Forkhead transcription factor FOXO3a mediates interferon-γ-induced MHC II transcription in macrophages

Abstract

Macrophages are professional antigen-presenting cells relying on the expression of class II major histocompatibility complex (MHC II) genes. Interferon-γ (IFN-γ) activates MHC II transcription via the assembly of an enhanceosome centred on class II trans-activator (CIITA). In the present study, we investigated the role of the forkhead transcription factor FOXO3a in IFN- γ-induced MHC II transcription in macrophages. Knockdown of FOXO3a, but not FOXO1 or FOXO4, diminished IFN-γ-induced MHC II expression in RAW cells. On the contrary, over-expression of FOXO3a, but neither FOXO1 nor FOXO4, enhanced CIITA-mediated trans-activation of the MHC II promoter. IFN-γ treatment promoted the recruitment of FOXO3a to the MHC II promoter. Co-immunoprecipitation and RE-ChIP assays showed that FOXO3a was a component of the MHC II enhanceosome forming interactions with CIITA, RFX5, RFXB and RFXAP. FOXO3a contributed to MHC II transcription by altering histone modifications surrounding the MHC II promoter. Of interest, FOXO3a was recruited to the type IV CIITA promoter and directly activated CIITA transcription by interacting with signal transducer of activation and transcription 1 in response to IFN-γ stimulation. In conclusion, our data unveil a novel role for FOXO3a in the regulation of MHC II transcription in macrophages.

Keywords: macrophage; FOXO; epigenetics; transcriptional regulation.

Figure 1

FOXO3a contributes to interferon‐γ (IFN‐γ) ‐induced MHC II transcription in macrophages. (a) RAW cells were transfected with small interfering RNAs (siRNAs) targeting specific FOXO or scrambled siRNA (SCR) followed by treatment with IFN‐γ. Gene expression levels were examined by quantitative PCR and Western blotting. (b) Bone marrow‐derived macrophages were transfected with siRNAs targeting specific FOXO or scrambled siRNA (SCR) followed by treatment with IFN‐γ. Gene expression levels were examined by quantitative PCR. (c) An MHC II promoter luciferase construct (DRA300) was transfected into HEK293 cells with FOXO and CIITA expression constructs. Luciferase activities were normalized by both protein concentration and GFP fluorescence. (d) An MHC II promoter luciferase construct (DRA300) was transfected into HEK293 cells with FOXO3a and CIITA expression constructs followed by treatment with a Jun N‐terminal kinase (JNK) inhibitor (SP600125). Luciferase activities were normalized by both protein concentration and GFP fluorescence. (e) An MHC II promoter luciferase construct (DRA300) was transfected into HEK293 cells with FOXO3a, JNK and CIITA expression constructs. Luciferase activities were normalized by both protein concentration and GFP fluorescence. (f) An MHC II promoter luciferase construct (DRA300) was transfected into HEK293 cells with FOXO3a and CIITA expression constructs followed by treatment with a SIRT1 agonist (resveratrol). Luciferase activities were normalized by both protein concentration and GFP fluorescence. (g) An MHC II promoter luciferase construct (DRA300) was transfected into RAW cells with FOXO3a, SIRT1 and CIITA expression constructs. Luciferase activities were normalized by both protein concentration and GFP fluorescence. N.S., not statistically significant. Data represent averages of three independent experiments and error bars represent SEM. *P ≤ 0·05.

Figure 2

FOXO3a is a component of the CIITA‐centred MHC II enhanceosome. (a) RAW cells were treated with interferon‐γ (IFN‐γ) and harvested at the indicated time‐points. ChIP assay was performed with anti‐FOXO3a or IgG. (b) HEK293 cells were transfected with indicated expression constructs. Immunoprecipitation was performed with anti‐GFP. (c) RAW cells were treated with IFN‐γ for 12 hr. Re‐ChIP assay was performed with the indicated antibodies. (d, e) RAW cells were transfected with small interfering RNA (siRNA) targeting RFX5 or scrambled siRNA (SCR) followed by treatment with IFN‐γ for 12 hr. Knockdown efficiency was examined by Western blotting. ChIP assay was performed with anti‐FOXO3a. (f) RAW cells were transfected with siRNA targeting FOXO3a or SCR followed by treatment with IFN‐γ for 12 hr. Re‐ChIP assay was performed with the indicated antibodies. Data represent averages of three independent experiments and error bars represent SEM. *P ≤ 0·05.

Figure 3

FOXO3a contributes to MHC II transcription by modulating histone modification. (a–g) RAW cells were transfected with small interfering RNA targeting FOXO3a or SCR followed by treatment with interferon‐γ (IFN‐γ) for 12 hr. ChIP assay was performed with anti‐acetyl histone H3 (a), anti‐trimethyl H3K4 (b), anti‐symmetrical dimethyl H3R2 (c), anti‐CBP (d), anti‐ASH2 (e), anti‐WDR5 (f), or anti‐PRMT5 (g). Data represent averages of three independent experiments and error bars represent SEM. *P ≤ 0·05.

Figure 4

FOXO3a directly regulates type IV CIITA transcription. (a) A CIITA type IV promoter luciferase construct (DRA300) was transfected into HEK293 cells with FOXO followed by treatment with interferon‐γ (IFN‐γ). Luciferase activities were normalized by both protein concentration and GFP fluorescence. (b) RAW cells were treated with IFN‐γ and harvested at the indicated time‐points. ChIP assay was performed with anti‐FOXO3a or IgG. (c) RAW cells were treated with IFN‐γ for 12 hr. Re‐ChIP assay was performed with the indicated antibodies. (d, e) RAW cells were transfected with small interfering RNA (siRNA) targeting STAT1 or scrambled siRNA (SCR) followed by treatment with IFN‐γ for 12 hr. Knockdown efficiency was examined by Western blotting. ChIP assay was performed with anti‐FOXO3a. (f–h) RAW cells were transfected with siRNA targeting FOXO3a or SCR followed by treatment with IFN‐γ for 12 hr. Re‐ChIP assay was performed with indicated antibodies. Data represent averages of three independent experiments and error bars represent SEM. (i) A schematic model. *P ≤ 0·05.