IFR-9/STAT2 Functional Interaction Drives Retinoic Acid–Induced Gene G Expression Independently of STAT1

Published OnlineFirst April 7, 2009; DOI: 10.1158/0008-5472.CAN-08-4922 Published Online First on April 7, 2009 as 10.1158/0008-5472.CAN-08-4922

Research Article

Ye-Jiang Lou, Xiao-Rong Pan, Pei-Min Jia, Dong Li, Shu Xiao, Zhang-Lin Zhang, Sai-Juan Chen, Zhu Chen, and Jian-Hua Tong

Shanghai Institute of Hematology and State Key Laboratory of Medical Genomics, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

Abstract transcription (2). Besides, some other potential cis-acting elements for transcription factors [such as CAAT/enhancer binding protein Retinoic acid–induced gene G (RIG-G), a gene originally identified in all-trans retinoic acid–treated NB4 acute (C/EBP) and PU.1] have been also noted in the RIG-G gene promyelocytic leukemia cells, is also induced by IFNA in promoter. Therefore, the question of what are the transcription various hematopoietic and solid tumor cells.Our previous factors essential for RIG-G gene induction remains open. work showed that RIG-G possessed a potent antiproliferative The first identified multimeric complex to recognize the ISRE activity.However, the mechanism for the transcriptional consensus sequence is the IFN-stimulated gene (ISG) factor 3 regulation of RIG-G gene remains unknown.Here, we report (ISGF3), which is composed of the phosphorylated forms of signal that signal transducer and activator of transcription (STAT) 2 transducer and activator of transcription (STAT) 1 and STAT2, together with IFN regulatory factor (IRF)-9 can effectively together with IFN regulatory factor (IRF)-9 (4). The IRF-9 protein is drive the transcription of RIG-G gene by their functional a member of the IRF family characterized by a well-conserved interaction through a STAT1-independent manner, even helix-turn-helix DNA-binding motif, which can recognize the ISRE. without the tyrosine phosphorylation of STAT2.The complex The mammalian IRF family contains nine members to date, IRF-1 IRF-9/STAT2 is both necessary and sufficient for RIG-G gene to IRF-9, which have diverse roles in host defense, cell cycle expression.In addition, IRF-1 is also able to induce RIG-G regulation, apoptosis, oncogenesis, and immune cell development gene expression through an IRF-9/STAT2–dependent or IRF-9/ (5). STAT1 and STAT2 represent two of the seven mammalian STAT STAT2–independent mechanism.Moreover, the induction of proteins that share several structurally and functionally conserved RIG-G by retinoic acid in NB4 cells resulted, to some extent, domains, including an NH2-terminal coiled-coil region for inter- from an IFNA autocrine pathway, a finding that suggests a actions with other transcription factors and chaperone proteins, novel mechanism for the signal cross-talk between IFNA and a phosphotyrosine-binding Src homology 2 domain required for retinoic acid.Taken together, our results provide for the first receptor binding and STAT dimerization, and a COOH-terminal time the evidence of the biological significance of IRF-9/STAT2 transcription activation domain (TAD) within which a highly complex, and furnish an alternative pathway modulating the conserved tyrosine residue is present. Importantly, the phosphor- expression of IFN-stimulated genes, contributing to the diver- ylation of this tyrosine allows a functional interaction between sity of IFN signaling to mediate their multiple biological prop- activated STAT proteins (6). The mature ISGF3 complex serves as a erties in normal and tumor cells. [Cancer Res 2009;69(8):3673–80] transcriptional regulator in type I IFN signaling pathways by translocating into the nucleus and then binding to ISRE of certain ISGs. Noteworthy, it is generally admitted that within the ISGF3 Introduction complex, STAT1 and IRF-9 mediate the DNA binding, whereas The retinoic acid–induced gene G (RIG-G), also known as IFN- STAT2 does not seem to interact directly with DNA but provides a inducible gene 60, was originally identified in all-trans retinoic acid potent TAD (7). (ATRA)–treated NB4 acute promyelocytic leukemia (APL) cells In addition to the ISGF3, other types of STAT complexes with (1) and plays an important role in cell growth inhibition through IRF-9 can also be formed to bind the ISRE. It was reported that a up-regulation of p21 and p27 (2). RIG-G gene expression can be complex containing IRF-9 and the phosphorylated STAT1, but induced not only by ATRA along with the differentiation of NB4 lacking STAT2, could be directed to ISRE-containing genes in cells but also by IFNa in a series of hematopoietic cell lines as well response to IFNg (8, 9). In mature B cells, Gupta and colleagues as various types of solid carcinoma cells (1, 3), which pointed to a (10) found that IFNa was able to activate the formation of an possible role of RIG-G in signal cross-talk between ATRA and IFNa. ISGF3-like complex comprising STAT2, STAT6, and IRF-9. Whereas However, at present, little is known about the regulation control of IRF-9 functions as an adaptor for tethering the different STAT RIG-G gene expression. We previously reported two well-conserved dimers to ISRE, other members of IRF family have also been shown IFN-stimulated response elements (ISRE) in the promoter region to be involved in the regulation of ISRE-containing genes. It was of RIG-G gene, these two ISREs being likely involved in RIG-G shown that IRF-1 could regulate expression of ISGs by directly binding to ISRE on its own or by dimerizing with other proteins, including IRF-3, IRF-7, IRF-8, or STAT1 (11–13). Recently, accumulating observations support the notion that STAT proteins Note: Y-J. Lou and X-R. Pan contributed equally to this work. Requests for reprints: Jian-Hua Tong, Shanghai Institute of Hematology, Rui-Jin can drive gene expression without tyrosine phosphorylation Hospital, 197 Rui-Jin Road II, Shanghai 200025, People’s Republic of China. Phone/Fax: (14–19). It strongly suggests that some not yet identified 86-21-64743206; E-mail: [email protected]. mechanisms could exist in gene transcription regulation in I2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-4922 response to these cytokines. www.aacrjournals.org 3673 Cancer Res 2009; 69: (8). April 15, 2009

