The Type I Interferon-IRF7 Axis Mediates Transcriptional Expression of Usp25 Gene*

Viral infection or lipopolysaccharide (LPS) treatment induces expression of a large array of genes, the products of which play a critical role in host antipathogen immunity and inflammation. We have previously reported that the expression of ubiquitin-specific protease 25 (USP25) is significantly up-regulated after viral infection or LPS treatment, and this is essential for innate immune signaling. However, the mechanism behind this phenomenon is unclear. In this study, we found that viral infection-induced up-regulation of Usp25 is diminished in cells lacking interferon regulatory factor 7 (IRF7) or interferon α receptor 1 (IFNAR1) but not p65. Sendai virus- or type I interferon-induced up-regulation of Usp25 requires de novo protein synthesis of IRF7. Furthermore, IRF7 directly binds to the two conserved IRF binding sites on the USP25 promoter to drive transcription of Usp25, and mutation of these two sites abolished Sendai virus-induced IRF7-mediated activation of the USP25 promoter. Our study has uncovered a previously unknown mechanism by which viral infection or LPS induces up-regulation of USP25.


Viral infection or lipopolysaccharide (LPS) treatment induces expression of a large array of genes, the products of which play a critical role in host antipathogen immunity and inflammation.
We have previously reported that the expression of ubiquitinspecific protease 25 (USP25) is significantly up-regulated after viral infection or LPS treatment, and this is essential for innate immune signaling. However, the mechanism behind this phenomenon is unclear. In this study, we found that viral infectioninduced up-regulation of Usp25 is diminished in cells lacking interferon regulatory factor 7 (IRF7) or interferon ␣ receptor 1 (IFNAR1) but not p65. Sendai virus-or type I interferon-induced up-regulation of Usp25 requires de novo protein synthesis of IRF7. Furthermore, IRF7 directly binds to the two conserved IRF binding sites on the USP25 promoter to drive transcription of Usp25, and mutation of these two sites abolished Sendai virus-induced IRF7-mediated activation of the USP25 promoter. Our study has uncovered a previously unknown mechanism by which viral infection or LPS induces up-regulation of USP25.
Host pattern recognition receptors recognize pathogen-associated molecular patterns and initiate a series of signaling cascades that lead to activation of transcription factors including NF-B and interferon regulatory factor 3 (IRF3) 2 (1)(2)(3). It has been well documented that activation of NF-B (p65/p50 heterodimer) is dependent on inhibitors of B kinase (IKK) complex (IKK␣/␤/␥)-mediated phosphorylation and degradation of IB␣, whereas activation of IRF3 requires phosphorylation by TBK1 or IKK⑀ (4 -9). The activated NF-B and IRF3 enter into nucleus, bind to the conserved B or IRF binding sites of promoters, and recruit co-activators to activate the transcription of target genes.
Viral nucleic acid and lipopolysaccharide (LPS) of Gramnegative bacteria are two common pathogen-associated molecular patterns that trigger signaling cascades to activate NF-B and IRF3 and induce the production of type I interferons (IFNs) (3,10). Type I IFNs further induce the expression of hundreds of downstream genes in an autocrine or paracrine manner, and the products of these genes including interferon-induced GTPbinding protein (Mx), 2Ј-5Ј-oligoadenylate synthase (OAS), double-stranded RNA-activated protein kinase (PKR), ISG56, and ISG15 orchestrate inhibition of pathogen replication and spread and promote apoptosis and clearance of the infected cells (11). In addition to the direct effect on innate immune cells for antipathogen responses, type I IFNs also regulate adaptive immunity including T cell activation and differentiation and antitumor immunity (12,13).
