Functional Relevance of the Conserved DNA-binding Domain of STAT2*

Several distinct type I interferon (IFN)-inducible STAT2-containing complexes have been identified. For the IFN-stimulated gene factor 3 (ISGF3), STAT1 and IRF-9 mediate IFN-stimulated response element (ISRE) binding, whereas STAT2 provides a potent transactivational domain. ISGF3-independent STAT2-containing complexes, specifically STAT2:1 and STAT2:3, bind a γ-activated sequence (GAS)-like element, yet the contribution of each STAT to DNA binding is unknown. Moreover, the contribution of these ISGF3-independent STAT2-containing complexes to IFN-inducible responses is not defined. Accordingly, we generated mutant cDNAs, targeting the DNA-binding domain in STAT2. These cDNAs were introduced by transfection into U6A cells lacking STAT2, resulting in a panel of cell lines expressing mutant STAT2 proteins. Studies assessed the sensitivity of U6A cells reconstituted with intact STAT2 (U6A-2) and cells expressing mutant STAT2s (U6A-2E426A,E427A (EE-AA), U6A-2V453I, U6A-2V454I, U6A-2V454A, U6A-2V453I,V454I(VV-II), U6A-2N458A) to IFN-inducible responses. Our data reveal that none of the mutations in the STAT2 DNA-binding domain affected IFN-inducible ISGF3 activation, and only the VV-II mutation restricted antiviral and growth inhibitory responses to IFN. Indeed, U6A-2VV-II cells are refractory to these IFN-inducible biological activities and also exhibit impaired IFN-inducible GAS-driven transcriptional activation and subsequent gene expression. Chromatin immunoprecipitation assays revealed that the VV-II mutation in STAT2 does not abrogate, but reduces the DNA binding activity of STAT2:1 heterodimers. Taken together, these data suggest a role for the conserved DNA-binding domain of STAT2 specific to the activity of ISGF3-independent STAT2-containing complexes.

Within the ISGF3 complex, STAT2 contributes its potent transcriptional activation domain, whereas STAT1 and IRF-9 mediate DNA binding (17)(18)(19). The carboxyl-terminal transactivation domain of STAT2 also contributes to the transcriptional activation potential of ISGF3-independent complexes (10). However, the role of STAT2 in ISGF3-independent STAT2:1 heterodimers is not fully defined and the contribution of each STAT protein to DNA binding remains unknown.
In the present study we provide evidence that IFN-inducible ISGF3-independent STAT2-containing complexes contribute to IFN-inducible biologic responses in target cells. Specifically, we show that in cells where IFN-inducible ISGF3 activation is intact, it is possible to disrupt IFN-inducible transcriptional activation and ISG expression mediated by GAS-like gene elements, effected by STAT2:1-DNA interactions, resulting in blunted antiviral and antiproliferative responses. Using a panel of STAT2 mutants, we provide evidence that specific residues in the putative DNA-binding domain of STAT2 influence STAT2:1-DNA binding activity and transcriptional activation of ISGs.
Cell Lysis and Immunoblotting-Cells were treated with 5 ng/ml IFN alfacon-1 for 15 min and lysed in phosphorylation lysis buffer as previously described (25). Immunoblotting using an enhanced chemiluminescence was performed as previously described (25). Antiviral Assays-The assay for IFN-inducible antiviral activity against encephalomyocarditis virus (EMCV) has been described. Briefly, cells were either left untreated or treated with the indicated concentrations of IFN alfacon-1 for 16 h, then challenged with EMCV for 24 h. The extent of the viral cytopathic effect is determined using a crystal violet colorimetric assay (26).
Cell Proliferation Assays-Cells were seeded in flat bottom 96-well plates at a concentration of 2 ϫ 10 5 cells/ml in the presence or absence of the indicated concentrations of IFN alfacon-1 and were incubated at 37°C for 72 h. Cell proliferation was assessed as previously described (27).