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Figure 1. A, time courses of expression of RIG-G, STAT1, STAT2, and IRF-9 were analyzed by Western blot. NB4 cells were treated for indicated times with IFNa or ATRA, respectively. The expression of h-actin was used as a loading control. *, nonspecific bands. B, reverse transcription-PCR detection of RIG-G mRNA. NB4 cells were treated with IFNa and/or cycloheximide (CHX) as indicated. The expression of b-actin was used as internal control. C and D, luciferase reporter gene assay on the transcription factors for RIG-G gene expression. The reporter gene construct pXP2 (À310) was cotransfected with indicated expression plasmids into STAT1-null U3A cells (C) or IRF-9–deficient U2A cells (D). Twelve hours after transfection, cells were treated with or without 1,000 units/mL IFNa. The luciferase activity was measured 36 h after transfection.

In this work, we intensively explored the mechanisms underlying DMEM supplemented with 10% calf serum, 2 mmol/L L-glutamine, and the expression regulation of RIG-G gene and found that STAT2, antibiotics. ATRA and cycloheximide (Sigma) were dissolved in ethanol as À2 together with IRF-9, was sufficient for RIG-G expression, even stock solution at 10 mol/L and 10 mg/mL, respectively, and stored at À20jC. Human IFNa was purchased from Schering-Plough Co. and stored without the phosphorylation of STAT2, and provided a novel at 4jC. mechanism modulating the expression of ISGs that accounts for Plasmids. The expression constructs for wild-type (wt) STAT1 and the signal cross-talk between IFNa and ATRA. mutant STAT1-Y701F (converting tyrosine 701 to phenylalanine) were kindly provided by Dr. J.E. Darnell, Jr. (Rockefeller University, New York, NY). STAT2 full-length cDNA expression plasmid was obtained by subcloning the Materials and Methods insert of pBlueScript-STAT2 (from Dr. J.E. Darnell, Jr.) to the vector Cell culture and reagents. The NB4 cells (gift from Dr. Lanotte, INSERM pcDNA3.1. The mutant STAT2-Y690F (converting tyrosine 690 to phenyl- U685, Paris, France) were cultured in RPMI 1640 (Life Technologies) alanine) was prepared by Mutagenesis kit (Stratagene). The luciferase- supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, and containing reporter plasmids pXP2 (À310) and pXP2 (À87) were antibiotics. The U3A, U2A, and HT1080 cells were kindly provided by G.R. constructed as previously described (2). The mutant reporter constructs Stark (Lerner Research Institute, Cleveland, OH; refs. 20, 21) and cultured in pXP2-mut-1, pXP2-mut-2, and pXP2-mut-3 were generated from pXP2