The type I IFN family is composed of 13 functional IFNA genes in humans (14 in mice), a single IFNB gene, and others. The IFN␣ family shares 80% sequence homology among them, whereas the homology between various IFN␣ and IFN␤ is 30% (14,15). However, all the type I IFNs bind to the same receptors, IFNAR1 and IFNAR2, with affinities varying from picomolar to micromolar orders to recruit tyrosine kinase 2 (TYK2) and Janus kinase 1 (JAK1) for signal transduction, respectively. TYK2 and JAK1 are cross-phosphorylated and activated to phosphorylate several conserved tyrosine residues on IFNAR1 and IFNAR2, which provides docking sites for the downstream effector proteins including STAT1 (16,17). It has been shown that STAT2 interacts with IFNAR2 constitutively, whereas STAT1 is recruited to IFNAR2-IFNAR1 receptor complex in both STAT2-dependent and -independent manners (18 -20). TYK2 and JAK1 further phosphorylate Tyr-701 of STAT1 and Tyr-690 of STAT2, which form the ISGF3 transcription factor complex together with IRF9 to bind to the IFN-stimulated response elements on the promoters of and activate the transcription of ISGs (16). Type I IFN treatment also results in Tyr(P)-STAT1 homodimers that are responsible for the regulation of IFN␥-activated sequence elements (21,22). In addition to phosphorylation of STAT1 at Tyr-701, the phosphorylation of STAT1 at Ser-708 by IKK⑀ accounts for transcriptional activation of about 30% of the ISGF3 target genes (23). Thus, it is conceivable that type I IFN-triggered transcription of ISGs is regulated at multiple steps ranging from the ligand subtypes to the modifications of transcription factors. IRF7 is strongly induced by type I IFN-mediated signaling in a manner that is dependent on the TYK2-mediated phosphorylation of Tyr-701 of STAT1 but independent of IKK⑀-mediated phosphorylation of Ser-708 of STAT1 (23,24). Although IRF3 and IRF7 share a similar structure to bind the conserved IRF binding sites and are activated by TBK1-or IKK⑀-mediated phosphorylation, studies with Irf3 Ϫ/Ϫ and Irf7 Ϫ/Ϫ mice or cells suggest that IRF3 is required for early induction of IFN␤ and IFN␣4, whereas IRF7 is a master transcription factor essential for later induction of IFN␣ subsets (25)(26)(27)(28)(29)(30). Whether and how IRF3 and IRF7 differentially regulate transcription of other genes are of great interest.
We have previously observed that LPS or viral infection substantially up-regulates the expression of Usp25 gene (31,32). In this study, we found that virus-or LPS-induced expression of Usp25 was significantly abolished in cells lacking IRF7 or IFNAR1. Importantly, type I IFN-triggered signaling indirectly induces up-regulation of Usp25 by inducing expression of IRF7. Furthermore, we have identified two conserved IRF7 binding sites on the promoter of Usp25 gene, and mutation of these two sites impaired SeV-induced or IRF7-mediated activation of the USP25 promoter. Our study has uncovered the type I IFN-IRF7 axis-mediated expression of Usp25 gene.

Experimental Procedures
Mice-Ifnar1 ϩ/Ϫ mice were purchased from The Jackson Laboratory and maintained and crossed to obtain Ifnar1 ϩ/ϩ and Ifnar1 Ϫ/Ϫ littermates in the specific pathogen-free facility of Wuhan University. Age-and sex-matched Ifnar1 ϩ/ϩ and Ifnar1 Ϫ/Ϫ littermates were used for all experiments. All animal experiments were in accordance with protocols approved by the Institutional Animal Care and Use Committee of Wuhan University.
Real Time Quantitative PCR-Cells treated with various stimuli were harvested in TRIzol (Invitrogen), and first strand cDNA was synthesized with a reverse transcription kit (Biotool). Gene expression was examined with a Bio-Rad CFX Connect system with a SYBR Green One Step Real-Time PCR kit (Biotool). Data were normalized to the expression of ␤-actin. Real time quantitative PCR primers were described previously (31) and are as follows: Irf3: forward, CGG AAA GAA GTG TTG CGG TT; reverse, TTT TCC TGG GAG TGA GGC AG; Irf7: forward, AGA GGG CGT TTT ATC TTG CG; reverse, TGG AGC CCA GCA TTT TCT CT; and Ifnan: forward, TCA AAG GAC TCA TCT GCT GC; reverse, GGT TCC TGC ACC CCC ACC TG.
Viral Infection-Cells were seeded into 24-well plates (2 ϫ 10 5 cells/well) or 6-well plates (10 6 -10 7 cells/well). Twentyfour hours later, cells were treated with LPS or infected with SeV or HSV-1 for the indicated time points. The cells were collected for quantitative PCR (qPCR) or immunoblotting assays.
Reporter Gene Assays-HEK293 cells (4 ϫ 10 4 cells/well) cultured in 24-well plates were transfected with the reporter plasmid (100 ng) and an internal control vector, phRL-TK-Renilla luciferase (Promega) (2.5 ng). The pGL3-Basic vector served as a negative control, and empty vector was used to equalize the total amount of DNA. Twenty-four hours after transfection, cells were lysed in passive lysis buffer, and the firefly and Renilla luciferase activities were determined using a Dual-Luciferase reporter assay kit (Promega). The firefly luciferase activity was normalized by Renilla luciferase activity and expressed as the -fold stimulation relative to the activity in vector-transfected cells.