Luciferase Gene Reporter Assay-Cells were transfected with a ␤-galactosidase expression vector and either an ISRE luciferase construct (28) or a luciferase reporter gene containing eight GAS elements (8ϫGAS) (29) as previously described (30).
Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSAs)-Cells were concentrated to 2 ϫ 10 7 and treated with 5 ng/ml IFN alfacon-1 for 15 min. Nuclear extracts were prepared as previously described (9). 10 g of nuclear protein from untreated and IFN alfacon-1-treated cells were analyzed using EMSAs as previously described (9). For supershift experiments, 2 g of antibody was incubated with extracts for 30 min at 4°C prior to addition of DNA.
Chromatin Immunoprecipitation (ChIP) Assay-Cells were un-treated or treated with 5 ng/ml IFN alfacon-1 for 30 min. ChIP assays were performed according to the manufacturer's protocol (Upstate). 1 l of input material (collected prior to addition of antibody) and 5 l of output material (purified product of ChIP) was analyzed by PCR using primers to the 6 -16 promoter (ISRE (33)), to the IRF-1 promoter (GASlike pIRE) and to the ␤-actin gene (control). The following primers were used: 6 -16 (5Ј-GGTGAAAGGCCTGTGTGCC and 5Ј-CAGCGAGTAAA-CGGTTCTCCG), IRF-1 (5Ј-GGACAAGGCGGAGTGAGAGGACA and 5Ј-TTGCCTCGACTAAGGAGTGGC), ␤-actin (5Ј-CGTGGCCATCTCTT-GCTCGAAGT and 5Ј-ACTGGCATCGTGATGGACTCCGGT). Genomic DNA harvested from 2fTGH cells and water was used as positive and negative PCR controls, respectively. PCR products were resolved in a 1.2% agarose gel and visualized using a Gel Doc apparatus (Bio-Rad). RNA Preparation, Complementary DNA Synthesis, and Real-time PCR-To harvest RNA, cells were either left untreated or treated with 5 ng/ml IFN alfacon-1 for 6 h at 37°C. Cells were then lysed and homogenized using Qiagen QIA-shredder columns and RNA isolation was performed using the Qiagen RNeasy mini kit according to the manufacturer's protocol. The complementary DNA (cDNA) was synthesized using 1 g of RNA in the presence of random primers and avian myeloblastosis virus reverse transcriptase for 1 h at 42°C (Promega).
Reaction components for real-time PCR were obtained from the LightCycler® FastStart Plus DNA Master SYBR Green I kit (Roche) and the LightCylcer® instrument (Roche) and Relative Quantification Software were used for all reactions. The PCR was performed in a final volume of 20 l containing 0.5 M of each primer and 5 l of template cDNA (concentration 100 ng/l). The following primer sets were employed: 6 -16 (5Ј-TAAGAAAAAGTGCTCGGAGAGCTC and 5Ј-CCGAC-GGCCATGAAGGT), ISG15 (5Ј-TCCTGGTGAGGAATAACAAGGG and 5Ј-CTCAGCCAGAACAGGTCGTC), IRF-1 (5Ј-CTTCCACCTCTCACCA-AGAAC and 5Ј-CCATCAGAGAAGGTATCAGGGC), and ␤-actin (5Ј-C-GTGGCCATCTCTTGCTCGAAGT and 5Ј-ACTGGCATCGTGATGGAC-TCCGGT). Standard curves were established for each primer set and both reference (␤-actin) and target reactions were performed for each sample.

