Interferon-stimulated transcription and innate antiviral immunity require deacetylase activity and histone deacetylase 1

Abstract

The use of histone deacetylase (HDAC) inhibitors has revealed an essential role for deacetylation in transcription of IFN-responsive genes. The HDAC1 protein associates with both signal transducer and activator of transcription (STAT) 1 and STAT2, and IFN-alpha stimulation induces deacetylation of histone H4. Inhibition of HDAC1 by small interfering RNA (siRNA) decreases IFN-alpha responsiveness whereas expression of HDAC1 augments the IFN-alpha response, demonstrating that HDAC1 modulates IFN-alpha-induced transcription. Importantly, the innate antiviral response is inhibited in the absence of deacetylase activity. The requirement for deacetylase is shared by IFN-gamma transcription response and may represent a general requirement for STAT-dependent gene expression.

Fig. 2.

IFN-responsive STAT activation, dimerization, nuclear translocation, and DNA binding activity are intact in TSA-treated cells. (A) 2fTGH cells were treated with IFN-α alone or together with TSA for the indicated times. Protein extracts were analyzed by Western blot (WB) by using antibodies specific for tyrosine phosphorylated STATs (pSTAT1, pSTAT2), or total STATs (STAT1, STAT2). (B) 2fTGH cells were treated as in A, but protein extracts were subject to immunoprecipitation (IP) with STAT2 antiserum. Immune complexes were analyzed by Western blot for coprecipitated STAT1α and STAT1β. (C) 2fTGH cells were treated with IFN-α alone or together with TSA and processed for indirect immunofluorescence to detect STAT1, STAT2, and IRF9. (D) 2fTGH cells were treated with IFN-α and TSA for the indicated times, and lysates were subjected to EMSA by using a ³²P-labeled ISG15-ISRE element probe. ISGF3 complex identity was confirmed by a STAT2 antibody supershift (αS2).

Fig. 1.

HDAC inhibition impairs IFN-α-stimulated gene activation. (A) 2fTGH cells were treated with IFN-α for the indicated amounts of time in the presence (+) or absence (–) of simultaneous TSA. RT-PCR was carried out with primers specific for the indicated genes. (B) Similar to A, but NaB was used. (C) 2fTGH cells were transfected with ISRE-luciferase reporter together with CMV-LacZ, treated as indicated, and analyzed by luciferase assay. Luciferase units were normalized to β-galactosidase. (Inset) β-Galactosidase levels, normalized to total protein concentration. (D Left) 2fTGH cells were transfected with empty vector or expression vectors for IRF9 or IRF9-S2C along with ISRE-luciferase reporter and Renilla luciferase control. *, P < 0.05. (Right) Lysates were tested for DNA binding by EMSA by using a ³²P-labeled ISG15-ISRE oligonucleotide as probe. Supershift was performed with STAT2 antibody (αS2). (E) Similar to C, but cells were transfected with IFN-γ-responsive M67-luciferase reporter gene along with Renilla luciferase reporter plasmid for normalization, and treated as indicated. Luciferase values were normalized to Renilla luciferase values. (Inset) Renilla luciferase values normalized to total protein concentration. (F) 2fTGH cells were treated as indicated, and total RNA was analyzed by RT-PCR using primers specific for IP-10 and GAPDH.

Fig. 3.

IFN deacetylates histone H4 and requires essential STAT–HDAC1 associations. (A Left) 2fTGH cells were treated with IFN-α for the times indicated and subjected to a ChIP assay using antibody specific for acetylated histone H4. Coprecipitated DNA was amplified by PCR by using primers specific for the ISG54 ISRE promoter element. (Right) HeLa cells were used in a similar assay in the presence or absence of TSA. (B) 2fTGH cells were transfected with expression vectors for FLAG epitope-tagged GFP, HDAC1, HDAC4, or HDAC5, stimulated with (+) or without (–) IFN-α as indicated, and lysates were precipitated with FLAG affinity resin. Eluted STAT1 and STAT2 were detected by Western blotting. Control FLAG immunoblot demonstrates equivalent precipitation of expressed proteins in the eluates; bottom, STAT2 immunoblot of unprecipitated extracts (Input) demonstrates equivalent loading. (C) Coimmunoprecipitation of STAT2 and HDAC1 from 2fTGH cell extracts. RαM, rabbit anti-mouse IgG; ns, nonspecific band. (D) Similar to B, but 2fTGH cells were transfected with IRF9-S2C and FLAG-HDAC1 or Flag-GFP expression plasmids before Flag affinity precipitation and analysis by Western blotting with STAT2 (Top) and Flag antibodies (Middle). Input panel demonstrates similar expression and loading levels. (E) 2fTGH cells were transfected with siRNA specific for HDAC1 or a scrambled siRNA control (Con) along with ISRE-luciferase reporter gene and Renilla luciferase control for normalization. *, P < 0.005. (F) Similar to E, except total RNA was isolated from cells and subjected to RT-PCR for ISG54 mRNA. IFN-α treatments were performed for 3 h. con = scrambled siRNA duplex. (G) 293T cells were transfected with ISRE-luciferase and Renilla luciferase in the absence (–) or presence of 1, 2, or 6 μg of HDAC1 expression plasmid, and treated with IFN-α as indicated. (H) 2fTGH cells were treated with (+) or without (–) IFN-α and/or TSA for 4 h followed by infection of duplicate wells with serially diluted vesicular stomatitis virus (VSV). Pfu, plaque-forming units.