STAT1 is an indispensable component of a heterotrimer (ISGF3) and a STAT1 homodimer (GAF) that function as transcription regulators in type 1 and type 2 interferon signaling, respectively. To investigate the importance of STAT1-cooperative DNA binding, we generated gene-targeted mice expressing cooperativity-deficient STAT1 with alanine substituted for Phe77. Neither ISGF3 nor GAF bound DNA cooperatively in the STAT1F77A mouse strain, but type 1 and type 2 interferon responses were affected differently. Type 2 interferon–mediated transcription and antibacterial immunity essentially disappeared owing to defective promoter recruitment of GAF. In contrast, STAT1 recruitment to ISGF3 binding sites and type 1 interferon–dependent responses, including antiviral protection, remained intact. We conclude that STAT1 cooperativity is essential for its biological activity and underlies the cellular responses to type 2, but not type 1 interferon.
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We thank M. Mee, C. Pelzel and N. Wenta for immunoblot or EMSA results; N. Salhat for data on Listeria-infected macrophages; V. Ruppert and J. Staab for animal data; U. Kalinke (Universität Hannover) for VSV; L. Olohan (University of Liverpool) for microarray scanning; and K. Weber, E. Louis and I. Macdonald for manuscript review. Supported by University of Nottingham Pump Priming (U.V.), Deutsche Forschungsgemeinschaft (VI 218/3 to U.V. and ME 1648/4-1 to T.M.), Austrian Science Foundation (SFB28 to T.D.), Biotechnology and Biological Sciences Research Council (BB/G019290/1 to U.V. and BB/I532353/1 to M.B.), Pfizer (M.B.) and the Wellcome Trust (A.B.).
The authors declare no competing financial interests.
Integrated supplementary information
(a) Schematic diagram of the targeting construct used for homologous recombination in ES cells (top), the murine Stat1 gene locus encompassing exons 1-6 (second), a depiction of the targeted allele with insertion of a loxP-flanked neomycin gene (third), and the resulting Stat1 locus after Cre-induced deletion of the neomycin gene (bottom). The horizontal rectangle represents the position of probe P used for Southern blotting. (b) Southern blot analysis of genomic DNA after digestion with BamHI and hybridization with probe P. Homologous recombination with the targeting vector introduces an additional BamHI site that produces a 5.7 kB band representing the modified allele. (c) RFLP analyses using genomic DNA from wild-type (WT/WT), hetero- (WT/KI) and homozygous (KI/KI) STAT1F77A mice. Due to the loss of a Tsp509I site caused by mutation F77A, STAT1F77A-derived DNA showed a 234 bp fragment, which was cleaved into 110 bp and 120 bp fragments with wild-type-derived DNA. The undigested PCR amplicon (282 bp) is shown in lane 6; std, sizing standard. (d) Electropherograms showing DNA sequencing results for the non-coding strand from wild-type (WT/WT), hetero- (WT/KI) and homozygous (KI/KI) STAT1F77A animals.
Supplementary Figure 2 Type-2, but not type-1 IFN-stimulated NO production is defective in STAT1F77A-derived macrophages.
(a-c). Nitric oxide production of bone marrow-derived macrophages (BMMs) from wild-type (left) or STAT1F77A mice (right). Macrophages were seeded in 96-well plates at a density of 1 × 104 cells per well and kept in L cell-conditioned medium (10% L cell-supplemented DMEM with 10% calf serum). For the indicated times the medium was supplemented with different concentrations of IFN-γ (a) or IFN-α (b,c) alone or in combination with LPS. The concentrations of IFN and LPS are given in the keys. Nitrite concentrations in the culture supernatants were subsequently determined by Griess assay. Data are representative of two independent experiments done in duplicates
(a) Gene induction in immortalized MEFs after 6 h IFN-α (1000 U/ml) or IFN-γ (50 U/ml) as determined by qRT-PCR. (b) Validation of IFN-mediated gene repression. Cells were treated with IFNs for 6 h unless indicated otherwise. Note that Jun expression was below the 4-fold expression limit at the 6 h time point and hence was not detected in the microarray studies. For Pvrl4, qRT-PCR and microarray results (Supplementary Table 1) were contradictory. Bars represent the mean ± s.d. of three independent experiments. The 45 min time point for Jun was done once.
Gels show recruitment of complexes to promoters for Gapdh (a), Tgtp (b), Igtp (c), Socs1 (d), Cxcl10 (e), Irf9 (f), and AldoB (g), determined by ChIP analysis of nuclear preparations from immortalized wild-type (WT) and STAT1F77A MEFs (KI), before or after treatment with 1,000 U/ml IFN-α or 50 U/ml IFN-γ for 45 min. Anti-STAT1 (S1), anti-STAT2 (S2), anti-IRF9 (IRF9), anti-acetylated histone 3 (Ac-H3), or anti-unspecific IgG (IgG) was used to immunoprecipitate (IP) the complexes, followed by PCR with primers designed to detect the promoters. The position and configuration of GAS and ISRE in each promoter sequence are indicated below the gels. bp, base pairs. Data are representative of one (g) or at least two independent experiments.