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IRF-9/STAT2 Drives the Expression of RIG-G Gene

(À310) by Mutagenesis kit. All the constructs were verified by DNA extracts were prepared in the buffer containing 150 mmol/L NaCl, sequencing. 50 mmol/L Tris-HCl (pH 8.0), and 0.5% NP40 and then mixed with protein Reverse transcription-PCR. Total RNA was extracted with Trizol A-agarose and rabbit anti–IRF-9 antibody (Santa Cruz Biotechnology) at reagent (Invitrogen). After reverse transcription, cDNA was amplified under 4jC overnight with rotation. The precipitated proteins were eluted by the following conditions: 94jC for 5 min; 25 cycles of 94jC for 30 s, 58jC boiling beads in SDS-loading buffer and analyzed by Western blotting. for 45 s, and 70jC for 50 s; and 70jC for 10 min. The primers Chromatin immunoprecipitation assay. Chromatin immunoprecipi- 5¶-ACCTCGAGACACAGAGGGCAGTC-3¶ and 5¶-ACGGATCCGCCTTGTAG- tation (ChIP) assay was performed using EZ ChIP kit (Millipore) according CAGCACCCAATC-3¶ were used to detect RIG-G transcripts. The amplifi- to the instruction of the manufacturer. The primers specific to the promoter cation of b-actin gene was used as an internal control for cDNA loading region of RIG-G gene were as follows: 5¶-TGGTGAGTATGGCCTTAGAATT-3¶ with the primers 5¶-CATCCTCACCCTGAAGTACCCC-3¶ and 5¶-AGCCTG- and 5¶-TTCCTGTCTGCCTCAAGTAAAT-3¶. Normal rabbit IgG (Millipore) GATGCAACGTACATG-3¶. was used to control the nonspecific immunoprecipitation of chromatin by Western blot analysis. The performance of Western blot and the immunoglobulins. generation of rabbit polyclonal anti-RIG-G sera were described in our Quantitative measurement of human IFNA in medium. The cell previous work (2). Other primary antibodies were from Santa Cruz culture supernatant was collected and concentrated if necessary, and then Biotechnology (STAT1, STAT2, IRF-9, and IRF-1), Sigma (h-actin), and Cell 100 AL aliquots were used for detection with ELISA kit (PBL Biomedical Signaling (pTyr701-STAT1 and pTyr690-STAT2). Laboratories) following the protocol of the manufacturer. The ratio unit/pg Transfection and luciferase reporter assay. The cells were cotrans- for human IFNa is approximately 3 to 5. Data were shown as means F SD fected with the reporter gene constructs and related expression plasmids as of three independent experiments. indicated by using SuperFect (Qiagen) followed by transcriptional activity assay using Dual-Luciferase Assay System (Promega) according to the manufacturer’s instruction. Ten nanograms pRL-TK vector (Promega) were Results used as an internal control for normalizing the transfection efficiency. Data RIG-G is not a primary target of IFNA-activated ISGF3 were shown as means F SD of three independent experiments. complex. By Western blot analysis, we first examined whether the RNA interference. To make small interfering RNA (siRNA) against IRF-1, Janus-activated kinase (JAK)-STAT pathway–activated ISGF3 com- ¶ ¶ 19-bp sequence (5 -CTTCCAGGTGTCACCCATG-3 ) within the coding region plex was involved in IFNa-induced RIG-G up-regulation. We found of IRF-1 gene was selected and cloned into the pTER+ vector (22). The that in NB4 cells the total amounts of STAT1 and STAT2 continued plasmid pTER-si-IRF-1 was then transfected into U3A cells by using a SuperFect. to increase throughout the IFN treatment in parallel with the RIG- Coimmunoprecipitation. The expression plasmid encoding green G induction. In contrast, the level of tyrosine-phosphorylated fluorescent protein (GFP)-IRF-9 hybrid protein was transfected with the STAT1 increased rapidly, peaking at 10 minutes to 1 hour after indicated STAT2 constructs into U3A cells. Twelve hours after transfection, IFNa treatment and then returning to the levels characteristic of cells were treated with or without IFNa for another 24 h. The protein untreated cells, whereas the level of tyrosine-phosphorylated

