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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

STAT2a and STAT2b localize in the cytoplasm, whereas IRF9 reside in the nucleus TO cells. (A) GFP-STAT2a non-stimulated (upper panel) and stimulated with IFNa1 (lower panel). (B) GFP-STAT2a non-stimulated (upper panel) and stimulated with IFNγ (lower panel). (C) GFP-STAT2b non-stimulated (upper panel) and stimulated with IFNa1 (lower panel). (D) GFP-STAT2b non-stimulated (upper panel) and stimulated with IFNγ (lower panel). (E) GFP-IRF9 non-stimulated and (F) stimulated with IFNa1. Cultured TO cells was transfected with the indicated constructs for 48 h. Subsequently, the cells were stimulated either with IFNa1 or IFNγ before fixation and counterstaining with Draq5 DNA dye to visualize nuclei and nucleic acids. Laser scanning confocal microscopy was done using Zeiss LSM 510 and high-magnification image analysis was done using a Zeiss LSM image browser.
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f0035: STAT2a and STAT2b localize in the cytoplasm, whereas IRF9 reside in the nucleus TO cells. (A) GFP-STAT2a non-stimulated (upper panel) and stimulated with IFNa1 (lower panel). (B) GFP-STAT2a non-stimulated (upper panel) and stimulated with IFNγ (lower panel). (C) GFP-STAT2b non-stimulated (upper panel) and stimulated with IFNa1 (lower panel). (D) GFP-STAT2b non-stimulated (upper panel) and stimulated with IFNγ (lower panel). (E) GFP-IRF9 non-stimulated and (F) stimulated with IFNa1. Cultured TO cells was transfected with the indicated constructs for 48 h. Subsequently, the cells were stimulated either with IFNa1 or IFNγ before fixation and counterstaining with Draq5 DNA dye to visualize nuclei and nucleic acids. Laser scanning confocal microscopy was done using Zeiss LSM 510 and high-magnification image analysis was done using a Zeiss LSM image browser.

Mentions: In order to study the sub-cellular localization, TO cells were transiently transfected with expression plasmids coding for GFP-tagged STAT2a, STAT2b, or IRF9 (Fig. 8A–F) and subjected to confocal microscopy. The results showed that both STAT2a and STAT2b resided in the cytoplasm of both stimulated and unstimulated cells (Fig. 7A–D). Even though IRF9 was mainly located in the nucleus, in some of the transfectants it was detectable in cytoplasm of both unstimulated and IFNa1 stimulated cells (Fig. 7E–F).


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)

STAT2a and STAT2b localize in the cytoplasm, whereas IRF9 reside in the nucleus TO cells. (A) GFP-STAT2a non-stimulated (upper panel) and stimulated with IFNa1 (lower panel). (B) GFP-STAT2a non-stimulated (upper panel) and stimulated with IFNγ (lower panel). (C) GFP-STAT2b non-stimulated (upper panel) and stimulated with IFNa1 (lower panel). (D) GFP-STAT2b non-stimulated (upper panel) and stimulated with IFNγ (lower panel). (E) GFP-IRF9 non-stimulated and (F) stimulated with IFNa1. Cultured TO cells was transfected with the indicated constructs for 48 h. Subsequently, the cells were stimulated either with IFNa1 or IFNγ before fixation and counterstaining with Draq5 DNA dye to visualize nuclei and nucleic acids. Laser scanning confocal microscopy was done using Zeiss LSM 510 and high-magnification image analysis was done using a Zeiss LSM image browser.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

f0035: STAT2a and STAT2b localize in the cytoplasm, whereas IRF9 reside in the nucleus TO cells. (A) GFP-STAT2a non-stimulated (upper panel) and stimulated with IFNa1 (lower panel). (B) GFP-STAT2a non-stimulated (upper panel) and stimulated with IFNγ (lower panel). (C) GFP-STAT2b non-stimulated (upper panel) and stimulated with IFNa1 (lower panel). (D) GFP-STAT2b non-stimulated (upper panel) and stimulated with IFNγ (lower panel). (E) GFP-IRF9 non-stimulated and (F) stimulated with IFNa1. Cultured TO cells was transfected with the indicated constructs for 48 h. Subsequently, the cells were stimulated either with IFNa1 or IFNγ before fixation and counterstaining with Draq5 DNA dye to visualize nuclei and nucleic acids. Laser scanning confocal microscopy was done using Zeiss LSM 510 and high-magnification image analysis was done using a Zeiss LSM image browser.
Mentions: In order to study the sub-cellular localization, TO cells were transiently transfected with expression plasmids coding for GFP-tagged STAT2a, STAT2b, or IRF9 (Fig. 8A–F) and subjected to confocal microscopy. The results showed that both STAT2a and STAT2b resided in the cytoplasm of both stimulated and unstimulated cells (Fig. 7A–D). Even though IRF9 was mainly located in the nucleus, in some of the transfectants it was detectable in cytoplasm of both unstimulated and IFNa1 stimulated cells (Fig. 7E–F).

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