Chromatin Immunoprecipitation Assays-Briefly, 5 ϫ 10 6 cells were fixed with 1% formaldehyde and quenched by gly-cine. The cells were washed three times with PBS and then harvested in chromatin immunoprecipitation (ChIP) lysis buffer (50 mM Tris⅐HCl, pH 8.0, 1% SDS, 5 mM EDTA) followed by sonication until the sizes of DNA were 400 -600 bp. The lysate was centrifuged at 4°C for 15 min, and ChIP dilution buffer (20 mM Tris⅐HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) was added to the supernatant (4:1 volume). The resulted lysate was then incubated with anti-FLAG at 4°C overnight. The protein G beads were added into the lysate on the next morning and incubated at 4°C for 3 h. DNA was eluted using ChIP elution buffer (0.1 M NaHCO 3 , 1% SDS, 30 g/ml proteinase K) through incubation at 65°C overnight, and DNA was purified with a DNA purification kit (TIANGEN). The purified DNA was assayed by quantitative PCR with an CFX Connect system with a SYBR Green One Step Real-Time PCR kit.
Statistical Analysis-Differences between experimental and control groups were determined by Prism software with twoway analysis of variance and Bonferroni test. p values less than 0.05 were considered statistically significant.

IRF7 Plays an Essential Role of LPS-or Virus-induced Expression of Usp25
-In our previous studies, we have observed that the expression of Usp25 is up-regulated by LPS treatment or viral infection in various types of cells (31,32). Interestingly, treatment with actinomycin D, a compound that inhibits transcription, almost abolished the up-regulation of Usp25 and the increase of USP25 protein by LPS or SeV or HSV-1 infection in BMDCs (Fig. 1A). Sequence analysis of the promoter of mouse Usp25 gene identified two IRF binding sites (Ϫ1399 to Ϫ1375 and Ϫ4648 to Ϫ4624), two ISGF3 binding sites (Ϫ3154 to Ϫ3130 and Ϫ3345 to Ϫ3321), and at least five NF-B binding sites (Fig. 1B). However, p65 deficiency did not affect SeV-induced expression of Usp25 but did inhibit SeV-induced expression of Ifnb and Il6 (Fig. 1C). We reconstituted either IRF3 or IRF7 into Irf3 Ϫ/Ϫ Irf7 Ϫ/Ϫ MEFs and examined SeV-induced expression of Usp25. We found that reconstitution of IRF7 into Irf3 Ϫ/Ϫ Irf7 Ϫ/Ϫ MEFs promoted SeV-induced up-regulation of Usp25 more robustly than did reconstitution of IRF3 (Fig. 1D). In addition, SeV-induced up-regulation of Usp25 was substantially inhibited by knockdown of IRF7 and to a lesser extent by knockdown of IRF3 in MEFs (Fig. 1E). These data suggest that IRF7 and IRF3 (to a lesser extent) but not p65 are essential transcription factors for virus-induced up-regulation of USP25.

LPS-and Virus-induced Up-regulation of Usp25
Depends on Type I IFN-triggered Signaling-We next examined the effects of various kinase inhibitors on virus-or LPS-induced up-regulation of Usp25 in BMDCs or MLFs. Consistent with the notion that IRF7 and IRF3 are essential for transcriptional up-regulation of Usp25, inhibition of the upstream kinases TBK1 and IKK⑀ by amlexanox but not IMD0354 (inhibitor for IKK␤) or the p38 kinase inhibitor impaired LPS-or virus-induced expression of Usp25 ( Fig. 2A). Interestingly, we also found that ZM449828 (a JAK1 inhibitor) strongly inhibited up-regulation of Usp25 induced by LPS or viral infection, indicating that JAK1-mediated signaling is critical for the induction of USP25.
Because JAK1 is critical for type I IFN-triggered signaling, we reasoned that LPS or virus up-regulates the expression of Usp25 through type I IFN-triggered signaling. To test this hypothesis, we treated BMDCs with anti-IFN␣, anti-IFN␤, or both followed by LPS stimulation or viral infection. As shown in Fig. 2B, blocking IFN␣, IFN␤, or both strongly inhibited LPS-or virus-triggered induction of Usp25 and Irf7. Furthermore, LPSor virus-induced up-regulation of Usp25 was substantially diminished in Ifnar1 Ϫ/Ϫ MEFs and almost completely abolished in Ifnar1 Ϫ/Ϫ BMDCs compared with the wild-type controls (Fig. 2C). These data together suggest that LPS-or virusinduced expression mainly depends on type I IFN-triggered signaling.
