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. 2014 Jun;34(12):2235-48.
doi: 10.1128/MCB.00295-14. Epub 2014 Apr 7.

STAT1β is not dominant negative and is capable of contributing to gamma interferon-dependent innate immunity

Christian Semper  1 , Nicole R Leitner  1 , Caroline Lassnig  2 , Matthias Parrini  1 , Tanel Mahlakõiv  3 , Michael Rammerstorfer  1 , Karin Lorenz  1 , Doris Rigler  1 , Simone Müller  1 , Thomas Kolbe  4 , Claus Vogl  1 , Thomas Rülicke  5 , Peter Staeheli  6 , Thomas Decker  7 , Mathias Müller  2 , Birgit Strobl  8
Affiliations

STAT1β is not dominant negative and is capable of contributing to gamma interferon-dependent innate immunity

Christian Semper et al. Mol Cell Biol. 2014 Jun.

Abstract

The transcription factor STAT1 is essential for interferon (IFN)-mediated immunity in humans and mice. STAT1 function is tightly regulated, and both loss- and gain-of-function mutations result in severe immune diseases. The two alternatively spliced isoforms, STAT1α and STAT1β, differ with regard to a C-terminal transactivation domain, which is absent in STAT1β. STAT1β is considered to be transcriptionally inactive and to be a competitive inhibitor of STAT1α. To investigate the functions of the STAT1 isoforms in vivo, we generated mice deficient for either STAT1α or STAT1β. As expected, the functions of STAT1α and STAT1β in IFN-α/β- and IFN-λ-dependent antiviral activity are largely redundant. In contrast to the current dogma, however, we found that STAT1β is transcriptionally active in response to IFN-γ. In the absence of STAT1α, STAT1β shows more prolonged IFN-γ-induced phosphorylation and promoter binding. Both isoforms mediate protective, IFN-γ-dependent immunity against the bacterium Listeria monocytogenes, although with remarkably different efficiencies. Our data shed new light on the potential contributions of the individual STAT1 isoforms to STAT1-dependent immune responses. Knowledge of STAT1β's function will help fine-tune diagnostic approaches and help design more specific strategies to interfere with STAT1 activity.