Mutational Analysis of Conserved DNA-binding Domain of STAT2-
The DNA binding activity of STAT1 has been characterized and mutagenesis studies have identified residues in STAT1 that are critical for DNA binding activity (22,23,34). We undertook a comprehensive analysis of the crystal structure of STAT1 bound to DNA, using molecular modeling and visualization software (SYBYL, version 6.9, Tripos Inc.). Using SYBYL we imported the STAT1 RCSB Protein Data Bank coordinates (Protein Data Bank code 1BF5) and rendered the protein to highlight the DNA-binding domain and examine key residues involved in mediating DNA binding. This approach allowed us to identify several residues that appear crucial for mediating DNA binding and for maintaining the structure of the DNA-binding domain (Fig. 1). Specifically, Glu 429 lies in the long loop between ␤8 and ␤9, that interacts with the minor groove and makes phosphate contacts with the major groove of the DNA molecule. This residue forms a hydrogen bond with His 431 , an interaction that contributes to the stability of this region. Similarly, Glu 428 stabilizes this area by forming a hydrogen bond back onto itself. Another key segment is the region between ␤-sheet 11 and ␣-helix 6 wherein Asn 460 is located. Asn 460 directly forms a hydrogen bond to DNA and mediates specific recognition of the DNA sequence. Residues Val 455 -Val 456 are also important for DNA binding as they lie within ␤-sheet 11 and provide stability for the loop containing Asn 460 .
Each of these residues is conserved in STAT2 and occupies positions Glu 426 -Glu 427 , Val 453 -Val 454 , and Asn 458 . Based on crystal structure analysis, we infer that substitution of Glu 426 -Glu 427 to alanines (AA) may disrupt DNA binding by reducing the rigidity and support of DNA-contacting loops. This mutation may abolish hydrogen bonds stabilizing the loop between ␤8 and ␤9. Additionally, mutation of one or both Val 453 -Val 454 to isoleucines (II) may alter the structure of ␤-sheet 11, destabilizing the loop between ␤-sheet 11 and ␣-helix 6 and perhaps preventing Asn 458 from making direct contact with the DNA molecule. A single Val 454 to Ala mutation targets the second, more critical residue of this VV motif. Also, mutation of Asn 458 to Ala may have a detrimental effect on DNA binding by impairing specific STAT-DNA interactions.
Therefore, to assess the function of the conserved putative DNA-binding domain of STAT2, the following mutations were introduced into the STAT2 protein: E426A,E427A (EE-AA), V453I, V454I, V454A, V453I,V454I (VV-II), and N458A (Table  I). cDNAs encoding the STAT2 mutants were introduced by stable transfection into U6A cells lacking STAT2 to generate a panel of cell lines expressing these mutant STAT2 proteins. A cDNA construct encoding intact STAT2 was also stably introduced into U6A cells, generating the U6A-2 cell line.
IFN Alfacon-1 Treatment of U6A-2 Cells Induces GAS-driven Luciferase Activity-In earlier studies, we reported that treat-ment of cells with 5 ng/ml IFN alfacon-1 resulted in ISGF3independent STAT2-containing complexes (9,12). To establish a dosage appropriate for further studies, we treated U6A cells reconstituted with intact STAT2 (U6A-2) with varying doses of IFN alfacon-1 (data not shown). The data show that 0.5 ng/ml is sufficient for maximal levels of IFN-inducible STAT2 tyrosine phosphorylation. Dose-response studies for IFN-inducible GAS-mediated transcriptional activation were performed and the results indicate a dose-dependent increase in luciferase activity. Indeed, a dose of 5 ng/ml IFN alfacon-1 is required to achieve optimal GAS-driven transcriptional activity in U6A-2 cells (data not shown). Accordingly, to assess the relevance of IFN-inducible, GAS-binding ISGF3-independent STAT2-containing complexes, a dose of 5 ng/ml IFN was routinely used in all subsequent experiments.
Mutations in the STAT2 DNA-binding Domain Do Not Disrupt IFN-inducible STAT Tyrosine Phosphorylation-Following introduction of the STAT2 cDNA constructs into U6A cells, Western blot analysis was performed to determine cellular levels of STAT proteins. All transfectants express comparable levels of both STAT1 and STAT2, with the exception of cells expressing the N458A form of STAT2, in which STAT2 levels are lower (Fig. 2). To confirm that the mutations introduced into STAT2 have not affected the ability of STAT2 to be inducibly phosphorylated upon IFN stimulation, lysates of either untreated or IFN-treated transfectants were prepared. Lysates from IFN-treated cells were resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted with an antibody against the phosphorylated/activated form of STAT2. The data reveal that mutations in the putative DNA-binding domain of STAT2 do not interfere with IFN-inducible tyrosine phosphorylation of STAT2 ( Fig. 2A).