Type I IFN-induced Expression of Usp25 Is Dependent on TBK1/IKK⑀ and de Novo Synthesized IRF7-Considering that Usp25 gene promoter contains two ISGF3 binding sites and that type I IFN-triggered signaling is critical for the up-regulation of Usp25, we hypothesized that type I IFNs activate transcription of Usp25 though ISGF3. However, treatment with cycloheximide, a compound that inhibits mRNA translation, impaired IFN␣-or IFN␤-induced expression of Usp25. In contrast, IFN␣-or IFN␤-induced expression of Irf7, a direct target of ISGF3, was not affected by cycloheximide treatment (Fig.  3A), indicating that virus-triggered type I IFN-mediated upregulation of Usp25 requires de novo protein synthesis. In addition, IFN␣-triggered up-regulation of Usp25 was impaired by knockdown of IRF7 but not IRF3 in MEFs and restored by reconstitution of IRF7 but not IRF3 into Irf3 Ϫ/Ϫ Irf7 Ϫ/Ϫ MEFs (Fig. 3, B and C), indicating that the de novo synthesized IRF7 is required for type I IFN-induced up-regulation of Usp25.

The Type I IFN-IRF7 Axis-mediated Transcription of Usp25
nant negative mutant regulating type I IFN-induced up-regulation of Usp25. In addition, knockdown of IKK⑀ in Tbk1 Ϫ/Ϫ MEFs reconstituted with the empty vector but not in those reconstituted with TBK1 significantly impaired IFN␣-induced expression of Usp25 (Fig. 4C). We further transfected siRNA-resistant TBK1 (rTBK1) or IKK⑀ (rIKK⑀) into wild-type MEFs followed by simultaneous knockdown of endogenous TBK1 and IKK⑀. Interestingly, IFN␣-induced up-regulation of Usp25 was not affected in MEFs transfected with either rTBK1 or rIKK⑀ (Fig. 4D). Furthermore, simultaneous knockdown of TBK1 and IKK⑀ by siRNA significantly inhibited IFN␣-induced expression of Usp25 in BMDCs (Fig. 4E). Taken together, these data suggest that TBK1 and IKK⑀ function redundantly to regulate type I IFN-induced expression of Usp25. IRF7 Binds to the USP25 Promoter-To further confirm that IRF7 drives transcription of Usp25, we cloned the upstream 5000 bp starting from the transcription start site of Usp25 into the pGL3-Basic luciferase vector (USP25 promoter), made con-structs with various mutations in the IRF or NF-B binding sites, and performed luciferase reporter assays (Fig. 5A). Interestingly, IRF7 or SeV potently activated the luciferase activity of USP25 promoter, which was substantially impaired by muta- were left untreated or treated with IFN␣ (20 units/ml) or IFN␤ (100 ng/ml) in the presence or absence of various kinase inhibitors for 12 h followed by qPCR analysis. B, Tbk1 Ϫ/Ϫ MEFs were reconstituted with Vec, TBK1, or TBK1(K38A) through lentivirus-mediated gene transfer. The reconstituted TBK1 or TBK1(K38A) in the cells was examined by immunoblotting analysis (lower panels). Cells were left untreated or treated with IFN␣ (20 units/ml) for 12 h followed by qPCR analysis (upper graph). C, Tbk1 Ϫ/Ϫ MEFs were reconstituted with Vec or TBK1 through lentivirus-mediated gene transfer. Cells were further transfected with control or siRNA targeting IKK⑀ followed by immunoblotting analysis (lower panels) or qPCR analysis after IFN␣ treatment (20 units/ml) (upper graph). D, wild-type MEFs were stably transfected with Vec, rTBK1, or rIKK⑀ followed by transfection of control or siRNAs targeting endogenous TBK1 and IKK⑀. Cells were subjected to immunoblotting analysis (right panels) or qPCR analysis after IFN␣ treatment (20 units/ml) (left graph). E, wild-type BMDCs were transfected control, siTBK1, or siIKK⑀. Twenty-four hours later, cells were stimulated with IFN␣ (20 units/ml) for 8 h followed by qPCR analysis. Data shown are representatives of at least three independent experiments. *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001. Error bars represent S.D. Rel., relative.