Figure 2. A, the interaction between STAT2 and IRF-9 was analyzed by coimmunoprecipitation experiments. U3A cells were cotransfected with GFP-IRF-9 and wt STAT2 or mutant STAT2-Y690F. Twelve hours after transfection, cells were treated with or without IFNa for another 24 h. Cell lysates (Input) were then immunoprecipitated (IP) with anti–IRF-9 antibody followed by Western blot (WB) analysis with anti-STAT2 antibody. B, effect of IRF-9 with wt STAT2 or mutant STAT2-Y690F on pXP2 (À310) reporter gene in U3A cells. C, ChIP assay was used to test the binding of exogenous IRF-9 to RIG-G gene promoter. U3A cells were cotransfected with STAT2 and IRF-9 followed by the treatment of IFNa or medium for 24 h. The immunoprecipitated chromatin by anti–IRF-9 antibody was analyzed by PCR using primers specific for RIG-G promoter sequences (+11 to À288). www.aacrjournals.org 3675 Cancer Res 2009; 69: (8). April 15, 2009

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STAT2 followed a similar time course but with a much slower decline (Fig. 1A). The differential expression kinetics between RIG- G and the activated ISGF3 hardly exhibited a possibility that RIG-G could be a direct target of ISGF3. In addition, no tyrosine- phosphorylated STAT1 was detected in NB4 cells treated either by IFNa for 6 hours or by ATRA for 72 hours (Fig. 1A), further suggesting that the RIG-G gene expression was most likely activated through a novel mechanism. Using the protein synthesis inhibitor cycloheximide, we ques- tioned whether RIG-G was an IFNa primary responsive gene. In comparison with the direct transcriptional response of IRF-1 to IFNa,theRIG-G mRNA induction was entirely blocked by cycloheximide (Fig. 1B), indicating that the RIG-G was not a primary responsive gene in IFNa-induced JAK-STAT pathway. Both IRF-9 and STAT2 are key factors required for RIG-G expression. We then conducted a survey of transcription factors essential to the expression of RIG-G. Several IFN signaling pathway– related key regulators, including STAT1, STAT2, IRF-1, and IRF-9, were transfected individually or in combination into STAT1-deficient U3A cells, together with a luciferase reporter gene containing RIG-G promoter. Consequently, STAT1, STAT2, or IRF-9 alone exhibited no obvious effect on the reporter gene expression, whereas the combination of IRF-9 and STAT2 could cooperatively cause an up to 7-fold induction in luciferase activity, as the IRF-1 alone (Fig. 1C). Some important factors implicated in myelopoiesis, such as C/EBPs and PU.1, were also evaluated for their abilities to transactivate the reporter gene. No significant change was observed when these expression plasmids were transfected into U3A cells, except that C/EBPa had a potent repressive activity (data not shown). We found that enforced expression of STAT1 in U3A cells could rescue the response of RIG-G gene to IFNa, but mutant STAT1- Y701F had no such an effect (Fig. 1C), suggesting that phosphorylation of STAT1 was required for RIG-G expression in IFNa-treated U3A cells. However, when we coexpressed mutant STAT2-Y690F with wt STAT1 in U3A cells, IFNa-induced luciferase activity was Figure 3. A, IFNa secretion in ATRA-treated NB4 cells. NB4 cells were effectively inhibited by mutant STAT2-Y690F in a dose-dependent treated with or without 1 Amol/L ATRA for 24, 48, 72, and 96 h, respectively. The manner (Fig. 1C). Furthermore, we observed no induction of culture supernatants corresponding to every time point were used for IFNa concentration quantification. B, U3A cells were cotransfected with STAT2 and luciferase activity in IRF-9–deficient U2A cells after IFNa treatment, IRF-9. Twelve hours after transfection, the cells were incubated for another unless the cells were reconstituted by IRF-9 (Fig. 1D). Taken 24 h in the culture supernatants of NB4 cells treated with or without ATRA. The transfected U3A cell extracts were then analyzed by Western blot. Lanes I, together, these results provided an indication that IRF-9 and STAT2, II, III, IV, and V represent U3A cells cultured in the supernatants of NB4 cells rather than STAT1, were key factors necessary for RIG-G expression. treated with 1 Amol/L ATRA for 0, 24, 48, 72, and 96 h, respectively. The total To investigate how IRF-9 and STAT2 synergistically regulate the STAT2 level was used to control the transfection efficiency. C, U3A cells were transfected with IRF-1 or empty vector. Thirty-six hours after transfection, expression of RIG-G gene, we detected whether these two proteins the culture supernatants were collected and used for IFNa titration. could interact. Unlike IFNa-activated ISGF3 complex in which tyrosine-phosphorylated STAT proteins are required, IRF-9 could successfully interact with either wt STAT2 or mutant STAT2-Y690F supported by the fact that ATRA could not only induce the total in the absence of IFNa, although the interaction between IRF-9 and amounts of STAT2 and IRF-9 proteins but also increase the tyrosine wt STAT2 could be obviously enhanced by IFNa via tyrosine phosphorylation level of STAT2 in NB4 cells (Fig. 1A). Because ATRA phosphorylation of STAT2 (Fig. 2A). In agreement with this, both could cause IFNa synthesis and secretion in NB4 cells by up- wt STAT2 and mutant STAT2-Y690F displayed a similar effect on regulating IRF-1 (23), we thus quantitated the IFNa in culture the luciferase reporter gene expression in U3A cells without IFNa. supernatant of ATRA-treated NB4 cells. We noted that the IFNa level By contrast, in IFNa-treated U3A cells, the luciferase activity was undetectable until the NB4 cells were treated with ATRA for showed an additional 7-fold increase by the complex of IRF-9 with 72 hours when the IFNa titer was f7 pg/mL. After a 96-hour treat- wt STAT2, but not in the case of mutant STAT2-Y690F (Fig. 2B). ment,afurther9-foldincreaseinIFNa level could be measured Noticeably, no obviously increased binding ability of IRF-9 to (Fig. 3A). RIG-G promoter was observed in the presence of IFNa after nor- To test whether the IFNa secreted from ATRA-treated NB4 cells malizing the amounts of the detected samples according to input was able to induce the STAT2 phosphorylation and RIG-G (Fig. 2C). expression, we incubated the IRF-9/STAT2–cotransfected U3A Dramatic induction of RIG-G by ATRA is related to the IFNA cells in the supernatant of ATRA-treated NB4 cells. The results secretion in NB4 cells. The idea that both STAT2 and IRF-9 were revealed that the supernatant of 72-hour ATRA-treated NB4 cells basic components necessary for RIG-G expression was also was sufficient to induce the tyrosine phosphorylation of STAT2

