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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/IRF9 and ISGF3 regulate expression of a common set of ISGs with different kinetics(A) 2fTGH and hST2-U3C or (B) MEF WT and mST2-MS1KO were treated with IFNα for 0 h, 4 h, 8 h and 24 h and subjected to microarray analysis. Common up-regulated genes were selected by comparing transcriptomes of individual cell lines. Statistically significant up-regulated genes in human (A) and mouse (B) cell-line data sets were compared by Venn diagram analysis. Average expression profiles of common up-regulated genes between (C) 2fTGH and hST2-U3C and (D) MEF WT and mST2-MS1KO are displayed in centroid view. Expression values are shown as log2 ratio; error bars=S.D.
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Figure 4: STAT2/IRF9 and ISGF3 regulate expression of a common set of ISGs with different kinetics(A) 2fTGH and hST2-U3C or (B) MEF WT and mST2-MS1KO were treated with IFNα for 0 h, 4 h, 8 h and 24 h and subjected to microarray analysis. Common up-regulated genes were selected by comparing transcriptomes of individual cell lines. Statistically significant up-regulated genes in human (A) and mouse (B) cell-line data sets were compared by Venn diagram analysis. Average expression profiles of common up-regulated genes between (C) 2fTGH and hST2-U3C and (D) MEF WT and mST2-MS1KO are displayed in centroid view. Expression values are shown as log2 ratio; error bars=S.D.

Mentions: To characterize IFNα-mediated transcriptional responses and identify the genes being regulated by STAT2/IRF9 in relation to ISGF3, we performed microarray experiments comparing human and mouse STAT1 KO cells overexpressing STAT2 with their WT counterparts treated with IFNα for 4 h, 8 h and 24 h. After quality check and data analysis, we only focused on the up-regulated genes. By comparing the expression profiles of hST2-U3C with 2fTGH, we identified 303 up-regulated genes in hST2-U3C of which 117 were in common with 2fTGH (Figure 4A). Similarly, by comparing the expression profiles of mST2-MS1KO with MEF-WT, we identified 295 up-regulated genes with 126 genes commonly induced between the two cell lines (Figure 4B). To characterize these commonly up-regulated genes in more detail, first we performed hierarchical cluster analysis (based on average linkage clustering of ratios) comparing human 2fTGH with hST2-U3C and mouse MEF-WT with mST2-MS1KO (Figures 5A and 5B, respectively). Strikingly, among the commonly induced genes in both human and mouse cell lines many known ISGs could be recognized, including IFITs, IFIs, ISGs, OASs, MX, radical S-adenosylmethionine domain-containing (RSAD2) and HECT and RLD domain-containing E3 ubiquitin protein ligase 5 (HERC5). In general, the induction level of these genes was lower in the STAT1 KO cells overexpressing STAT2 as opposed to WT cells. The centroid view, representing the average gene expression pattern in human (Figure 4C) and mouse (Figure 4D) cells unveiled a prolonged profile in hST2-U3C and MST2-MS1KO cells in response to IFNα. In contrast, in the WT cells, this was early and transient. In order to validate the microarray data, qRT-PCR was performed for a selection of these genes. Indeed, IFIT1, IFIT2, IFIT3, ISG15 and MX1 exhibited a prolonged IFNα-induced expression profile in hST2-U3C as compared with the 2fTGH cells (not shown). The same was true for the expression of Mx2, Ifit3, Isg15, Oas1b and RSAD2 when compared with MST2-MS1KO versus MEF-WT (not shown). Collectively, our results reveal that STAT2/IRF9 and ISGF3 regulate expression of a common set of ISGs, however, with a different kinetics.


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/IRF9 and ISGF3 regulate expression of a common set of ISGs with different kinetics(A) 2fTGH and hST2-U3C or (B) MEF WT and mST2-MS1KO were treated with IFNα for 0 h, 4 h, 8 h and 24 h and subjected to microarray analysis. Common up-regulated genes were selected by comparing transcriptomes of individual cell lines. Statistically significant up-regulated genes in human (A) and mouse (B) cell-line data sets were compared by Venn diagram analysis. Average expression profiles of common up-regulated genes between (C) 2fTGH and hST2-U3C and (D) MEF WT and mST2-MS1KO are displayed in centroid view. Expression values are shown as log2 ratio; error bars=S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: STAT2/IRF9 and ISGF3 regulate expression of a common set of ISGs with different kinetics(A) 2fTGH and hST2-U3C or (B) MEF WT and mST2-MS1KO were treated with IFNα for 0 h, 4 h, 8 h and 24 h and subjected to microarray analysis. Common up-regulated genes were selected by comparing transcriptomes of individual cell lines. Statistically significant up-regulated genes in human (A) and mouse (B) cell-line data sets were compared by Venn diagram analysis. Average expression profiles of common up-regulated genes between (C) 2fTGH and hST2-U3C and (D) MEF WT and mST2-MS1KO are displayed in centroid view. Expression values are shown as log2 ratio; error bars=S.D.
Mentions: To characterize IFNα-mediated transcriptional responses and identify the genes being regulated by STAT2/IRF9 in relation to ISGF3, we performed microarray experiments comparing human and mouse STAT1 KO cells overexpressing STAT2 with their WT counterparts treated with IFNα for 4 h, 8 h and 24 h. After quality check and data analysis, we only focused on the up-regulated genes. By comparing the expression profiles of hST2-U3C with 2fTGH, we identified 303 up-regulated genes in hST2-U3C of which 117 were in common with 2fTGH (Figure 4A). Similarly, by comparing the expression profiles of mST2-MS1KO with MEF-WT, we identified 295 up-regulated genes with 126 genes commonly induced between the two cell lines (Figure 4B). To characterize these commonly up-regulated genes in more detail, first we performed hierarchical cluster analysis (based on average linkage clustering of ratios) comparing human 2fTGH with hST2-U3C and mouse MEF-WT with mST2-MS1KO (Figures 5A and 5B, respectively). Strikingly, among the commonly induced genes in both human and mouse cell lines many known ISGs could be recognized, including IFITs, IFIs, ISGs, OASs, MX, radical S-adenosylmethionine domain-containing (RSAD2) and HECT and RLD domain-containing E3 ubiquitin protein ligase 5 (HERC5). In general, the induction level of these genes was lower in the STAT1 KO cells overexpressing STAT2 as opposed to WT cells. The centroid view, representing the average gene expression pattern in human (Figure 4C) and mouse (Figure 4D) cells unveiled a prolonged profile in hST2-U3C and MST2-MS1KO cells in response to IFNα. In contrast, in the WT cells, this was early and transient. In order to validate the microarray data, qRT-PCR was performed for a selection of these genes. Indeed, IFIT1, IFIT2, IFIT3, ISG15 and MX1 exhibited a prolonged IFNα-induced expression profile in hST2-U3C as compared with the 2fTGH cells (not shown). The same was true for the expression of Mx2, Ifit3, Isg15, Oas1b and RSAD2 when compared with MST2-MS1KO versus MEF-WT (not shown). Collectively, our results reveal that STAT2/IRF9 and ISGF3 regulate expression of a common set of ISGs, however, with a different kinetics.

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