Fig 2: Model describing transcriptional regulation of Signal Integrated genes by STAT1-mediated preceding of p65 and PolII to acetylated GAS/NFκB or ISRE/NFκB composite sites. 1st wave of stimulation: After initial cell exposure to IFNα or IFNγ, receptors dimerize, and facilitate transphosphorylation of receptor-bound JAK1/TYK2 kinases for IFNα and JAK1/JAK2 kinases for IFNγ. Next STAT proteins are recruited, phosphorylated and dimerized, either in a form of ISGF3 complex (STAT1-STAT2 together with IRF9), GAF (STAT1 homodimers) or STAT1/IRF9 complex. Activated transcription factors supply a platform for 2nd wave of stimulation: LPS stimulates TLR4 receptor associated with adapter molecules MyD88 and TRAM and activates NFκB, as well as STAT1-containing transcriptional complexes. IFNα stimulation results in recruitment of ISGF3 to ISRE sites and GAF to GAS sites present in ISGs promoters. IFNγ initiates binding of GAF to GAS sites as well as ISGF3 and possibly STAT1/IRF9 to ISRE elements. Initial binding of STAT1-containing complexes followed by subsequent p65-p50 heterodimers binding (indicated by a violet curved arrow) to NFκB sites closely spaced to ISRE and GAS sites (~200 bp), together results in histone acetylation enrichment and PolII recruitment to ISG promoters. For a detailed explanation, see the text.

Fig 3: IRF9 regulates IL-6 transcription. (A) Transcript expression of Il6, Tnf, Cxcl1 (Kc), and Il1b in the colon tissues of wild type (WT) (n = 8) and Irf9−/− (n = 7) mice14 days after AOM injection and (B) in LPS-stimulated WT and Irf9−/− bone marrow-derived macrophages (BMDMs) at 1 and 4 h post-stimulation. (C) ChIP-Seq analysis of IRF9 binding to the Il6 (top) and Il1b (bottom) promoter. Green highlights the IRF9 binding site. Each symbol represents an individual mouse in A. ns, not statistically significant. * p < 0.05. Two-way ANOVA was used in A. Data are represented as mean ± SEM in (A,B).

Fig 4: IRF9 does not regulate inflammatory signaling but does regulate IL-6 production in the colon. (A) Immunoblot analysis of phosphorylated and total IκBα (pIκBα and IκBα, respectively), phosphorylated and total p38 (p-p38 and p38, respectively), IRF9, and GAPDH (loading control) in colon lysates from wild type (WT) and Irf9−/− mice. Each lane corresponds to an individual mouse. (B) Levels of inflammatory cytokines in the colon of WT (n = 7–9) and Irf9−/− (n = 7–8) mice 14 days after AOM injection. (C) Levels of inflammatory cytokines in the serum of WT (n = 8–10) and Irf9−/− (n = 8–10) mice 14 days after AOM injection. Each symbol represents an individual mouse in (B,C). ns, not statistically significant. *** p < 0.001, **** p < 0.0001. Two-tailed t-test was used in (B,C). Data are represented as mean ± SEM in (B,C).

Fig 5: IRF9-deficient mice are resistant to the development of colorectal cancer. (A) Body weight change of wild type (WT) (n = 10) and Irf9−/− (n = 8) littermate mice from one experiment, representative of three independent experiments. (B) Representative images of colon tumors in littermate WT and Irf9−/− mice 80 days after injection of azoxymethane (AOM). (C) Number and (D) size of colon tumors in WT (n = 10) and Irf9−/− (n = 8) mice. (E) Representative hematoxylin and eosin staining of colon tumors; scale bar, 2 mm for whole scan and 500 μm for 10×. (F) Histological scores 80 days after injection of AOM in WT (n = 8) and Irf9−/− (n = 7) mice. (G) Representative images of colon tumors in 120-day-old littermate ApcMin/+ and ApcMin/+Irf9−/− mice. (H) Number of colon tumors in 120-day-old ApcMin/+ (n = 5) and ApcMin/+Irf9−/− mice (n = 5) mice. (I) Percentage of tumors of various sizes in 120-day-old littermate ApcMin/+ (n = 5) and ApcMin/+Irf9−/− (n = 5) mice. Each symbol represents 1 individual mouse (C,F,H). * p < 0.05; ** p < 0.01; **** p < 0.0001. Two-tailed t-test (C,F,H) and one-way ANOVA (A) were used. Data are represented as mean ± SEM in (A,C,F,H).