Induction of interferon-stimulated gene expression and antiviral responses require protein deacetylase activity - PubMed (original) (raw)

Induction of interferon-stimulated gene expression and antiviral responses require protein deacetylase activity

Hao-Ming Chang et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Histone deacetylase (HDAC) activity, commonly correlated with transcriptional repression, was essential for transcriptional induction of IFN-stimulated genes (ISG). Inhibition of HDAC function led to global impairment of ISG expression, with little effect on basal expression. HDAC function was not required for signal transducer and activator of transcription tyrosine phosphorylation, nuclear translocation, or assembly on chromatin, but it was needed for full activity of the signal transducer and activator of transcription transactivation domain. HDAC function was also required for gene induction driven by the IFN regulatory factor 3 transcription factor activated by virus infection, and it was essential for establishment of an antiviral response against Flaviviridae, Rhabdoviridae, and Picornaviridae. Requirement for HDAC function in transcriptional activation may represent a general mechanism for rapid stimulation of ISG transcription.

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Figures

Fig. 1.

Fig. 1.

Global impairment of IFN-stimulated gene expression by HDAC inhibition. (A) U2OS cells were stimulated with IFNα in the absence (filled bars) or presence of TSA (open bars) or were treated with TSA alone (hatched bars) for 8 h. Isolated RNA was analyzed by two-color microarray hybridization in comparison with RNA isolated from untreated cells. A Upper represents genes induced by IFNα >2.5-fold; A Lower includes genes induced >2-fold. (B) HeLa cells were treated with IFNα, TSA, and VPA, as indicated, for 6 h before isolation of RNA. Expression of ISG54 was quantified by real-time RT-PCR and represented as fold induction over untreated cells. (C) Expression of additional ISG transcripts was analyzed as in B, except that HeLa cells were treated overnight with IFNγ; cells were treated with IFNα (filled bars), TSA (hatched bars), or IFNα plus TSA (open bars) for 4.5 h before isolation of RNA and analysis by quantitative RT-PCR with primers specific for the indicated genes. (D) As in B, except human fibroblast FS2 cells were treated for 1 h with IFNα and/or TSA, as indicated, before quantitation of ISG54 transcript levels. (E) Western blot analysis of IRF9 and β-tubulin expression in HeLa cells treated with TSA (T), IFNγ (γ), and IFNα (α), as indicated. (F) As in C, except expression of ISG54 nuclear RNA precursors rather than mature mRNA was quantified by using an intron/exon primer pair.

Fig. 2.

Fig. 2.

HDAC function is required for transcription without affecting Janus kinase–Stat signaling. (A) HeLa cells were untreated or treated with IFNα with or without TSA for 1 h, before isolation of nuclei for in vitro run-on transcription reactions. Labeled RNA was hybridized with filter-bound cDNA for the indicated sequences and quantified by PhosphorImager analysis. (B) HeLa cells transfected with ISG54-luciferase together with CMV-LacZ were treated with TSA and IFNα, as indicated, and cell extracts were analyzed for luciferase activity, normalized to β-galactosidase activity, and reported as fold induction over untreated cells. (C) HeLa cells were untreated or treated with IFNα in the absence or presence of TSA, as indicated, before isolation of nuclear extracts. Western blots were probed with antibodies against phospho-Stat1 and Stat2, total Stat1 and Stat2, IRF9, and RPA-2, as indicated. (D) Extracts from HeLa cells treated with IFNα with or without TSA were analyzed for the presence of ISGF3 by electrophoretic mobility-shift assay. (E) ISG54 transcript levels were quantified from HeLa cells treated with IFNα, TSA, and Cx, as indicated. (F) HeLa cells were transfected with UAS-luciferase along with Gal4 DNA-binding domain alone or fused with the transactivation domain of Stat2 or VP16. Fold change in luciferase activity in the presence of TSA or VPA relative to untreated samples is shown.

Fig. 3.

Fig. 3.

HDAC function is not required for ISGF3 assembly on chromatin but is essential for IFN suppression of HCV replication. (A) ChIP assays were performed on HeLa cells treated with IFNα, TSA, and Cx, as indicated, with antibodies against Stat2. Amounts of ISG54, ISG15, and actin promoter sequences recovered in immunoprecipitates relative to input levels were quantified by real-time PCR and reported as fold induction relative to untreated cells. (B) As in A, except promoter sequences of OAS2, gene 9–27, and gene 6–16 were quantified and reported relative to maximal amount recovered from IFNα-treated cells. (C) Induction of ISG15 and ISG54 transcript levels was quantified in Vero cells infected with NDV in the absence or presence of TSA. (D) NDV transcripts from Vero cells infected in the absence (filled bar) or presence of TSA (open bar) were quantified by RT-PCR. (E) Inhibition of HCV replication by IFNα treatment in the absence or presence of HC toxin was monitored by RT-PCR.

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