The Type I IFN-IRF7 Axis-mediated Transcription of Usp25
tion of either the proximal or distal IRF binding site (IB) (USP25 promoter ⌬IB1 or USP25 promoter ⌬IB2) of USP25 promoter and abolished by simultaneous mutation of both IRF binding sites (USP25 promoter ⌬IB1ϩ2), indicating that the two IRF binding sites cooperatively mediate transcription of Usp25 (Fig. 5B). In addition, IRF7 but not IRF3 was sufficient to activate the IB1-, IB2-, or IB1ϩ2-driven reporters in a dose-dependent manner (Fig. 5C). In contrast, IRF3 (at a high dosage) but not p65, p50, or p52-RelB complex activated the USP25 promoter ϳ2-4-fold, and mutation of all five NF-B binding sites (⌬5B) did not affect SeV-, IRF7-, or IRF3-mediated activation of USP25 promoter (Fig. 5B), indicating that the NF-B binding sites are dispensable for USP25 transcription after viral infection and that sequences other than the B sites in USP25 promoter may facilitate IRF3-mediated activation of USP25 promoter. Results from ChIP analysis demonstrated that IRF7 directly bound to the two IRF binding sites (Usp25 IB1 and Usp25 IB2) but not a nonspecific site (Usp25 NS) of USP25 promoter ( Fig. 5D and Table 1). Together, these data suggest that IRF7 directly binds to the distal and proximal IRF binding sites in the USP25 promoter and drives transcriptional activation of Usp25 gene.

Discussion
We have previously shown that LPS and viral infection strongly induce up-regulation of USP25. In this study, we further confirmed that LPS and viral infection activated transcription of Usp25 through type I IFN-triggered signaling. In addition, type I IFN-triggered signaling induced expression and protein synthesis of IRF7, which was activated by TBK1 and IKK⑀ and bound to the USP25 promoter to activate transcription of Usp25 gene (Fig. 6).
Sequence analysis of USP25 promoter indicated that multiple NF-B binding sites exist in the USP25 promoter. In our study, p65 deficiency did not affect virus-induced expression of Usp25. Mutation of the five NF-B binding sites did not affect basal, SeV-triggered, or IRF7/3-medaited activation of USP25 promoter. In addition, overexpression of p65, p50, or p52-RelB complex did not activate USP25 promoter, indicating that the NF-B sites on USP25 promoter are dispensable for virus-or LPS-induced up-regulation of Usp25. However, whether the NF-B sites are involved in the induction of Usp25 by other stimuli is unknown. The USP25 promoter also contains two potential ISGF3 binding sites. We found that cycloheximide treatment impaired type I IFN-triggered induction of Usp25, indicating that ISGF3 does not directly regulate transcription of Usp25 but instead activates de novo synthesis of other transcription factor(s) to mediate transcription of Usp25.
IRF3 and IRF7 are two structurally related transcription factors that bind to the conserved IRF binding site (5Ј-GAAANNGAAA-3Ј) on the promoters and are essential for induction of hundreds of genes involved in innate immunity and inflammation. IRF3 exhibits more restricted DNA binding site specificity compared with IRF7. Mutation of a single nucleotide in either of the two GAAA core sequences impairs IRF3 binding and transcription activity, whereas the G and the third A in the GAAA core sequence are variable for IRF7 binding activity (36). According to this standard, IB1 "gaaacataaa" and IB2 "gaatgagaag" in USP25 promoter are preferentially recognized and bound by IRF7 but not IRF3. Consistent with this notion, we observed that (i) IRF7 but not IRF3 was sufficient to activate the IB1-or IB2-driven reporters and required for virustriggered type I IFN-mediated up-regulation of Usp25, (ii) IRF7 activated USP25 promoter more potently than did IRF3 in luciferase reporter assays, and (iii) IRF7 bound to the USP25 promoter more potently than did IRF3. However, it should be noted that IRF3 activated USP25 promoter (ϳ2-4-fold) when transfected at a high dosage and partially rescued USP25 induction in Irf3 Ϫ/Ϫ Irf7 Ϫ/Ϫ MEFs after viral infection. In addition, we observed that IFNAR1 deficiency in MEFs partially inhibited virus-triggered up-regulation of Usp25, whereas IRFAR1 deficiency in BMDCs completely abolished up-regulation of Usp25 after viral infection, indicating that virus-induced expression of Usp25 might be differentially regulated by IRF3 and IRF7 in distinct types of cells. Taken together, it is likely that IRF3 is responsible for minimal expression of Usp25 in MEFs after viral infection, whereas the de novo synthesized IRF7 induced by type I IFNs is a master transcription factor for USP25 expression in MEFs and BMDCs.
Unlike IRF3, which is constitutively expressed and resides in the cytosol, IRF7 is expressed at a low level and strongly induced by type I IFN-triggered signaling. Both IRF3 and IRF7 undergo TBK1-or IKK⑀-mediated phosphorylation, dimerization, and nuclear translocation after LPS treatment or viral infection. We found that treatment with TBK1 and IKK⑀ inhibitor severely abolished type I IFN-triggered induction of Usp25. Furthermore, reconstitution of TBK1(K38A) into Tbk1 Ϫ/Ϫ MEFs inhibited