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Figures

FIG 1
FIG 1
Generation of STAT1α and STAT1β isoform-specific knock-in mice. (A) Schematic illustration of the genomic organization of the Stat1 locus from exon 14 to exon 23 (black boxes). Knock-in (KI) constructs and the structure of recombined alleles after insertion are shown below. Restriction endonuclease sites used for cloning and Southern blot analysis and the resulting fragment sizes are indicated. B, BamHI; P, PmlI; S, SmaI; H, HindIII; β-globin, human β-globin splice; Neo, neomycin cassette flanked by loxP sites (triangles); TK, herpes simplex virus thymidine kinase cassette; α, end of Stat1α cDNA (exons 19 to 23); β, end of Stat1β cDNA (exons 19 to 21); stop, stop codon. (B) Southern blot analysis after digestion with BsrGI of either two Stat1+/β or two Stat1+/α heterozygous ES cell clones and a Stat1+/+ control. Restriction sites and fragment sizes are depicted in panel A. WT, WT allele; mut, mutated allele. (C) Total RNA was extracted from BMMϕ and the indicated organs from Stat1+/+, Stat1β/β, and Stat1α/α mice and subjected to RT-PCR. Cyclophilin was used as the endogenous control. (D) Total-protein extracts of BMMϕ, liver, and spleen from WT (+/+), Stat1β/β (β/β), Stat1α/α (α/α), and Stat1−/− (−/−) mice were used for Western blot analysis. Protein expression of STAT1α and STAT1β was detected with a STAT1 antibody against the N-terminal region of the protein. A pan-ERK antibody was used as a loading control (p85 is depicted).
FIG 2
FIG 2
STAT1β shows prolonged tyrosine 701 phosphorylation. (A to D) BMMϕ derived from WT (+/+), Stat1β/β (β/β), and Stat1α/α (α/α) mice were stimulated with IFN-β (A) or IFN-γ (B to D) for the times indicated or left untreated (w/o). (A to C) Total-protein extracts were used for detection of Tyr701-phosphorylated STAT1 (A and B) or Ser727-phosphorylated STAT1 (C) by Western blotting. (A to C) Membranes were reprobed with N-terminus-specific STAT1 and pan-ERK antibodies. (D) Total cell extracts were used for EMSA using a GAS consensus sequence-containing oligonucleotide (IRDye 700 labeled). α/α, STAT1α-STAT1α; α/β, STAT1α-STAT1β; β/β, STAT1β-STAT1β. (A to D) Results are representative of three independent experiments. (E and F) BMMϕ isolated from WT (+/+) and Stat1β/β (β/β) (E) and WT (+/+) and Stat1α/α (α/α) (F) mice were stimulated with IFN-γ for 1 h or left untreated (−), followed by incubation with or without (−) staurosporine (stauro) (500 nM) for the times indicated. Total-protein extracts were used for detection of Tyr701-phosphorylated STAT1 by Western blotting. The data are representative of four independent experiments. x, empty lane.
FIG 3
FIG 3
STAT1β shows prolonged nuclear localization and prolonged promoter binding after IFN-γ treatment compared to STAT1α. (A) BMMϕ derived from WT (+/+), Stat1β/β (β/β), and Stat1α/α (α/α) mice were stimulated with IFN-γ (100 U/ml) for the times indicated or left untreated (w/o). Phosphorylation of Tyr701 STAT1 was detected with a specific phospho-STAT1 primary antibody and an IRDye 800-labeled secondary antibody (green). DAPI (100 ng/ml) was used for nuclear staining (blue). Fluorescence signals were analyzed with a Leica DM5500B microscope. (B) BMMϕ derived from WT (+/+) and Stat1β/β (β/β) mice were stimulated with IFN-γ as described for panel A. Cytoplasmic and nuclear proteins were isolated and used for Western blotting. Membranes were probed with an anti-phosphotyrosine 701 STAT1 antibody (pTyr701) and an antibody against the STAT1 N-terminal region (STAT1α/β). The purity of fractions was determined by detection of GAPDH (cytoplasm specific) and SP1 (nucleus specific). (C and D) STAT1 binding to the endogenous distal (dist) Gbp2 (C) or Irf1 (D) promoter region was examined by ChIP analysis using an antibody against the N-terminal portion of STAT1 and qPCR. The values were normalized to the input control and calculated relative to untreated WT cells. Mean values ± standard errors (SE) from at least three independent experiments are shown; *, P ≤ 0.05; **, P ≤ 0.01.
FIG 4
FIG 4
STAT1β is transcriptionally active in response to IFN-β and IFN-γ. BMMϕ isolated from WT (+/+), Stat1β/β (β/β), Stat1α/α (α/α), and Stat1−/− (−/−) mice were stimulated with IFN-β (A and B) or IFN-γ (C to F) for the times indicated or left untreated (w/o). Total RNA was extracted and used for RT-qPCR analysis for the genes indicated. Ube2d2 was used for normalization, and expression levels were calculated relative to untreated WT cells. Mean values ± SE are given (log scale), and data from at least three independent experiments are shown; x, not detectable; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