Gels show recruitment of complexes to promoters for Pvalb (a), Npas (b), Pvrl4 (c), and Jun (d), determined by ChIP analysis of nuclear preparations from immortalized wild-type (WT) and STAT1F77A MEFs (KI), before or after treatment with 1,000 U/ml IFN-α or 50 U/ml IFN-γ for 45 min. Anti-STAT1 (S1), or anti-STAT2 (S2) was used to immunoprecipitate (IP) the complexes, followed by PCR with primers designed to detect the promoters. The position and configuration of GAS and ISRE in each promoter sequence are indicated below the gels. bp, base pairs. Data are representative of one (a) or at least two independent experiments.
(a) Sequence logo with a graphical representation of the binding preferences of STAT1 as derived from ChIP-seq data28 Letter sizes indicate the frequency of the respective bases in the binding sequences. (b) Representative examples of sequences obtaining the corresponding PWM scores.
(a) Results of mathematical modeling of STAT1-DNA binding using the ‘single GAS polymer model’. Promoter recruitment was tested over a range of different affinities of STAT1 for non-GAS sites (key). The solid lines depict results for high cooperativity (Kd = 0.0017). For comparison, the dotted line shows the result for low cooperativity (Kd = 0.1) and a 50-fold difference in GAS to non-GAS STAT-DNA binding affinity, as displayed in Figure 6a. As the affinity of STAT1 for non-GAS sites decreases, the curves shift to the right of the plot, indicating that a greater STAT1 concentration would be needed to achieve the same level of recruitment. However, the dotted line shows that cooperativity facilitated promoter recruitment at all binding affinities tested. (b) Results of mathematical modeling of STAT1 DNA binding in a tetramer model. This model operates with two binding sites, either two GAS sites (blue), or a GAS and a non-GAS site (red). These configurations can be compared to the black lines (key) that show STAT1 recruitment to a single GAS site in the more realistic ‘single GAS polymer model’ as displayed in Figure 6a. The curves are shifted to the right slightly in the ‘single GAS polymer model’, indicating that the tetrameric model would overestimate the STAT1 concentration needed to achieve any particular level of STAT1 recruitment.
(a) Wide-field microscopy images of HeLa cells expressing the wild-type STAT1 or STAT1-NES. Shown is the GFP fluorescence, nuclei were stained with Hoechst dye. In unstimulated cells, STAT1-NES localizes predominantly in the cytoplasm (i). Addition of 10 ng/ml NES inactivatorleptomycin B (LMB, Sigma)50 had little effect on wild-type, but caused STAT1-NES to adopt a pancellular distribution (ii). Treatment with IFN-γ (50 U/ml) for 60 min expectedly resulted in nuclear accumulation of wild-type, but did not visibly change the distribution of STAT1-NES (iii). However, IFN-γ-induced nuclear accumulation of STAT1-NES occurred upon NES inactivation by LMB (iv), indicating that its nuclear import was functioning. Next, phosphatase inhibitor vanadate (0.8 mM)32 was used together with IFN-γ (v), which resulted in the nuclear accumulation of wild-type STAT1, demonstrating that vanadate did not inhibit STAT1 nuclear import. Vanadate, on the other hand, did not trigger nuclear accumulation of STAT1-NES in the IFN-γ-treated cells (v). A possible explanation for this outcome could have been that vanadate inhibited nuclear import. However, this was ruled out since wild-type STAT1 was imported normally in the presence of vanadate. As STAT1-NES remained predominantly cytoplasmic in the vanadate-treated cells, it was concluded furthermore that vanadate did not prevent the nuclear export of STAT1-NES either. Thus, vanadate did not preclude nuclear import and export of activated STAT1-NES. Since nuclear accumulation was not observed in the presence of phosphatase inhibitor vanadate, we concluded that NES-fusion enabled STAT1 to exit from the nucleus in the tyrosine-phosphorylated state.To test this hypothesis, the NES was inactivated in cells treated with both IFN-γ and vanadate (vi), which resulted in the indistinguishable nuclear accumulation of wild-type STAT1 and the NES fusion variant. Together, these results indicated that tyrosine phosphorylation was not sufficient for nuclear retention of STAT1-NES, contrary to wild-type STAT1 (ref. 30). (b) Immunoblot analysis of STAT1 phosphorylated at Tyr701 (pS1Y) and total STAT1 (S1) in HeLa cells transfected with GFP-tagged wild-type STAT1 or STAT1-NES. The cells were treated with 50 U/ml IFN-γ for the times indicated. (c) Representative quantitative confocal microscopy images of optical slices through the median of HeLa cells before (US) and after 1 h stimulation with IFN-γ (50 U/ml) or IFN-α (1000 U/ml). The cells expressed GFP-tagged STAT1-NES (WT) or the indicated mutants; amino acid changes are listed. Shown is the GFP fluorescence intensity in a color-coded rendering based on the scale shown at the bottom. Data are representative of three or more independent experiments.
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Begitt, A., Droescher, M., Meyer, T. et al. STAT1-cooperative DNA binding distinguishes type 1 from type 2 interferon signaling. Nat Immunol 15, 168–176 (2014). https://doi.org/10.1038/ni.2794
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