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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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STAT2 and IRF9 complex and mediate an IFNα response in the absence of STAT1(A) The interaction between STAT2 and IRF9 was analyzed by immunoprecipitation. hSTAT2-U3C were treated with IFNα for the indicated times. Cell lysates were immunoprecipitated with anti-HA antibody followed by Western blotting with IRF9, tSTAT2 and pSTAT2 antibodies. (B) Two different clones of hST2-U3C (hST2-U3Ca and hST2-U3C) varying in hSTAT2 expression level and their control Migr1-U3C; (C) ΔmST2-MS1KO, mST2-MS1KO and their control Migr1-MS1KO; (D) Migr1-U3C, IRF9-U3C and hST2-U3C; (E) hST2-U3C transiently transfected with Migr1-IRF9 (500 ng); (F) U3C cells transiently transfected with STAT2-Y690F or STAT2 plasmid (2.5 μg) were all treated with or without 200 U/ml IFNα for 8 h (B–E) or 24 h (F). Total RNA was extracted and OAS2, Ifit1, STAT2 or IRF9 relative fold inductions were 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 except in (E) where a Student's t-test, two-tailed, was used. *P≤0.05, **P≤0.01.
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Figure 3: STAT2 and IRF9 complex and mediate an IFNα response in the absence of STAT1(A) The interaction between STAT2 and IRF9 was analyzed by immunoprecipitation. hSTAT2-U3C were treated with IFNα for the indicated times. Cell lysates were immunoprecipitated with anti-HA antibody followed by Western blotting with IRF9, tSTAT2 and pSTAT2 antibodies. (B) Two different clones of hST2-U3C (hST2-U3Ca and hST2-U3C) varying in hSTAT2 expression level and their control Migr1-U3C; (C) ΔmST2-MS1KO, mST2-MS1KO and their control Migr1-MS1KO; (D) Migr1-U3C, IRF9-U3C and hST2-U3C; (E) hST2-U3C transiently transfected with Migr1-IRF9 (500 ng); (F) U3C cells transiently transfected with STAT2-Y690F or STAT2 plasmid (2.5 μg) were all treated with or without 200 U/ml IFNα for 8 h (B–E) or 24 h (F). Total RNA was extracted and OAS2, Ifit1, STAT2 or IRF9 relative fold inductions were 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 except in (E) where a Student's t-test, two-tailed, was used. *P≤0.05, **P≤0.01.

Mentions: To prove that a STAT2/IRF9-containing complex is responsible for the IFNα response in the STAT1 KO cells overexpressing STAT2, we performed additional experiments. First, by using protein extracts from hST2-U3C cells treated with IFNα for increasing times in combination with anti-HA antibodies to immunoprecipitate STAT2, we were able to observe specific STAT2/IRF9 complex formation even after 24 h of IFNα treatment (Figure 3A; input control is shown in Figure 2A). Interestingly, the STAT2/IRF9 complex could already be detected in the absence of IFNα treatment (lane 1, Figure 3A), and was not affected by increased STAT2 phosphorylation. On the other hand, the phosphorylation kinetics of STAT2 correlated with the prolonged expression pattern of OAS2 (Figures 2A and 2C). We also checked the level of ISG expression in response to IFNα in two different clones of hST2-U3C with varying STAT2 mRNA levels. In hST2-U3C, the STAT2 mRNA level was 75-fold higher than in Migr1-U3C control, whereas in hST2-U3Ca there was a 30-fold difference (Figure 3B). This correlated with the difference in expression of OAS2 in these two cell lines in response to IFNα, being 9-fold higher in hST2-U3C (46-fold) as opposed to hST2-U3Ca (5-fold), when compared with untreated cells (Figure 3B). In addition to mST2-MS1KO cells, we generated a MS1KO stable cell line overexpressing a C-terminally truncated form of mSTAT2 (ΔmST2-MS1KO), which lacks the trans-activation domain of STAT2 and acts as a dominant negative. As shown in Figure 3(C), the levels of STAT2 in mST2-MS1KO cells correlated with the high induction of mouse Ifit1. ΔmST2-MS1KO facilitated no significant induction of the mouse Ifit1 gene in response to IFNα. Subsequently, we investigated in more detail the role of IRF9 in the IFNα response in the absence of STAT1. We generated a U3C cell line stably overexpressing IRF9 (IRF9-U3C). Interestingly, OAS2 expression increased only 3-fold as compared with Migr1-U3C cells after 8 h of IFNα treatment (Figure 3D). However, hST2-U3C cells transiently transfected with IRF9 showed a 10-fold increase in OAS2 gene expression in comparison with the hST2-U3C IFNα-treated cells and a 57-fold increase in contrast with Migr1-U3C cells (Figure 3E). Finally, we compared expression of IFIT2 and OAS2 in U3C cells transiently transfected with STAT2 or the tyrosine mutant STAT2Y690F (mutant form of STAT2 that cannot be phosphorylated on tyrosine). U3C-ST2 showed a 10-fold increase upon IFN treatment, whereas U3C-ST2Y690F exhibited no response, implying that the STAT2/IRF9-mediated IFNα-response is dependent on STAT2 phosphorylation. Together, these results point to the importance of the STAT2/IRF9 complex in the prolonged IFNα response in the absence of STAT1 and suggest an ISGF3-like function.