The extent of IFN-inducible STAT1 phosphorylation was also examined in the different transfectants. Studies have demonstrated that STAT2 is required for IFN-induced STAT1 tyrosine phosphorylation, suggesting there is a sequential activation of STAT proteins at the IFN receptor (7,17). However, STAT1 activation by IFN has been detected in STAT2-deficient cells (35). Therefore, Western immunoblot analysis was performed to determine whether the mutations in the DNA-binding domain of STAT2 affected the ability of STAT1 to be tyrosine phosphorylated upon IFN stimulation (Fig. 2B). IFNinduced STAT1 tyrosine phosphorylation was detected in all transfectants, albeit more weakly in cells expressing the N458A STAT2 mutant. Interestingly, in U6A cells lacking STAT2, the extent of STAT1 tyrosine phosphorylation was similar to that observed in other cell types.   (Fig. 3A). By contrast, cells expressing intact STAT2 respond to IFN with the appropriate antiviral response. U6A-2EE-AA, U6A-2V453I, U6A-2V454I, U6A-2V454A, and U6A-2N458A cells are also able to mount a full antiviral response upon IFN stimulation (Fig. 3A). However, cells expressing the VV-II STAT2 mutant fail to mount a robust IFN-inducible antiviral response (Fig. 3A). Transfectants were also examined for IFN-inducible growth inhibitory responses using the cell proliferation assay. STAT2deficient U6A cells do not exhibit a growth inhibitory response to IFN (Fig. 3B). In contrast, IFN treatment of U6A-2 cells expressing intact STAT2 results in growth inhibition (Fig. 3B). Cells expressing the EE-AA, V453I, V454I, V454A, and N458A mutant forms of STAT2, likewise exhibit normal levels of IFNinducible growth inhibition (Fig. 3B). Similar to our results in the antiviral assays, cells expressing the VV-II STAT2 mutant are impaired in their ability to respond to the growth inhibitory effects of IFN (Fig. 3B). Together, these data suggest that the VV-II mutation disrupts STAT2-mediated signal transduction such that IFN-inducible antiviral and growth inhibitory responses are diminished.

Cells Expressing the VV-II STAT2 Mutant Exhibit Diminished IFN-inducible Responses-To
Cells Expressing the VV-II Mutant Form of STAT2 Exhibit Diminished IFN-inducible GAS-dependent Luciferase Activity- To examine IFN-inducible transcriptional activation in cells expressing the mutant STAT2 proteins, ISRE-and GAS-luciferase gene reporter assays were conducted. ISRE-luciferase or 8ϫGAS-luciferase constructs were introduced into the different cell types by transfection. Following a 6-h 5 ng/ml IFN alfacon-1 treatment, luciferase activity was measured. In U6A cells, IFN-stimulated STAT2-dependent luciferase activity mediated by ISRE or GAS elements was absent (Figs. 3, C and D). In cells expressing either the intact or any of the mutant forms of STAT2, IFN-inducible ISRE-mediated luciferase activity was detected (Fig. 3C). In cells expressing the various mutant forms of STAT2, except U6A-2VV-II, IFN-inducible GASdriven luciferase activity was detected (Fig. 3D). However, IFN-stimulated luciferase activity mediated by the 8ϫGAS element was significantly reduced in cells expressing the VV-II mutant form of STAT2.

