Differential requirement of histone acetylase and deacetylase activities for IRF5-mediated proinflammatory cytokine expression

Abstract

Recent evidence indicates a new role for histone deacetylases (HDACs) in the activation of genes governing the host immune response. Virus, along with other pathogenic stimuli, triggers an antiviral defense mechanism through the induction of IFN, IFN-stimulated genes, and other proinflammatory cytokines. Many of these genes have been shown to be regulated by transcription factors of the IFN regulatory factor (IRF) family. Recent studies from IRF5 knockout mice have confirmed a critical role for IRF5 in virus-induced type I IFN expression and proinflammatory cytokines IL-6, IL-12, and TNF-α; yet, little is known of the molecular mechanism of IRF5-mediated proinflammatory cytokine expression. In this study, we show that both HDACs and histone acetyltransferases (HATs) associate with IRF5, leading to alterations in its transactivation ability. Using the HDAC inhibitor trichostatin A, we demonstrate that ISRE, IFNA, and IL6 promoters require HDAC activity for transactivation and transcription, whereas TNFα does not. Mapping the interaction of corepressor proteins (HDAC1, silencing mediator of retinoid and thyroid receptor/nuclear corepressor of retinoid receptor, and Sin3a) and HATs to IRF5 revealed distinct differences, including the dependence of IRF5 phosphorylation on HAT association resulting in IRF5 acetylation. Data presented in this study support a mechanism whereby virus triggers the dynamic conversion of an IRF5-mediated silencing complex to that of an activating complex on promoters of target genes. These data provide the first evidence, to our knowledge, of a tightly controlled transcriptional mechanism whereby IRF5 regulates proinflammatory cytokine expression in conjunction with HATs and HDACs.

FIGURE 1

TSA selectively impairs IRF5-mediated proinflammatory cytokine expression. A, 2fTGH or 2fTGH/IRF5 V3/V4 stable expressing cells were transiently transfected with ISRE, IFNA1, IL6, or TNFA luciferase promoter reporters and left uninfected or infected with NDV. Fold relative activity is shown compared with untreated controls after normalization to protein and thymidine Renilla-luciferase activity. Results are representative of at least three independent experiments run in triplicate. B, Same as in A except cells were also treated with TSA. C, Levels of endogenous IFNA, IL6, and TNFA transcripts were detected by semiquantitative PCR in 2fTGH/IRF5-expressing cells left uninfected (0 h) or infected with NDV (6 h) in the presence or absence of TSA. Levels of β-actin are shown as a loading control and for RNA integrity. Results from the endogenous ChIP assay in THP-1 cells left uninfected (0 h) or infected with NDV over the indicated time course are shown in the bottom panels. Cell lysates were immunoprecipitated with anti-IRF5 Abs and bound DNA amplified with primers recognizing PRD/ISRE/IRF-E regions in each promoter. Results are representative of three independent experiments. D, Same as in A except increasing levels of HDAC1 were cotransfected with the indicated promoters to 2fTGH cells. *p < 0.05 compared with IRF5 expressing (2f/5) uninfected (cont) cells.

FIGURE 2

IRF5 interacts with corepressor proteins to repress IFNA/IFN-α expression. A, 2fTGH and 2fTGH/IRF5 V3/V4-expressing cells were cotransfected with an IFNA1 luciferase reporter and equal amounts of individual corepressor proteins (HDAC1, SMRT, and Sin3a) or the combination of HDAC1, SMRT, and Sin3a. Cells were left uninfected or infected with virus for 16 h and relative luciferase activity determined after normalization to protein and thymidine Renilla-luciferase activity. Supernatants were assayed for endogenous biologically active type I IFNs by the cytopathic effect assay in HF cells. Levels of synthesized type I IFNs are shown as units/ml. B, Coimmunoprecipitations were performed in 2fTGH and 2fTGH/IRF5 expressing cells left uninfected or infected with NDV for 6 h. Endogenous proteins were immunoprecipitated as indicated and bound IRF5 detected with anti-Flag Abs. Levels of proteins expressed are shown in the bottom panels. Results are representative of three independent experiments. *p < 0.05 compared with 2fTGH/IRF5 cells lacking corepressors.

