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STAT2/IRF9 directs a prolonged ISGF3-like transcriptional response and antiviral activity in the absence of STAT1.

Blaszczyk K, Olejnik A, Nowicka H, Ozgyin L, Chen YL, Chmielewski S, Kostyrko K, Wesoly J, Balint BL, Lee CK, Bluyssen HA - Biochem. J. (2015)

Bottom Line: However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFNα signalling as compared with interferon-stimulated gene factor 3 (ISGF3).The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3.Moreover, the existence of 'STAT2/IRF9-specific' target genes predicts a novel role of STAT2 in IFNα signalling.

View Article: PubMed Central - PubMed

Affiliation: *Department of Human Molecular Genetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland.

ABSTRACT
Evidence is accumulating for the existence of a signal transducer and activator of transcription 2 (STAT2)/interferon regulatory factor 9 (IRF9)-dependent, STAT1-independent interferon alpha (IFNα) signalling pathway. However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFNα signalling as compared with interferon-stimulated gene factor 3 (ISGF3). In STAT1-defeicient U3C cells stably overexpressing human STAT2 (hST2-U3C) and STAT1-deficient murine embryonic fibroblast cells stably overexpressing mouse STAT2 (mST2-MS1KO) we observed that the IFNα-induced expression of 2'-5'-oligoadenylate synthase 2 (OAS2) and interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) correlated with the kinetics of STAT2 phosphorylation, and the presence of a STAT2/IRF9 complex requiring STAT2 phosphorylation and the STAT2 transactivation domain. Subsequent microarray analysis of IFNα-treated wild-type (WT) and STAT1 KO cells overexpressing STAT2 extended our observations and identified ∼120 known antiviral ISRE-containing interferon-stimulated genes (ISGs) commonly up-regulated by STAT2/IRF9 and ISGF3. The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3. In addition, we identified a group of 'STAT2/IRF9-specific' ISGs, whose response to IFNα was ISGF3-independent. Finally, STAT2/IRF9 was able to trigger an antiviral response upon encephalomyocarditis virus (EMCV) and vesicular stomatitis Indiana virus (VSV). Our results further prove that IFNα-activated STAT2/IRF9 induces a prolonged ISGF3-like transcriptome and generates an antiviral response in the absence of STAT1. Moreover, the existence of 'STAT2/IRF9-specific' target genes predicts a novel role of STAT2 in IFNα signalling.

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The IFNα response in STAT1 KO cells is abrogated(A, C) 2fTGH and U3C; (B, D) MEF WT and MS1KO were treated with IFNα for indicated times. For (A) and (B), protein lysates were isolated and analyzed by Western blot analysis. Total STAT2 (tSTAT2), phosphorylated STAT2 (pSTAT2), total STAT1 (tSTAT1), phosphorylated STAT1 (pSTAT1) and IRF9 were analyzed using specific antibodies. Equal loading was verified using anti-tubulin. For (C) and (D), total RNA was extracted and OAS2 and Ifit1 relative fold induction was quantified using qRT-PCR. Statistical significance is presented as compared with the non-treated control (results are means ±S.E.M.). Statistical analysis was conducted using one-way ANOVA with Tukey's post hoc test. *P≤0.05, ***P≤0.01.
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Figure 1: The IFNα response in STAT1 KO cells is abrogated(A, C) 2fTGH and U3C; (B, D) MEF WT and MS1KO were treated with IFNα for indicated times. For (A) and (B), protein lysates were isolated and analyzed by Western blot analysis. Total STAT2 (tSTAT2), phosphorylated STAT2 (pSTAT2), total STAT1 (tSTAT1), phosphorylated STAT1 (pSTAT1) and IRF9 were analyzed using specific antibodies. Equal loading was verified using anti-tubulin. For (C) and (D), total RNA was extracted and OAS2 and Ifit1 relative fold induction was quantified using qRT-PCR. Statistical significance is presented as compared with the non-treated control (results are means ±S.E.M.). Statistical analysis was conducted using one-way ANOVA with Tukey's post hoc test. *P≤0.05, ***P≤0.01.

Mentions: First, we characterized IFNα responses of the human 2fTGH (WT) and U3C (STAT1-deficient) cell lines, and the mouse MEF (MEF WT) and MS1KO cells. Both human and mouse WT cells were treated with IFNα for increasing times, which resulted in a similar phosphorylation pattern of STAT1 and STAT2. Phosphorylation of both proteins increased after 4 h of treatment and diminished to near basal levels after 8 and 24 h (Figures 1A and 1B). Expression of STAT1 and STAT2 clearly increased in time in 2fTGH and MEF WT in response to IFNα. The expression of IRF9, on the other hand, only increased in 2fTGH. The IFNα response in both U3C and MS1KO cells exhibited diminished STAT2 phosphorylation, despite the normal expression of STAT2 and IRF9 proteins (Figures 1A and 1B). STAT2 phosphorylation in IFNα-treated human U3C cells was not detectable (Figure 1A), even after 1 h and 2 h of treatment (not shown). In mouse MS1KO cells diminished phosphorylation of STAT2 could be detected with more prolonged kinetics as compared with MEF WT cells (Figure 1B). Expression of STAT2 and IRF9 did not increase over time in response to IFNα (Figures 1A and 1B). However, the IFNα-induced expression of the classical ISGs human 2′-5′-oligoadenylate synthase 2 (OAS2) and mouse interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) still slowly increased over time, but at a much lower level as compared with the WT cells (Figures 1C and 1D). Together these results show that the decrease in STAT2 phosphorylation correlated with the diminution of OAS2 and Ifit1 gene expression, suggesting the involvement of STAT2 in IFNα-induction of the latter genes.


