Acetylation modulates cellular distribution and DNA sensing ability of interferon-inducible protein IFI16

Abstract

Detection of pathogenic nucleic acids is essential for mammalian innate immunity. IFN-inducible protein IFI16 has emerged as a critical sensor for detecting pathogenic DNA, stimulating both type I IFN and proinflammatory responses. Despite being predominantly nuclear, IFI16 can unexpectedly sense pathogenic DNA in both the cytoplasm and the nucleus. However, the mechanisms regulating its localization and sensing ability remain uncharacterized. Here, we propose a two-signal model for IFI16 sensing. We first identify an evolutionarily conserved multipartite nuclear localization signal (NLS). Next, using FISH and immunopurification, we demonstrate that IFI16 detects HSV-1 DNA primarily in the nucleus, requiring a functional NLS. Furthermore, we establish a localization-dependent IFN-β induction mediated by IFI16 in response to HSV-1 infection or viral DNA transfection. To identify mechanisms regulating the secondary cytoplasmic localization, we explored IFI16 posttranslation modifications. Combinatorial MS analyses identified numerous acetylations and phosphorylations on endogenous IFI16 in lymphocytes, in which we demonstrate an IFI16-mediated IFN-β response. Importantly, the IFI16 NLS was acetylated in lymphocytes, as well as in macrophages. Mutagenesis and nuclear import assays showed that NLS acetylations promote cytoplasmic localization by inhibiting nuclear import. Additionally, broad-spectrum deacetylase inhibition triggered accumulation of cytoplasmic IFI16, and we identify the acetyltransferase p300 as a regulator of IFI16 localization. Collectively, these studies establish acetylation as a molecular toggle of IFI16 distribution, providing a simple and elegant mechanism by which this versatile sensor detects pathogenic DNA in a localization-dependent manner.

Fig. 1.

Nuclear localization of IFI16 requires a multipartite NLS. (A) Schematic of IFI16 and alignment of the multipartite NLS of IFI16, IFIX, and MNDA. (B) Alignment of NLS sequences of mammalian IFI16 homologs. (C) Fluorescent microscopy of IFI16-EGFP WT and NLS deletion mutants (transient transfections in U2OS cells) with 20× objective. The nucleus is stained with DAPI, and the cytoplasm is stained with CellMask. (D) Quantification of relative nuclear abundances by an Operetta screen (mean ± SD of 3 biological replicates).

Fig. 2.

IFI16 sensing of viral DNA is localization-dependent. Cells were infected with HSV-1 for the indicated times (A, B, and D) or transfected with a mixture of four HSV-1 DNA fragments (C) or VACV 70mer DNA (E) for 3 h. (A) FISH assays demonstrate colocalization of HSV-1 genomic DNA with WT IFI16 but not with NLS mutants at 2 hpi of HSV-1 infection in U2OS cells 63× oil objective. (B) At 2 hpi, nuclear IFI16 binds more HSV-1 DNA than the NLS mutants in U2OS cells, as measured by co-IP of protein–DNA complexes and qPCR with four HSV-1 primer sets. DNA levels were normalized to isolated protein levels. (C) Cytoplasmic NLS mutants bind more transfected HSV-1 DNA fragments. (D and E) IFN-β expression following HSV-1 infection (2 and 6 hpi) or VACV 70mer transfection in FlpIn293 cells was quantified by qPCR and normalized to corresponding mock treatments. Mean ± SD, n = 3. *P < 0.05; **P < 0.01.

Fig. 3.

Endogenous IFI16 is acetylated within the NLS and mediates a type I IFN response in lymphocytes. shRNA-mediated knockdown of IFI16 (A) compromises IFN-β expression in response to VACV 70mer transfection in CEM-T lymphocytes (B). (C) Combinatorial mass spectrometric approach to identify PTMs on endogenous IFI16 from CEM-T cells. (D) Coomassie-stained SDS/PAGE shows efficient IFI16 isolation; dotted lines indicate IFI16 isoforms. (E) Map of IFI16 phosphorylations (red pins) and acetylations (green pins). (F) Identification of NLS acetylations (ac) using ETD and CID MS/MS.

Fig. 4.

IFI16 NLS acetylation prevents nuclear import. (A) Confocal microscopy of K99 and K128 mutants 40× objective. (B) Relative nuclear abundance of IFI16 mutants quantified by an Operetta screen. (C) (Left) Direct fluorescence images illustrate the nuclear import levels for peptidyl GST-EGFP proteins. DIC, differential interference contrast microscopy. Magnification, 20× objective. (Right) Fluorescence intensity histograms of 10⁴ nuclei measured by flow cytometry reflect import levels. The FlnIn293 IFI16-EGFP cell line was treated with trichostatin A (TSA) or mock for 6 h (D) or transfected with p300-myc or P/CAF-FLAG for 12 h (E). Localization of IFI16-EGFP, nuclear matrix protein p84, and acetyltransferases was visualized by confocal microscopy with a 63× objective. (F) IFI16 motif-1 peptide can be acetylated by the catalytic domain of p300 acetyltransferase in vitro.

Fig. 5.

Working model for the localization-dependent sensing activity of IFI16. Detection of herpes viral DNA occurs in the nucleus, and detection of transfected DNA or cytoplasmic viral DNA occurs in the cytoplasm. A multipartite NLS is required for nuclear import. NLS acetylation impedes nuclear import of newly synthesized IFI16 and is regulated by HDACs and p300. Ac, lysine acetylation.