Recruitment of Stat1 to chromatin is required for interferon-induced serine phosphorylation of Stat1 transactivation domain - PubMed (original) (raw)

Recruitment of Stat1 to chromatin is required for interferon-induced serine phosphorylation of Stat1 transactivation domain

Iwona Sadzak et al. Proc Natl Acad Sci U S A. 2008.

Abstract

The transcription factor Stat1 plays an essential role in responses to interferons (IFNs). Activation of Stat1 is achieved by phosphorylation on Y701 that is followed by nuclear accumulation. For full transcriptional activity and biological function Stat1 must also be phosphorylated on S727. The molecular mechanisms underlying the IFN-induced S727 phosphorylation are incompletely understood. Here, we show that both Stat1 Y701 phosphorylation and nuclear translocation are required for IFN-induced S727 phosphorylation. We further show that Stat1 mutants lacking the ability to stably associate with chromatin are poorly serine-phosphorylated in response to IFN-gamma. The S727 phosphorylation of these mutants is restored on IFN-beta treatment that induces the formation of the ISGF3 complex (Stat1/Stat2/Irf9) where Irf9 represents the main DNA binding subunit. These findings indicate that Stat1 needs to be assembled into chromatin-associated transcriptional complexes to become S727-phosphorylated and fully biologically active in response to IFNs. This control mechanism, which may be used by other Stat proteins as well, restricts the final activation step to the chromatin-tethered transcription factor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Y701 is required for S727 phosphorylation induced by type I or type II IFNs. (A) S1-WT cells were treated with IFN-γ for indicated times and phosphorylation of S727 was examined by Western blot analysis of whole-cell extracts with pS727-S1 antibody. The membrane was reprobed with antibody to phosphorylated Y701 (pY701-S1) and Stat1 (S1). (B) Same cells as in A were stimulated with IFN-γ for indicated times and localization of Stat1 was examined by immunofluorescence by using a Stat1 antibody. (C) Localization of S727-phosphorylated Stat1 was examined in untreated or IFN-γ-treated S1-WT cells by double immunofluorescence by using antibodies to pS727-S1 and total Stat1 (S1). (D) S1-WT and S1-Y701F cells were treated for 30 min with anisomycin (an), IFN-γ, IFN-β, or left untreated. Western blot analysis of whole-cell extracts was performed with pS727-S1 antibody, or with Stat1 antibody for loading control. (E) 293HEK cells stably expressing myc-tagged Stat1-WT (293, myc-S1-WT; Upper) or Stat1-Y701F (293, myc-S1-Y701F; Lower) were treated for 30 min with IFN-γ or anisomycin (an). Myc-tagged proteins were immunoprecipitated from extracts by using myc antibody, and analyzed by Western blot analysis with pS727-S1, or S1 (loading control) antibodies. (F) HepG2 cells stably expressing myc-tagged Stat1-WT (HepG2, myc-S1-WT; Upper) or Stat1-Y701F (HepG2, myc-S1-Y701F; Lower) were treated and analyzed as in E.

Fig. 2.

Fig. 2.

Nuclear import is required for IFN-induced S727 phosphorylation of Stat1. (A) Cells stably expressing GFP-S1-WT, GFP-S1-Δ27, or GFP-S1-L407 constructs were treated for 30 min with IFN-γ, IFN-β, or left untreated. Localization of Stat1 was monitored by GFP fluorescence. GFP-S1-WT (B) or GFP-S1-L407A and GFP-S1-Δ27 (C) cells were treated for 30 min with anisomycin (an), IFN-γ, or IFN-β. Phosphorylation of immunoprecipitated Stat1 was detected by Western blot analysis by using pS727-S1 and pY701-S1 antibodies. Membrane was reprobed with Stat1 antibody (S1).

Fig. 3.

Fig. 3.

Nuclear Stat1 is not phosphorylated on S727 unless it becomes Y701-phosphorylated in response to IFN-γ. (A and B) Stat1 was immunoprecipitated from extracts of GFP-NLS-S1 or GFP-NLS-S1-Y701F cells, respectively, treated for 30 min with IFN-γ or anisomycin (an). Phosphorylation of Stat1 on S727 and Y701 was assayed by Western blot analysis by using antibodies to phosphorylated S727 (pS727-S1) and Y701 (pY701-S1). The membrane was reprobed with Stat1 antibody (S1). (C and D) Localization of GFP-NLS-S1 or GFP-NLS-S1-Y701F (respectively) and their S727-phosphorylated forms was investigated by immunofluorescence microscopy by using double staining with antibodies to pS727-S1 and total Stat1 (S1). Cells were treated with IFN-γ or anisomycin (an).

Fig. 4.

Fig. 4.

Stat1 deficient in DNA binding displays strongly reduced S727-phosphorylation in response to IFN-γ. (A) Stat1 was immunoprecipitated from extracts of S1-K336A cells treated with anisomycin (an), IFN-γ, or IFN-β for times indicated. Phosphorylation of Stat1 was assayed by Western blot analysis by using antibodies to phosphorylated S727 (pS727-S1) and Y701 (pY701-S1). The membrane was reprobed with Stat1 antibody (S1). (B) Localization of the S1-K336A mutant in cells treated for 30 min with IFN-γ as revealed by immunofluorescence using S1 antibody. (C) S1-WT and S1-K336A cells were treated with IFN-γ or IFN-β for 1 h (for Irf1 and Mx2) or 4 h (for Tap1 and Gbp2), and total RNA was isolated for qRT-PCR. The graphs show induction of mRNA of Irf1, Tap1, Ggb2, and Mx2. (D) S1-K336A mutant is not recruited to the Irf1 GAS on treatment with IFN-γ or IFN-β (for 15 or 30 min) as revealed by ChIP (Upper) or qChIP (Lower).

Fig. 5.

Fig. 5.

Stable association of Stat1 with chromatin is required for efficient IFN-induced S727 phosphorylation. (A) Stat1 was immunoprecipitated from extracts of S1-K544A/E545A cells treated for 30 min with anisomycin (an), IFN-γ, or IFN-β. Phosphorylation of Stat1 was assayed by Western blot analysis by using antibodies to phosphorylated S727 (pS727-S1) and Y701 (pY701-S1). The membrane was reprobed with Stat1 antibody (S1). (B) Localization of the S1-K544A/E545A linker domain mutant in cells treated for 30 min with IFN-γ as revealed by immunofluorescence by using S1 antibody. (C) Recruitment of the S1- K544A/E545A mutant to the Irf1 GAS is strongly reduced compared with S1-WT in cells treated with IFN-γ or IFN-β (for 15 or 30 min) as revealed by ChIP (Upper) or qChIP (Lower).

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