FIGURE 3

Virus triggers interaction of IRF5 with HAT proteins. A, Coimmunoprecipitations were performed in 2fTGH- or 2fTGH/IRF5-expressing cells left uninfected or infected with NDV for 6 h. In the top panels, endogenous proteins were immunoprecipitated as indicated and bound IRF5 detected with anti-Flag Abs. In the bottom panels, Flag-tagged PCAF and myc-tagged IRF5 were transiently transfected to 2fTGH cells and infected as described above. Reciprocal immunoprecipitations were performed as indicated. Levels of proteins expressed are shown. Results are representative of three independent experiments. B, 2fTGH and 2fTGH/IRF5 V3/V4-expressing cells were cotransfected with an IFNA1 SAP promoter reporter and equal amounts of the indicated HATs. Cells were left uninfected or infected with NDV for 16 h. Relative SAP activity was determined after normalization to protein and β-galactosidase. Experiments were performed in triplicate and repeated three times. C, Same as in B except transactivation of the ISRE luciferase reporter was determined. D, 2fTGH/IRF5-expressing cells were left uninfected or infected with the indicated virus for 6 h and immunoprecipitated with Abs recognizing phosphorylated Ser⁴²⁷ and Ser⁴³⁰ of V3/V4. Interaction of IRF5 with p300 was determined with anti-p300 Abs. Results are representative of three independent experiments. *p < 0.05 compared with 2f/IRF5 NDV-infected cells lacking HATs.

FIGURE 4

HDAC corepressor proteins and HATs interact with distinct regions of IRF5. A, Interaction of ectopically expressed HDAC corepressor proteins or HATs with IRF5 was determined by the GST pulldown assay. 2fTGH cells were transiently transfected with the indicated expression plasmids and whole cell lysates applied to GST or GST-IRF5 Sepharose beads containing the N terminus (5 N) or the C terminus of IRF5 V3 (5C). Bound proteins were resolved by SDS-PAGE and detected with Abs recognizing Sin3a, SMRT, HDAC1, CBP, or p300. Levels of each GST fusion protein are shown after staining with Coomassie blue. Results are representative of three independent experiments. B, 2fTGH cells were transiently transfected with gfp vector control plasmid, gfp-IRF5N, or gfp-IRF5C and left uninfected or infected with NDV for 6 h. Immunoprecipitations were performed with Abs recognizing the indicated endogenous proteins and bound gfp-IRF5 was detected with anti-gfp Abs. Levels of ectopically expressed gfp fusion proteins are shown at the bottom. Results are representative of three independent experiments.

FIGURE 5

IRF5 is acetylated in vivo after NDV infection or coexpression with HATs. A, 2fTGH/IRF5-expressing cells were left uninfected, infected with NDV, or transiently transfected with PCAF, CBP, or p300 expression plasmids. Whole cell lysates were immunoprecipitated with either anti-acetyl lysine Abs or IRF5 Abs and acetylated IRF5 detected with the reciprocal Ab. Levels of transfected proteins are shown at the bottom. Results are representative of three independent experiments. B, NDV-induced acetylation of IRF5 occurs in both the amino- and C terminus. gfp-IRF5 fusion proteins were transiently transfected to 2fTGH cells and left uninfected or infected with NDV. Similar to A, whole cell lysates were immunoprecipitated with anti-acetyl lysine Abs and acetylated IRF5 detected with anti-gfp Abs. The same membrane was stripped and reprobed with anti-IRF3 Abs. Results are representative of three independent experiments. C, Treatment with TSA increases basal IRF5 acetylation. 2fTGH and 2fTGH/IRF5 expressing cells were left untreated or treated with TSA, immunoprecipitated with antiacetyl lysine Abs, and acetylated IRF5 detected with anti-IRF5. Results are representative of three independent experiments.

FIGURE 6

Alterations in IRF5-mediated IFNA promoter regulation are dependent on virus. A, In vivo binding of IRF5 in THP-1 cells was determined by the ChIP assay. THP-1 cells were left uninfected or infected with NDV for 3 h. DNA recovered from ChIP after immunoprecipitation with the indicated Abs was amplified using universal primers specific for all endogenous IFNA gene promoters. Input corresponds to the amplified IFNA promoter region from DNA-protein complexes before immunoprecipitation; IgG controls are shown for IRF5 Ab specificity. Results are representative of three independent experiments. B, Cellular localization of endogenous SMRT was determined by cytoplasmic (C)/nuclear (N) extraction from THP-1 cells left uninfected or infected with NDV for 3 h. Localization of SMRT, HDAC1, and Sp1 were determined by immunoblot with Abs specific for each. C, Schematic representation of how IRF5 switches from a silencing to an activating complex in cells expressing high levels of IRF5. In uninfected cells, the IFNA promoter is silenced by chromatin remodeling through the binding of a corepressor complex comprising of HDAC1, Sin3a, and SMRT to the N terminus of IRF5 V3; histones are deacetylated. Virus infection triggers a change not only in the IRF5 polypeptide leading to enhanced nuclear localization, but it also signals the export of SMRT out of the nucleus to the cytoplasm, resulting in the association of IRF5 with coactivator proteins p300/CBP and acetylation of histones resulting in gene transcription. AC, acetylated histones; P, IRF5 phosphorylation.