FIG 5
FIG 5
Transcriptional activities of STAT1α and STAT1β overlap but are nonredundant. BMMϕ isolated from WT (+/+), Stat1β/β (β/β), Stat1α/α (α/α), and Stat1−/− (−/−) mice were stimulated with IFN-γ for the times indicated or left untreated (w/o). Total RNA was extracted and subjected to microarray (A and B) or RT-qPCR (C to H) analysis. (A and B) Summary of data derived from three independent experiments. Transcripts at least 4-fold (significantly) induced in WT cells were selected for each time point. To specifically analyze STAT1-dependent genes, only transcripts that were differentially expressed between WT and Stat1−/− cells were included in the analysis. Expression of the transcripts (108 at 6 h and 135 at 24 h of treatment) was compared between Stat1β/β and WT (A) and Stat1α/α and WT (B) cells, and the percentages and numbers of transcripts that showed common expression patterns are depicted. No difference, <2-fold difference and/or P > 0.05; reduced, lower expression than in WT cells but still induced by treatment; not induced, lower expression than in WT cells and not induced by treatment; differential expression was defined as at least 2-fold difference and a P value of ≤0.05. (C to H) RT-qPCR validation of expression patterns was performed as described in the legend to Fig. 4 for CIIta (C), Ly6i (D), Isg20 (E), Isg15 (F), Plac8 (G), and probable Gpr33 (H). Expression levels of genes that were not detectable in untreated WT cells were calculated relative to 6-h-treated WT cells. Mean values ± SE are given (log scale), and data from at least three independent experiments (different from those used for microarray analysis) are shown; x or nd, not detectable; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. See Tables S1 and S2 in the supplemental material for lists of differentially expressed genes.
FIG 6
FIG 6
STAT1α and STAT1β can mediate type I and type III IFN-dependent antiviral immunity in vivo. (A) EMCV (50 PFU/mouse) was administered i.p. to WT (Stat1+/+), Stat1β/β, Stat1α/α, and Stat1−/− mice, and survival was monitored for 14 days. The data are derived from two independent experiments; n = 20 (Stat1+/+), n = 18 (Stat1β/β), n = 10 (Stat1α/α), and n = 18 (Stat1−/−). (B) VSV (104 PFU/mouse) was administered intranasally to mice of the genotypes indicated, and survival was monitored for 14 days. The data are from two independent experiments; n = 21 (Stat1+/+, Stat1β/β, and Stat1α/α) and n = 15 (Stat1−/−). Significances were as follows: Stat1−/− versus all others, P < 0.001; Stat1β/β versus WT, P < 0.01; Stat1β/β versus Stat1α/α, P < 0.001. (C) Rotavirus was administered orally to adult mice, and virus shedding in feces on days 4 and 5 postinfection was determined by enzyme-linked immunosorbent assay (ELISA). Pooled data from two independent experiments with 16 or 17 animals per genotype are shown. ***, P ≤ 0.001. (D) Influenza A virus strain SC35M (104 PFU/mouse) was administered intranasally to mice carrying one functional allele of the IFN-regulated Mx1 gene, and survival was monitored for 10 days. n = 6 (Stat1+/−), n = 7 (Stat1β/−), n = 8 (Stat1α/−), and n = 4 (Stat1−/−). Significance, Stat1−/− versus all others, P < 0.001.
FIG 7
FIG 7
STAT1α and STAT1β show differential efficiencies in immune defense against MCMV and L. monocytogenes infections. (A) WT (Stat1+/+), Stat1β/β, Stat1α/α, and Stat1−/− mice were infected i.p. with MCMV (4 × 105 PFU/mouse), and survival was monitored for 14 days. The data are derived from two independent experiments; n = 20 (Stat1+/+), n = 23 (Stat1β/β), n = 14 (Stat1α/α), and n = 18 (Stat1−/−). Significances were as follows: Stat1β/β versus all others, P < 0.01; Stat1−/− versus WT and Stat1α/α, P < 0.001. (B) WT (Stat1+/+), Stat1β/β, Stat1α/α, and Stat1−/− mice were infected i.p. with L. monocytogenes (2 × 105 CFU/mouse), and survival was monitored for 14 days. The data are derived from four independent experiments. n = 39 (Stat1+/+), n = 34 (Stat1β/β), n = 21 (Stat1α/α), and n = 17 (Stat1−/−). Significances were as follows: Stat1β/β versus all others, P < 0.0001; Stat1−/− versus all others, P < 0.0001. (C) Mice were infected as for panel B and killed 3 or 5 days postinfection. Whole spleens and livers were removed and homogenized, and bacterial loads were determined with standard CFU assays. The data are from two independent experiments. Days 3 and 5, n = 11 and 9 (Stat1+/+), n = 11 and 12 (Stat1β/β), n = 13 and 11 (Stat1α/α), and n = 8 and 0 (Stat1−/−); because of their early lethality after infection, Stat1−/− mice had to be excluded from the analysis at day 5. **, P ≤ 0.01; ***, P ≤ 0.001.
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