in IFN promoters. First, DrIRF1, DrIRF3, and presence of both IRFs/the sum of luciferase activity values obtained DrIRF7 possess highly conserved N-terminal DBD (36), guaran- with each alone, and the values . 1 indicate a synergistic activation teeing their binding to the IFN promoter. Second, like human (Fig. 9B, 9D). Based on this criterion, overexpression of DrIRF1 and IFNa gene promoters whose IRF-E motifs are selectively recog- DrIRF7 together results in a significantly synergistic activation of nized by IRF3 and IRF7 (6, 7), each fish IRF protein binds to the DrIFNF3 promoter and less activation of DrIFNF1promoter, cognate IRF-E/ISRE motifs of the DrIFNF1 and DrIFNF3 pro- but at higher amounts of DrIRF7 (Fig. 9, middle panels). This result moters (Fig. 5). Actually, the IRF-E/ISRE motifs are directly re- is consistent with the binding assays showing that DrIRF7 facili- sponsible for either binding or activation of crucian carp IRF3 and tates an enhanced IFN promoter binding of DrIRF1, particularly to zebrafish IRF1 to fish IFN promoters (28, 36). Third, DrIRF1, the DrIFNF3 promoter (Fig. 7G). The same is true for cooperative DrIRF3, and DrIRF7 are associated with one another in vitro activation of both IFN promoters by DrIRF3 together with DrIRF1 (Fig. 7A–F), and they bind to zebrafish IFN promoters through or DrIRF7, which correlates with enhanced promoter binding of the formation of homo- and heteroprotein complexes (Figs. 7G, DrIRF1 and DrIRF7 as a consequence of DrIRF3 recruitment (Fig. 8). 7H, 8). Finally and importantly, overexpression of DrIRF1, These results indicate a molecular mechanism underlying IRF DrIRF3, and DrIRF7 individually and collectively differentially combination-mediated differential activation of fish IFN pro- activates two fish promoters depending on their combination and moters. Generally, the best cooperation occurs when DrIRF3 or the relative amounts (Fig. 9). DrIRF7 is present at higher amounts, likely due to the fact that Although eukaryotically expressed DrIRF3 in HEK293T cells IRF3 and IRF7 are two essential transcription factors for distinct or prokaryotically expressed DrIRF3 binds to both zebrafish IFN IFN expression in most cell types (8, 29), with the exception that promoters by pull-down assays, the binding is not detectable with DrIRF1/7-dependent DrIFNF3 expression is most significantly in vitro–translated DrIRF3 protein alone (Fig. 4A). Interestingly, induced at equal amounts of DrIRF1 and DrIRF7. The IRF the same phenomenon is seen for in vitro–translated human IRF3 concentration-dependent IFN gene-expression patterns might re- by EMSA assays, and binding to the IFN-b promoter is observed flect an important physiological significance. In healthy or in- with a high concentration of recombinant human IRF3 in baculovirus- fected zebrafish tissues, DrIRF1/3/7 display various constitutive infected cells (4). However, the failure to bind the IFN promoter is or inducible expression levels; therefore, when these factors are not a result of fish IRF3 protein concentration, because a lower activated simultaneously in a given cell type, even downstream of concentration of prokaryotically expressed DrIRF3 than in vitro– different pattern recognition receptor–mediated signaling, they translated IRF3 used in our experiments still binds to fish IFN might work together to fine-tune the activation of DrIFNF1 and promoters (data not shown). The exact causes of such a phe- DrIFNF3 genes, depending on their relative expression levels. nomenon are unknown, but it is possible that a misfolded protein Although DrIRF3 alone does not activate the DrIFNF3 pro- conformation in in vitro–translated IRF3 restricts its binding to moter, it still regulates DrIRF1- or DrIRF7-mediated DrIFNF3 IFN promoters. Notably, in vivo assays showed that, in mammals, gene expression. Strikingly, DrIRF3 facilitates DrIRF7-mediated IRF3 binding to target gene promoters requires the activation of DrIFNF3 activation only at high concentrations (Fig. 9C, 9D, The Journal of Immunology 1903 bottom panels), and it exerts dual effects on DrIRF1-mediated IFN response, there might be unique fish IRF-mediated IFN regu- DrIFNF3 gene expression at different concentrations (Fig. 9C, lation awaiting further investigation. 9D, top panels). When DrIRF3 is present at lower amounts than DrIRF1, DrIRF3 significantly downregulates DrIRF1-mediated Disclosures DrIFNF3 expression; however, when DrIRF3 is expressed at The authors have no financial conflicts of interest. higher amounts, both transcriptional factors synergistically ac- F tivate DrIFN 3 gene expression. Similar results are observed References in mammals. Human IRF3 selectively binds to distinct IRF-E a b 1. Tamura, T., H. Yanai, D. Savitsky, and T. Taniguchi. 2008. The IRF family tran- motifs within IFN and IFN gene promoters (51); however, scription factors in immunity and oncogenesis. Annu. Rev. Immunol. 26: 535–584. this factor activates the expression of early-phase IFN genes like 2. Gurtler,€ C., and A. G. Bowie. 2013. Innate immune detection of microbial IFNb but has no effect on most IFNa gene expression (6, 7, 51). nucleic acids. Trends Microbiol. 21: 413–420. 3. Stein, C., M. Caccamo, G. Laird, and M. Leptin. 2007. Conservation and di- Similar to DrIRF3, human IRF3 selectively exerts a similarly vergence of gene families encoding components of innate immune response Downloaded from dual effect on IRF7-mediated IFNa gene expression (7) and an systems in zebrafish. Genome Biol. 8: R251. a 4. Wathelet, M. G., C. H. Lin, B. S. Parekh, L. V. Ronco, P. M. Howley, and inhibitory effect on IRF1-mediated IFN 4 gene expression (5). T. Maniatis. 1998. Virus infection induces the assembly of coordinately activated Obviously, the mechanism of IRF-mediated IFN expression in transcription factors on the IFN-beta enhancer in vivo. Mol. Cell 1: 507–518. humans is not identical to that in zebrafish, and we cannot ex- 5. Schafer, S. L., R. Lin, P. A. Moore, J. Hiscott, and P. M. Pitha. 1998. Regulation of type I gene expression by interferon regulatory factor-3. J. Biol. clude the possibility that there are dual effects of zebrafish IRF3 Chem. 273: 2714–2720. ´ on IRF7-mediated IFN responses, because two other zebrafish 6. Civas, A., P. Genin, P. Morin, R. Lin, and J. Hiscott. 2006. Promoter organization http://www.jimmunol.org/ IFN genes (DrIFNF2/4) are not included in the current study. of the interferon-A genes differentially affects virus-induced expression and responsiveness to TBK1 and IKKepsilon. J. Biol. Chem. 281: 4856–4866. Despite these differences, these findings suggest that zebrafish 7. Ge´nin, P., R. Lin, J. Hiscott, and A. 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