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Structural and functional characterization of salmon STAT1, STAT2 and IRF9 homologs sheds light on interferon signaling in teleosts.

Sobhkhez M, Skjesol A, Thomassen E, Tollersrud LG, Iliev DB, Sun B, Robertsen B, Jørgensen JB - FEBS Open Bio (2014)

Bottom Line: Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells.The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone.Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish.

View Article: PubMed Central - PubMed

Affiliation: The Norwegian College of Fishery Science, UiT The Arctic University of Norway, N-9037 Tromsø, Norway.

ABSTRACT
Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.

No MeSH data available.


Related in: MedlinePlus

IRF9, STAT2a and STAT2b stimulate GAS promoter activity upon IFNγ stimulation. CHSE-214 cells transiently transfected with STAT1a, STAT2a, STAT2b, RF9 alone or in combination with each other and GFP-EV (empty vector) was used as control. Transfectants were left non-stimulated or were stimulated with 500 ng/ml of IFNγ 24 h prior to reporter gene assay analysis. Firefly luciferase activity was normalized with Renilla luciferase levels (n = 5) and the data are presented as relative luciferase units (RLU). The results are representative of two independent experiments. ∗ Represents values where difference compared to EV control is statistically significant.
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f0055: IRF9, STAT2a and STAT2b stimulate GAS promoter activity upon IFNγ stimulation. CHSE-214 cells transiently transfected with STAT1a, STAT2a, STAT2b, RF9 alone or in combination with each other and GFP-EV (empty vector) was used as control. Transfectants were left non-stimulated or were stimulated with 500 ng/ml of IFNγ 24 h prior to reporter gene assay analysis. Firefly luciferase activity was normalized with Renilla luciferase levels (n = 5) and the data are presented as relative luciferase units (RLU). The results are representative of two independent experiments. ∗ Represents values where difference compared to EV control is statistically significant.

Mentions: The results presented in Fig. 11 show that addition of recombinant IFNγ induced a significant activation of the promoter compared to non-stimulated controls. Significant induction of luciferase activity was registered for overexpressed STAT2a, STAT2b and IRF9 alone, when compared to cells transfected with the plasmid control, and the most pronounced induction was detected for IRF9 transfected cells (p < 0.05). A modest induction of activity was also detected for cells transfected with STAT1a, although, the effect was not statistically significant when compared to the empty vector control. Of the combinations, STAT1a and STAT2b induced the highest GAS-promoter activity, which was significantly higher than each of the two alone, and the combination of STAT1a and STAT2a.


Structural and functional characterization of salmon STAT1, STAT2 and IRF9 homologs sheds light on interferon signaling in teleosts.

Sobhkhez M, Skjesol A, Thomassen E, Tollersrud LG, Iliev DB, Sun B, Robertsen B, Jørgensen JB - FEBS Open Bio (2014)

IRF9, STAT2a and STAT2b stimulate GAS promoter activity upon IFNγ stimulation. CHSE-214 cells transiently transfected with STAT1a, STAT2a, STAT2b, RF9 alone or in combination with each other and GFP-EV (empty vector) was used as control. Transfectants were left non-stimulated or were stimulated with 500 ng/ml of IFNγ 24 h prior to reporter gene assay analysis. Firefly luciferase activity was normalized with Renilla luciferase levels (n = 5) and the data are presented as relative luciferase units (RLU). The results are representative of two independent experiments. ∗ Represents values where difference compared to EV control is statistically significant.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215117&req=5

f0055: IRF9, STAT2a and STAT2b stimulate GAS promoter activity upon IFNγ stimulation. CHSE-214 cells transiently transfected with STAT1a, STAT2a, STAT2b, RF9 alone or in combination with each other and GFP-EV (empty vector) was used as control. Transfectants were left non-stimulated or were stimulated with 500 ng/ml of IFNγ 24 h prior to reporter gene assay analysis. Firefly luciferase activity was normalized with Renilla luciferase levels (n = 5) and the data are presented as relative luciferase units (RLU). The results are representative of two independent experiments. ∗ Represents values where difference compared to EV control is statistically significant.
Mentions: The results presented in Fig. 11 show that addition of recombinant IFNγ induced a significant activation of the promoter compared to non-stimulated controls. Significant induction of luciferase activity was registered for overexpressed STAT2a, STAT2b and IRF9 alone, when compared to cells transfected with the plasmid control, and the most pronounced induction was detected for IRF9 transfected cells (p < 0.05). A modest induction of activity was also detected for cells transfected with STAT1a, although, the effect was not statistically significant when compared to the empty vector control. Of the combinations, STAT1a and STAT2b induced the highest GAS-promoter activity, which was significantly higher than each of the two alone, and the combination of STAT1a and STAT2a.

Bottom Line: Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells.The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone.Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish.

View Article: PubMed Central - PubMed

Affiliation: The Norwegian College of Fishery Science, UiT The Arctic University of Norway, N-9037 Tromsø, Norway.

ABSTRACT
Mammalian IRF9 and STAT2, together with STAT1, form the ISGF3 transcription factor complex, which is critical for type I interferon (IFN)-induced signaling, while IFNγ stimulation is mediated by homodimeric STAT1 protein. Teleost fish are known to possess most JAK and STAT family members, however, description of their functional activity in lower vertebrates is still scarce. In the present study we have identified two different STAT2 homologs and one IRF9 homolog from Atlantic salmon (Salmo salar). Both proteins have domain-like structures with functional motifs that are similar to higher vertebrates, suggesting that they are orthologs to mammalian STAT2 and IRF9. The two identified salmon STAT2s, named STAT2a and STAT2b, showed high sequence identity but were divergent in their transactivation domain (TAD). Like STAT1, ectopically expressed STAT2a and b were shown to be tyrosine phosphorylated by type I IFNs and, interestingly, also by IFNγ. Microscopy analyses demonstrated that STAT2 co-localized with STAT1a in the cytoplasm of unstimulated cells, while IFNa1 and IFNγ stimulation seemed to favor their nuclear localization. Overexpression of STAT2a or STAT2b together with STAT1a activated a GAS-containing reporter gene construct in IFNγ-stimulated cells. The highest induction of GAS promoter activation was found in IFNγ-stimulated cells transfected with IRF9 alone. Taken together, these data suggest that salmon STAT2 and IRF9 may have a role in IFNγ-induced signaling and promote the expression of GAS-driven genes in bony fish. Since mammalian STAT2 is primarily an ISGF3 component and not involved in IFNγ signaling, our finding features a novel role for STAT2 in fish.

No MeSH data available.


Related in: MedlinePlus