Mutations in the Putative DNA-binding Domain of STAT2 Do Not Affect IFN-inducible STAT Complex Formation or DNA
Binding in Vitro-To examine whether the STAT2 mutations interfere with STAT complex formation and DNA binding, a series of EMSAs were performed. Data obtained from antiviral, growth inhibitory and luciferase gene reporter assays suggested only the VV-II mutation in STAT2 disrupted IFN signaling. Therefore, we analyzed IFN-induced STAT complex formation and DNA binding in U6A-2VV-II cells and in two transfectants that respond normally to IFN, namely U6A-2EE-AA and U6A-2V454A cells. At the outset, we conducted EMSAs using an ISRE probe. Within the ISGF3 complex, STAT2 associates with STAT1 via its phosphotyrosine-Src homology 2 domain and with IRF-9 by means of its coiled-coil domain, residues 138 -230 (36). Although we showed that our mutations to STAT2 did not affect IFN-inducible tyrosine phosphorylation of STAT1 or STAT2 (Fig.  2), we undertook experiments to examine the effects of these residue changes in STAT2 on the formation of ISGF3 complexes and their ability to bind ISRE. Cells were left untreated or treated with IFN alfacon-1 for 15 min. Nuclear extracts were prepared, incubated with an ISRE, and resolved by PAGE (Fig.  4A). The result in Fig. 4A shows that, as expected, IFN treatment of U6A cells lacking STAT2 fails to result in ISGF3 formation. In all cells expressing STAT2, either intact or mutant forms, IFNinducible ISGF3-ISRE complex formation is detectable. Supershifting experiments using antibodies against either STAT2 or IRF-9 confirmed the composition of the ISGF3 complex. Notably, the extent of ISGF3 complexed to ISRE varies among the differ-

FIG. 4. Chromatin DNA binding activity of IFN-inducible STAT2:1 heterodimers is reduced in cells expressing VV-II STAT2.
A, nuclear extracts from cells either left untreated (Ϫ) or treated (ϩ) for 15 min with 5 ng/ml IFN alfacon-1 were incubated with 32 P-labeled ISRE element probe. Untreated and IFNtreated nuclear extracts from human fibrosarcoma HT-1080 cells were used as a positive control. Complexes were resolved by native gel electrophoresis and visualized by autoradiography. B, similar to A, but extracts were incubated with 32 P-labeled pIRE probe to assess IFN-inducible ISGF3-independent STAT2:1 heterodimer formation and DNA binding. C, chromatin binding activity of complexes containing either intact STAT2 or the VV-II STAT2. Cells were untreated (Ϫ) or treated (ϩ) with 5 ng/ml IFN alfacon-1 for 30 min. Following cell lysis and DNA sonication, aliquots were collected and used as input samples. ChIPs were then performed when anti-STAT2 antibody was either added or omitted, as indicated. The precipitated chromatin was analyzed using primers specific for a 6 -16 ISRE and an IRF-1 GAS. Primers for ␤-actin were used to confirm the immunoprecipitation is specific for STAT2. D, the signal intensity of each band was determined. Histograms representing signal intensity ratios of each ChIP sample band to its corresponding input band for the 6 -16 and IRF-1 primer sets are provided. Data are representative of three independent experiments. ent transfectants. IFN treatment of cells expressing the EE-AA and V454A mutant forms of STAT2 results in levels of ISGF3-ISRE comparable with that for IFN-treated U6A cells expressing intact STAT2. IFN stimulation of cells expressing the VV-II STAT2 mutant results in lower levels of activated ISGF3.