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IRF-9/STAT2 Drives the Expression of RIG-G Gene and the endogenous RIG-G level in U3A cells, in comparison phosphorylation of STAT2 was then determined. The IFNa level in with the relative consistent level of total STAT2 (Fig. 3B). These the culture supernatant of IRF-1–transfected U3A cells was 3-fold data suggested that the effective IFNa secretion might be one elevated when compared with control cells (Fig. 3C). Similarly, of the causes resulting in RIG-G induction in ATRA-treated NB4 increased level of STAT2 tyrosine phosphorylation was detected as cells. well in IRF-1–transfected HT1080 cells (Fig. 4B). Together, these IRF-1 plays an important role in the modulation of RIG-G data indicated that IRF-1 could regulate RIG-G expression through expression. It was noteworthy that IRF-1 alone could significantly an IRF-9/STAT2–dependent mechanism. induce the expression of the reporter gene containing RIG-G However, because IRF-1 also has the ability to directly bind the promoter (Fig. 1C). To investigate the role of IRF-1 in RIG-G ISRE elements (24), an IRF-9/STAT2–independent mechanism in expression regulation, IRF-1 gene-specific siRNA treatments were which IRF-1 acted should not be ruled out. This notion was sustained included into the reporter gene assays. Consequently, we found by the fact that IRF-1 could activate the RIG-G gene promoter in IRF- that si-IRF-1 could significantly reduce IFNa-induced luciferase 9–deficient U2A cells and by our ChIP assay, confirming the ability of activity in STAT1-transfected U3A cells (Fig. 4A), showing that IRF- IRF-1 to bind the RIG-G gene promoter (Fig. 4D). Moreover, we found 1 was indeed involved in transcriptional activation of RIG-G. that IRF-9 could significantly inhibit the transactivity of IRF-1 in a Thus, we tried to better understand the mechanisms by which dose-dependent manner (Fig. 5A), reflecting again their competition IRF-1 induced RIG-G gene expression. An obvious up-regulation of for direct binding with RIG-G promoter. both STAT2 and IRF-9 proteins was found in HT1080 cells Two ISRE elements are molecular basis required for RIG-G transfected with IRF-1 (Fig. 4B), implying that the effect of IRF-1 expression. Because both IRF-1 and the complex IRF-9/STAT2 on RIG-G expression might also be attributed to IRF-9 and STAT2. could bind the RIG-G gene, we therefore conducted a detailed Moreover, we observed that the transactivity of IRF-9 complexed functional analysis on RIG-G promoter to precise the molecular with wt STAT2, rather than mutant STAT2-Y690F, could be basis for RIG-G expression. Several luciferase reporters with wt or enhanced 4-fold more when IRF-1 coexpressed in U3A cells mutant RIG-G promoters were constructed (Fig. 5B and C): pXP2 (Fig. 4C), suggesting a role of IRF-1 on STAT2 phosphorylation. (À87) was totally in lack of two ISRE elements; pXP2-mut-1 and Whether IRF-1 could also induce the IFNa synthesis as well as the pXP2-mut-2 contained mutations in the site of ISRE I and ISRE II,

Figure 4. Roles of IRF-1 in expression regulation of RIG-G gene. A, pTER-si-IRF-1 was transfected into U3A cells with the reporter construct pXP2 (À310) and the indicated expression plasmids. Twelve hours after transfection, cells were treated with or without 1,000 units/mL IFNa. The luciferase activity was measured 36 h after transfection. si-Control targeting EGFP sequence was used as a negative control. B, HT1080 cells were transiently transfected with IRF-1 or empty vector. After 36 h, cell lysates were analyzed by Western blot as indicated. *, nonspecific bands. C, effect of IRF-1 on the transactivities of IRF-9 associated with wt STAT2 or mutant STAT2-Y690F in reporter gene pXP2 (À310) induction in U3A cells. D, IRF-9–deficient U2A cells were transiently transfected with IRF-1 or empty vector, the binding of exogenous IRF-1 to RIG-G gene promoter was tested by ChIP assay, and the induction of reporter gene pXP2 (À310) was detected with luciferase assay.