STAT2/IRF9 directs a prolonged ISGF3-like transcriptional response and antiviral activity in the absence of STAT1.

Blaszczyk K, Olejnik A, Nowicka H, Ozgyin L, Chen YL, Chmielewski S, Kostyrko K, Wesoly J, Balint BL, Lee CK, Bluyssen HA - Biochem. J. (2015)

The IFNα response in STAT1 KO cells is abrogated(A, C) 2fTGH and U3C; (B, D) MEF WT and MS1KO were treated with IFNα for indicated times. For (A) and (B), protein lysates were isolated and analyzed by Western blot analysis. Total STAT2 (tSTAT2), phosphorylated STAT2 (pSTAT2), total STAT1 (tSTAT1), phosphorylated STAT1 (pSTAT1) and IRF9 were analyzed using specific antibodies. Equal loading was verified using anti-tubulin. For (C) and (D), total RNA was extracted and OAS2 and Ifit1 relative fold induction was quantified using qRT-PCR. Statistical significance is presented as compared with the non-treated control (results are means ±S.E.M.). Statistical analysis was conducted using one-way ANOVA with Tukey's post hoc test. *P≤0.05, ***P≤0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403947&req=5

Figure 1: The IFNα response in STAT1 KO cells is abrogated(A, C) 2fTGH and U3C; (B, D) MEF WT and MS1KO were treated with IFNα for indicated times. For (A) and (B), protein lysates were isolated and analyzed by Western blot analysis. Total STAT2 (tSTAT2), phosphorylated STAT2 (pSTAT2), total STAT1 (tSTAT1), phosphorylated STAT1 (pSTAT1) and IRF9 were analyzed using specific antibodies. Equal loading was verified using anti-tubulin. For (C) and (D), total RNA was extracted and OAS2 and Ifit1 relative fold induction was quantified using qRT-PCR. Statistical significance is presented as compared with the non-treated control (results are means ±S.E.M.). Statistical analysis was conducted using one-way ANOVA with Tukey's post hoc test. *P≤0.05, ***P≤0.01.
Mentions: First, we characterized IFNα responses of the human 2fTGH (WT) and U3C (STAT1-deficient) cell lines, and the mouse MEF (MEF WT) and MS1KO cells. Both human and mouse WT cells were treated with IFNα for increasing times, which resulted in a similar phosphorylation pattern of STAT1 and STAT2. Phosphorylation of both proteins increased after 4 h of treatment and diminished to near basal levels after 8 and 24 h (Figures 1A and 1B). Expression of STAT1 and STAT2 clearly increased in time in 2fTGH and MEF WT in response to IFNα. The expression of IRF9, on the other hand, only increased in 2fTGH. The IFNα response in both U3C and MS1KO cells exhibited diminished STAT2 phosphorylation, despite the normal expression of STAT2 and IRF9 proteins (Figures 1A and 1B). STAT2 phosphorylation in IFNα-treated human U3C cells was not detectable (Figure 1A), even after 1 h and 2 h of treatment (not shown). In mouse MS1KO cells diminished phosphorylation of STAT2 could be detected with more prolonged kinetics as compared with MEF WT cells (Figure 1B). Expression of STAT2 and IRF9 did not increase over time in response to IFNα (Figures 1A and 1B). However, the IFNα-induced expression of the classical ISGs human 2′-5′-oligoadenylate synthase 2 (OAS2) and mouse interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) still slowly increased over time, but at a much lower level as compared with the WT cells (Figures 1C and 1D). Together these results show that the decrease in STAT2 phosphorylation correlated with the diminution of OAS2 and Ifit1 gene expression, suggesting the involvement of STAT2 in IFNα-induction of the latter genes.

Bottom Line: However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFNα signalling as compared with interferon-stimulated gene factor 3 (ISGF3).The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3.Moreover, the existence of 'STAT2/IRF9-specific' target genes predicts a novel role of STAT2 in IFNα signalling.

View Article: PubMed Central - PubMed

Affiliation: *Department of Human Molecular Genetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland.

ABSTRACT
Evidence is accumulating for the existence of a signal transducer and activator of transcription 2 (STAT2)/interferon regulatory factor 9 (IRF9)-dependent, STAT1-independent interferon alpha (IFNα) signalling pathway. However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFNα signalling as compared with interferon-stimulated gene factor 3 (ISGF3). In STAT1-defeicient U3C cells stably overexpressing human STAT2 (hST2-U3C) and STAT1-deficient murine embryonic fibroblast cells stably overexpressing mouse STAT2 (mST2-MS1KO) we observed that the IFNα-induced expression of 2'-5'-oligoadenylate synthase 2 (OAS2) and interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) correlated with the kinetics of STAT2 phosphorylation, and the presence of a STAT2/IRF9 complex requiring STAT2 phosphorylation and the STAT2 transactivation domain. Subsequent microarray analysis of IFNα-treated wild-type (WT) and STAT1 KO cells overexpressing STAT2 extended our observations and identified ∼120 known antiviral ISRE-containing interferon-stimulated genes (ISGs) commonly up-regulated by STAT2/IRF9 and ISGF3. The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3. In addition, we identified a group of 'STAT2/IRF9-specific' ISGs, whose response to IFNα was ISGF3-independent. Finally, STAT2/IRF9 was able to trigger an antiviral response upon encephalomyocarditis virus (EMCV) and vesicular stomatitis Indiana virus (VSV). Our results further prove that IFNα-activated STAT2/IRF9 induces a prolonged ISGF3-like transcriptome and generates an antiviral response in the absence of STAT1. Moreover, the existence of 'STAT2/IRF9-specific' target genes predicts a novel role of STAT2 in IFNα signalling.

Show MeSH
Related in: MedlinePlus