We next examined the formation and DNA binding activity of ISGF3-independent STAT2:1 heterodimers. Using a PCRassisted binding site selection procedure, we have identified a pIRE, a GAS-like element containing the STAT binding consensus sequence TTCCCGGAA, as the preferential binding site for STAT2:1 heterodimers (12). Both STAT1:1 and STAT2:1 complexes will bind this DNA sequence. The results in Fig. 4B show that IFN treatment of U6A cells leads to STAT1:1 formation and DNA binding to this pIRE. The constituents of the IFN-induced DNA-binding complex were confirmed using anti-STAT1 antibodies in supershifting experiments (Fig. 4B). IFN treatment of 2fTGH and U6A-2 cells, both expressing intact STAT2, results in STAT1:1 and STAT2:1 complex formation and pIRE binding (Fig. 4B). Although the anti-STAT2 antibody was unable to supershift the STAT2:1 complex, an anti-STAT1 antibody affected the mobility of both the IFN-induced STAT1: 1-pIRE and STAT2:1-pIRE complexes. Because the anti-STAT2 antibody was able to associate with STAT2 in the ISGF3 and function as a supershifting antibody, we infer that the association of STAT2 with STAT1 and its interaction with DNA is distinct between ISGF3 and STAT2:1 complexes. Further evidence for the identity of the STAT2:1-pIRE complex is its absence in the EMSA of cell extracts from IFN-treated U6A cells. IFN treatment of cells expressing various mutant forms of STAT2 resulted in the formation of both STAT1:1-pIRE and STAT2:1-pIRE complexes (Fig. 4B), suggesting that the mutations in STAT2 have not altered the ability of ISGF3-independent STAT2-containing heterodimers to bind DNA. In all transfectants, the levels of STAT1:1 and STAT2:1 complexes are comparable. Notably, in cells expressing the VV-II STAT2 mutant, the pIRE binding activity of IFN-induced STAT2:1 complexes is intact.
The VV-II STAT2 Mutant Reduces the DNA Binding Activity of STAT2-containing Heterodimers to GAS Elements-To examine the DNA binding activity of IFN-inducible STAT2-containing complexes in the appropriate cellular context, ChIP analysis was performed. To assess ISGF3 DNA binding, a primer set specific for an ISRE-containing sequence of the ISG 6 -16 promoter was used. To examine pIRE interactions, primers were designed to amplify a region of the endogenous IRF-1 promoter containing this GAS-like element. Although not identical to the preferred STAT2:1 pIRE sequence, this element differs by only one base (TTCCCCGAA) and has been shown to bind the STAT2:1 heterodimer in vitro (10).
Cells expressing the EE-AA, V453I, V454I, V454A, and N458A mutant forms of STAT2 respond normally to IFN-inducible STAT activation, STAT-mediated transcriptional activity, and antiviral and antiproliferative responses. Consequently, these cells were not analyzed in ChIP assays. IFN treatment of 2fTGH and U6A-2 cells expressing intact STAT2 resulted in ISGF3-DNA binding on chromatin (Fig. 4C). Although ISGF3 activation measured by in vitro EMSA analysis suggested that the VV-II mutation in STAT2 affected IFNinducible ISGF3 levels, the ChIP results, representative of contextual transcription factor binding to DNA, indicate that ISGF3 levels and DNA binding are unaffected in the U6A-2VV-II cells. IFN-inducible STAT2:1-GAS binding on chromatin DNA was also detected (Fig. 4C). Notably, both of these complexes are formed following IFN treatment of cells carrying the VV-II mutant form of STAT2 (Fig. 4C). Compared with cells expressing intact STAT2, lower levels of STAT2:1-GAS binding on chromatin are observed in U6A-2VV-II cells (Fig. 4D). Thus, whereas the VV-II mutation in STAT2 does not inhibit DNA binding of ISGF3 to ISRE elements on chromatin, there is an effect on the DNA binding activity of STAT2-containing heterodimers to GAS elements on chromatin.