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Figure 5. A, inhibitory effect of IRF-9 on IRF-1–induced RIG-G promoter activities. IRF-1 (500 ng) expression plasmid was cotransfected with increasing amounts of IRF-9 plasmid (0–1 Ag) into U3A cells. B, partial 5¶-flanking genomic sequences of the RIG-G gene. Two conserved ISREs (ISRE I and II) are boxed. The transcriptional start site +1 is indicated by an arrow. The capital letters represent the first exon of the RIG-G gene. C, schematic representation of the wt or mutant RIG-G promoter-luciferase reporter gene constructs. D, different reporter gene constructs were, respectively, cotransfected with IRF-1 or IRF-9/STAT2 into U3A cells, as indicated. Luciferase activity was shown relative to the values obtained in the cells transfected with the same reporter gene construct and empty vectors. respectively; and pXP2-mut-3 possessed double mutations. Our expression in eukaryotic cells and their activities are dependent on results showed that both pXP2 (À87) and pXP2-mut-3 lost all distinct protein-protein interaction. In this regard, one of the best activities in response to indicated transcription factors. In contrast, models is IFN-stimulated JAK-STAT pathways, in which both pXP2-mut-1 could significantly reduce the transactivities of both STAT1 and STAT2 are activated by tyrosine phosphorylation in IRF-9/STAT2 complex and IRF-1, whereas pXP2-mut-2 had no response to IFNs (4). However, it has become apparent that the difference with the wt construct pXP2 (À310) (Fig. 5D). The similar activation of classic JAK-STAT pathways alone cannot account for results were also obtained in the presence of IFNa.These the pleiotropic biological effects of IFNs on target cells. Increasing observations clearly indicated that the two ISRE sequences in number of studies show that other types of STAT complexes can be RIG-G promoter were the molecular basis for RIG-G gene formed in response to IFNs (7). Several type I IFN-mediated STAT2- expression. Notably, it seems that both IRF-9 and IRF-1 preferen- dependent, but STAT1-independent, mechanisms were reported tially bind the ISRE I. (25–30). Because STAT2 is the only member of the STAT family without DNA-binding activity, it can act as a transcription factor only when complexed to other STATs or proteins (31). Two Discussion independent reports have pointed the existence of a complex This work intended to identify the transcription factors essential composed of STAT2 homodimers and IRF-9 without STAT1 in for RIG-G gene expression. The transcription factors usually IFNa-treated cells (32, 33), but this finding was tempered by the function as homodimers or heterodimers to regulate the gene facts that their authors mentioned that this complex displayed only

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IRF-9/STAT2 Drives the Expression of RIG-G Gene limited DNA-binding affinity for an ISRE sequence. Therefore, for a further indicating that the induction of RIG-G by ATRA in NB4 cells long time, the biological significance of IRF-9/STAT2 complex has was tightly related with an IFNa autocrine pathway. been difficult to assess. In summary, we propose here a novel mechanism for the In this study, we provide the first evidence that in STAT1-deficient transcriptional regulation of RIG-G,anIFNa-induced gene. As U3A cells, STAT2 forms a complex with IRF-9 on the ISRE regions of shown in Fig. 6, the binding of IFNa to its cognate receptors RIG-G promoter and effectively mediates the transcription of RIG-G activates the classic JAK-STAT pathway, which is required to gene, even without the tyrosine phosphorylation. Because IFNa initiate the transcription of a set of primary target genes, such as failed to induce RIG-G expression in STAT1-deficient U3A cells IRF-1. The expression of unphosphorylated STAT2 and IRF-9 is unless the cells were transfected with exogenous STAT1, we greatly increased in response to IFNa probably through an indirect previously concluded that STAT1 was a prerequisite to RIG-G manner. STAT2 interacts with IRF-9 to form a transcription factor expression. However, our conclusion no longer proved exact complex, which is both necessary and sufficient to transactivate because of our following new findings. First, mutant STAT2-Y690F the expression of RIG-G gene. On the other hand, IRF-1 can also could