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)

STAT2 and IRF9 complex and mediate an IFNα response in the absence of STAT1(A) The interaction between STAT2 and IRF9 was analyzed by immunoprecipitation. hSTAT2-U3C were treated with IFNα for the indicated times. Cell lysates were immunoprecipitated with anti-HA antibody followed by Western blotting with IRF9, tSTAT2 and pSTAT2 antibodies. (B) Two different clones of hST2-U3C (hST2-U3Ca and hST2-U3C) varying in hSTAT2 expression level and their control Migr1-U3C; (C) ΔmST2-MS1KO, mST2-MS1KO and their control Migr1-MS1KO; (D) Migr1-U3C, IRF9-U3C and hST2-U3C; (E) hST2-U3C transiently transfected with Migr1-IRF9 (500 ng); (F) U3C cells transiently transfected with STAT2-Y690F or STAT2 plasmid (2.5 μg) were all treated with or without 200 U/ml IFNα for 8 h (B–E) or 24 h (F). Total RNA was extracted and OAS2, Ifit1, STAT2 or IRF9 relative fold inductions were 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 except in (E) where a Student's t-test, two-tailed, was used. *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 3: STAT2 and IRF9 complex and mediate an IFNα response in the absence of STAT1(A) The interaction between STAT2 and IRF9 was analyzed by immunoprecipitation. hSTAT2-U3C were treated with IFNα for the indicated times. Cell lysates were immunoprecipitated with anti-HA antibody followed by Western blotting with IRF9, tSTAT2 and pSTAT2 antibodies. (B) Two different clones of hST2-U3C (hST2-U3Ca and hST2-U3C) varying in hSTAT2 expression level and their control Migr1-U3C; (C) ΔmST2-MS1KO, mST2-MS1KO and their control Migr1-MS1KO; (D) Migr1-U3C, IRF9-U3C and hST2-U3C; (E) hST2-U3C transiently transfected with Migr1-IRF9 (500 ng); (F) U3C cells transiently transfected with STAT2-Y690F or STAT2 plasmid (2.5 μg) were all treated with or without 200 U/ml IFNα for 8 h (B–E) or 24 h (F). Total RNA was extracted and OAS2, Ifit1, STAT2 or IRF9 relative fold inductions were 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 except in (E) where a Student's t-test, two-tailed, was used. *P≤0.05, **P≤0.01.
Mentions: To prove that a STAT2/IRF9-containing complex is responsible for the IFNα response in the STAT1 KO cells overexpressing STAT2, we performed additional experiments. First, by using protein extracts from hST2-U3C cells treated with IFNα for increasing times in combination with anti-HA antibodies to immunoprecipitate STAT2, we were able to observe specific STAT2/IRF9 complex formation even after 24 h of IFNα treatment (Figure 3A; input control is shown in Figure 2A). Interestingly, the STAT2/IRF9 complex could already be detected in the absence of IFNα treatment (lane 1, Figure 3A), and was not affected by increased STAT2 phosphorylation. On the other hand, the phosphorylation kinetics of STAT2 correlated with the prolonged expression pattern of OAS2 (Figures 2A and 2C). We also checked the level of ISG expression in response to IFNα in two different clones of hST2-U3C with varying STAT2 mRNA levels. In hST2-U3C, the STAT2 mRNA level was 75-fold higher than in Migr1-U3C control, whereas in hST2-U3Ca there was a 30-fold difference (Figure 3B). This correlated with the difference in expression of OAS2 in these two cell lines in response to IFNα, being 9-fold higher in hST2-U3C (46-fold) as opposed to hST2-U3Ca (5-fold), when compared with untreated cells (Figure 3B). In addition to mST2-MS1KO cells, we generated a MS1KO stable cell line overexpressing a C-terminally truncated form of mSTAT2 (ΔmST2-MS1KO), which lacks the trans-activation domain of STAT2 and acts as a dominant negative. As shown in Figure 3(C), the levels of STAT2 in mST2-MS1KO cells correlated with the high induction of mouse Ifit1. ΔmST2-MS1KO facilitated no significant induction of the mouse Ifit1 gene in response to IFNα. Subsequently, we investigated in more detail the role of IRF9 in the IFNα response in the absence of STAT1. We generated a U3C cell line stably overexpressing IRF9 (IRF9-U3C). Interestingly, OAS2 expression increased only 3-fold as compared with Migr1-U3C cells after 8 h of IFNα treatment (Figure 3D). However, hST2-U3C cells transiently transfected with IRF9 showed a 10-fold increase in OAS2 gene expression in comparison with the hST2-U3C IFNα-treated cells and a 57-fold increase in contrast with Migr1-U3C cells (Figure 3E). Finally, we compared expression of IFIT2 and OAS2 in U3C cells transiently transfected with STAT2 or the tyrosine mutant STAT2Y690F (mutant form of STAT2 that cannot be phosphorylated on tyrosine). U3C-ST2 showed a 10-fold increase upon IFN treatment, whereas U3C-ST2Y690F exhibited no response, implying that the STAT2/IRF9-mediated IFNα-response is dependent on STAT2 phosphorylation. Together, these results point to the importance of the STAT2/IRF9 complex in the prolonged IFNα response in the absence of STAT1 and suggest an ISGF3-like function.

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