The VV-II STAT2 Mutant Reduces the IFN-inducible Expression of the GAS-mediated IRF-1 Gene-To determine whether cells expressing the VV-II STAT2 mutant are able to induce ISG expression following treatment with IFN, a series of quantitative real-time PCR was performed. Following a 6-h 5 ng/ml IFN alfacon-1 treatment, ISRE-mediated gene expression was assessed using primers specific for the 6 -16 and ISG15 genes and GAS-driven gene expression was examined using primers designed for the IRF-1 gene. The data show that, upon IFN treatment, cells expressing the VV-II mutant form of STAT2 induce comparable levels of 6 -16 and ISG15 gene expression compared with IFN-treated U6A-2 cells (Fig. 5). In U6A cells, as expected, IFN-induced ISRE-mediated gene expression is abrogated (Fig. 5). IFN treatment of U6A cells induced low levels of IRF-1 gene expression, equivalent to a 1.3-fold induction. In U6A-2 cells a 4-fold induction of IRF-1 was observed following IFN treatment. We infer that this IRF-1 gene expression is regulated by STAT1 and STAT3 homo-and heterodimers, and by STAT2:1. Notably, only a 2.1-fold induction was reported in U6A-2VV-II cells, likely because of the absence of appropriate STAT2-STAT1 DNA interactions. Thus, in addition to reducing the DNA binding activity of STAT2-containing heterodimers to GAS elements (Fig. 4D), the VV-II mutation in STAT2 also has an effect on IFN-inducible GAS-driven gene expression. DISCUSSION IFN-␣ activation of STAT2 is pivotal for an IFN response in target cells. Considerable attention has focused on STAT2 in the context of ISGF3 complexes (7,8,17), yet little is understood about the role of STAT2 in ISGF3-independent complexes. Although STAT2 contains a conserved 170-residue putative DNA-binding domain, the functional activity of this domain has not been examined until now. In this report we provide evidence for IFN-activated STAT2 contributing to IFNinducible biological responses that are independent of ISGF3 activity. Specifically, our mutagenesis studies suggest that STAT2 forms a heterodimer with STAT1 that will bind to chromatin and determine transcriptional activation that contributes to IFN-inducible biological responses.
Our findings with the U6A cells that lack STAT2 indicate that STAT2 expression is a critical determinant for IFN responsiveness in target cells. In the absence of STAT2, cells are unresponsive to the antiviral and growth inhibitory effects of IFN (Fig. 3, A and B). IFN-inducible ISGF3 formation, DNA binding, and transcriptional activation were unaffected in cells expressing any of the STAT2 proteins carrying mutations in their DNA-binding domain. Moreover, these STAT2 mutations did not interfere with the formation of IFN-inducible ISGF3independent STAT2:1 heterodimers. Notably, the mutations that we introduced into STAT2 did not fully abrogate STAT2: 1-DNA interactions as gauged by EMSA or ChIP assay. A single mutant STAT2 carrying the VV-II mutation affected a decrease in IFN-inducible STAT2-GAS binding on chromatin, resulting in diminished GAS-driven transcriptional activation that was reflected in blunted antiviral and growth inhibitory responses to IFN. This STAT2 mutation reduced GAS-mediated gene expression of the IRF-1 gene. Notably, U6A-2VV-II cells exhibit diminished IFN-stimulated IRF-1 gene expression compared with U6A-2 cells. In U6A cells we also observed minimal IFN-induced IRF-1 expression. Studies have determined that, in addition to STAT2:1 heterodimers, both STAT1:1 and STAT3:3 complexes can bind the GAS-like element found within the promoter of the IRF-1 gene (9, 10). Our finding therefore suggests that although STAT1 and STAT3 homodimers may contribute to its transcriptional activation, STAT2 is required for full IRF-1 expression.