exert a dominant-negative effect in STAT1-reconstituted U3A induce RIG-G gene expression through an IRF-9/STAT2–dependent cells in the presence of IFNa (Fig. 1C). Second, in IRF-9–deficient or IRF-9/STAT2–independent mechanism. In NB4 cells, ATRA can U2A cells, IFNa-induced luciferase activity was detected only when up-regulate IRF-1, which was followed by the production and enforcing IRF-9 expression in these cells (Fig. 1D). Third, ectopic secretion of IFNa. Accordingly, the induction of RIG-G by ATRA is overexpression of wt STAT1 in U3A cells was unable to induce the reporter gene expression without IFNa, whereas mutant STAT1- Y701F remained responseless even under IFNa treatment (Fig. 1C), indicating that STAT1 activity depended on its phosphorylation. Taken together, data are currently convincing that STAT1 alone is not sufficient for the expression of RIG-G gene. The primary function of exogenous STAT1 transfected into U3A cells might be to restore the JAK-STAT pathways through which the downstream genes, including IRF-1,IRF-9,STAT2 , and RIG-G, can be directly or indirectly induced (Fig. 6). During the past decade, data on the putative roles of STAT proteins in mediating gene expression without tyrosine phosphorylation have been accumulating. For example, a complex of unphosphorylated STAT1 and IRF-1 was found to support the constitutive expression of low molecular mass polypeptide 2 gene coding for a subunit of 20S proteasome (15). Unphosphorylated STAT3 could drive overexpressions of certain oncogenes (such as M-RAS and MET), which contributed to cellular transformation (34). Similarly, unphosphorylated STAT6 was shown to be able to cooperate with p300 to enhance cyclooxygenase-2 gene expression in human non–small cell lung cancer (16). Here, we have shown for the first time that the unphosphorylated STAT2 could play an important role in RIG-G gene expression by interacting with IRF-9, further reinforcing the idea that STAT proteins could function as transcription factors in the absence of tyrosine phosphorylation. Interestingly, it was reported that unphosphorylated STAT2 was not static but dynamically shuttling between the nucleus and cytoplasm (31, 35, 36). The nuclear import of unphosphorylated STAT2 depended on its association with IRF-9, which contained a constitutive nuclear localization signal (35). These findings offer no doubt a prerequisite for the transactivation potential of unphosphorylated STAT2 on RIG-G expression. Another interesting question addressed by our investigation is how ATRA can induce RIG-G expression in NB4 cells. In addition that ATRA greatly increases the protein level of both STAT2 and Figure 6. Schematic illustration of signaling pathways on IFNa-or IRF-9, IFNa synthesis and secretion in NB4 cells after ATRA ATRA-induced RIG-G expression in NB4 cells. Through binding cell surface treatment for 72 hours and onward strongly suggests that ATRA- receptors [IFN receptor (IFNR)], IFNa activates classic JAK-STAT pathway, induced RIG-G may represent one of the key molecular nodes of which in turn leads to the formation of ISGF3 complex. The ISGF3 then translocates to the nucleus and initiates the transcription of IFNa target genes, cross-talk between ATRA and IFNa. This idea is supported by the such as IRF-1. IRF-1 can largely increase the expression of STAT2 and IRF-9, finding that the lack of IFNa synthesis in ATRA-resistant NB4-R1 which form a functional complex to drive the expression of RIG-G gene. The phosphorylation of STAT2 can greatly enhance the transactivities of the cells is exquisitely coincident with no induction of RIG-G by ATRA complex IRF-9/STAT2. In addition, IRF-1 itself can also recognize the ISRE in these cells (data not shown). Recently, some other cytokines sequences and directly bind RIG-G promoter to induce RIG-G gene expression. (such as tumor necrosis factor) or chemical drugs were also found ATRA has been generally known to exert its roles through their nuclear receptors. In NB4 cells, ATRA can induce IRF-1, which is a transcriptional to up-regulate several IFN-inducible gene family members activator of IFNa expression, suggesting that ATRA may induce the RIG-G (including RIG-G) through production of type I IFNs (37, 38), gene expression through an IFNa autocrine pathway. www.aacrjournals.org 3679 Cancer Res 2009; 69: (8). April 15, 2009