Analysis of the crystal structure of STAT1 bound to DNA identified valines 453 and 454 in STAT2 as important for mediating DNA binding (23,24). These VV residues do not mediate direct DNA contact, but provide structural support for the DNA-binding loop carrying Asn 458 . The Val to Ile mutations were designed to be structurally disruptive by virtue of the introduction of a larger aliphatic side chain. As observed, single mutations, V453I or V454I, were not sufficient to disturb the function of this region of the STAT2 protein in the context of DNA binding or transcriptional activation. The double VV-II mutation significantly impaired the ability of STAT2, within ISGF3-independent STAT2-containing complexes, to mediate IFN-inducible responses. Although the EMSA results do not suggest a difference in the STAT2:1-GAS DNA binding for the VV-II mutant compared with intact STAT2 (Fig. 4B), the ChIP data identify that binding of IFN-inducible STAT2-containing complexes that contain the VV-II mutant STAT2 to GAS elements on chromatin, is reduced compared with complexes containing intact STAT2 (Fig. 4D). Moreover, the VV-II mutation in STAT2 affected IFN-inducible GAS-driven transcriptional activation, in further support that this region of STAT2 is important for DNA interactions. The implications are that STAT2:1 complexes interact with chromatin DNA in a manner distinct from their interaction with the minimal pIRE in an EMSA, and that there is a region in STAT2, encompassing residues in the DNA-binding loop associated with Asn 458 , that is critical for these STAT-DNA interactions.
Studies examining the DNA binding activity of STATs have demonstrated that particular residues in the DNA-binding domain of different STAT proteins may have distinct roles. For instance, residues Glu 434 and Glu 435 are critical for STAT3-DNA binding but are non-essential for the DNA binding activity of STAT5b (20,37). Mutation of Val 461 -Val 462 -Val 463 to AAA in STAT3 disrupts DNA binding but does not have an effect on nuclear localization (20). However, alanine substitutions of the corresponding residues in STAT5b (V466I,V467I,V468I,V469I) prevent DNA binding and also abrogate growth hormone-induced nuclear import of the STAT5 complexes (21). A recent study suggested that although STAT-DNA binding activity regulates nuclear accumulation, it is not an absolute requirement for entry into the nucleus (34). Indeed, mutations in STAT1 (E428A, E429A, and N460A) abolish DNA binding but do not affect STAT1 nuclear translocation (38,39). Our data suggest that the VV-II mutation in STAT2 did not affect STAT2 nucleocytoplasmic transport. In the ISRE and pIRE EMSA series, nuclear extracts were used, indicating that entry of STAT2-containing complexes into the nucleus was intact in all cell types, including cells expressing the VV-II STAT2 mutant. ChIP data confirmed that IFN-inducible STAT2-containing complexes carrying the VV-II mutation enter the nucleus and bind chromatin. Moreover, we have evidence that nuclear import is unaffected by the VV-II mutation. 2 In the ISGF3 complex, STAT2 contributes its potent transactivation domain and thus has an important role in mediating transcription (17,18). The carboxyl-terminal STAT2 transactivation domain interacts with a number of different co-factors and mediators, including CBP/p300 and GCN5, which are required for ISRE-mediated transcriptional activation (19,40). Based on the structure of STAT proteins, it is unlikely that a disruption in the DNA-binding domain would prevent the STAT protein from interacting with co-activators. Indeed, we observed that in cells expressing the VV-II mutant form of STAT2, IFN-inducible ISRE-driven transcription and gene expression were intact, confirming this mutation did not affect transcriptional activation by restricting interactions with cofactors. We infer that it is unlikely that the VV-II mutation in STAT2 affected the transactivation domain and attribute the reduced GAS-mediated transcriptional activation and gene expression to the decreased binding we observed to GAS elements on chromatin.
These data establish that the putative DNA-binding domain of STAT2 has functional relevance and its role is specific to the activity of ISGF3-independent STAT2-containing complexes. Analysis of the crystal structure of STAT1 homodimer bound to DNA revealed that both STAT molecules contact DNA via their DNA-binding domains (20,21). In agreement with these data, our findings suggest that in STAT2:1 heterodimers, STAT2 will bind DNA. Indeed, the data suggest that, within ISGF3-independent STAT2-containing complexes, the function of this putative DNA-binding domain of STAT2 is to mediate interactions with GAS-like elements, driving the transcriptional activation of a subset of ISGs. The challenge of our ongoing experiments is to distinguish the contribution of this subset of ISGs to IFN-inducible responses.