Cancer Research also related to IFNa (Fig. 6). Our work deciphered the signal Acknowledgments a cross-talk between ATRA and IFN in APL NB4 cells, but our Received 12/23/08; revised 2/4/09; accepted 2/16/09; published OnlineFirst 4/7/09. finding should have broader implications in cancer. We furnished Grant support: Chinese National Key Program for Basic Research 973, Chinese here an alternative pathway for the diversity of IFN signaling to National High Tech Program 863 (2006AA02Z19A), National Natural Science Foundation of China (30570778 and 30670882), ‘‘Shu Guang’’ Program of Shanghai mediate its multiple biological properties in normal and tumor Municipal Commission for Education (03SG37), and Samuel Waxman Cancer Research cells. Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Disclosure of Potential Conflicts of Interest We thank Dr. Lanotte for help in reviewing the manuscript and constructive discussion and suggestion and all members of the Shanghai Institute of Hematology No potential conflicts of interest were disclosed. for their support.

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Cancer Res 2009; 69: (8). April 15, 2009 3680 www.aacrjournals.org

IFR-9/STAT2 Functional Interaction Drives Retinoic Acid− Induced Gene G Expression Independently of STAT1

Ye-Jiang Lou, Xiao-Rong Pan, Pei-Min Jia, et al.

Cancer Res Published OnlineFirst April 7, 2009.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-08-4922

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