Acetylation Modulates Cellular Distribution and DNA Sensing Ability of Interferon-Inducible Protein IFI16

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

Tuo Li, Benjamin A. Diner, Jin Chen, and Ileana M. Cristea1

Department of Molecular Biology, Princeton University, Princeton, NJ 08544

Edited* by Diane E. Griffin, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, and approved May 23, 2012 (received for review February 27, 2012) Detection of pathogenic nucleic acids is essential for mammalian in both the cytoplasm and the nucleus. Endogenous IFI16 was innate immunity. IFN-inducible protein IFI16 has emerged as a critical shown to colocalize with transfected vaccinia virus (VACV) sensor for detecting pathogenic DNA, stimulating both type I IFN and dsDNA in the cytoplasm of differentiated THP-1 monocytes (1). In proinflammatory responses. Despite being predominantly nuclear, contrast, nuclear IFI16 colocalized with KSHV DNA during early IFI16 can unexpectedly sense pathogenic DNA in both the cytoplasm infection, consequently activating inflammasome formation (3). and the nucleus. However, the mechanisms regulating its localiza- These findings contradict the canonical notion that sensing of tion and sensing ability remain uncharacterized. Here, we propose pathogenic DNA is solely a cytoplasmic process, suggesting that a two-signal model for IFI16 sensing. We first identify an evolution- both cytoplasmic and nuclear IFI16 may participate in viral DNA arily conserved multipartite nuclear localization signal (NLS). Next, surveillance. Hence, it is critical to understand the localization- using FISH and immunopurification, we demonstrate that IFI16 dependent DNA sensing properties of IFI16. As reported for other detects HSV-1 DNA primarily in the nucleus, requiring a functional innate sensors (e.g., Toll-like receptors), immune response may be NLS. Furthermore, we establish a localization-dependent IFN-β in- dictated by the sensing context and cell type (11, 12). However, duction mediated by IFI16 in response to HSV-1 infection or viral the precise molecular mechanisms regulating IFI16 localization DNA transfection. To identify mechanisms regulating the secondary remain uncharacterized. Furthermore, the roles of subcellular fi cytoplasmic localization, we explored IFI16 posttranslation modi - localization in its DNA sensing function have not been assessed. fi cations. Combinatorial MS analyses identi ed numerous acetyla- Here, we used an integrative multidisciplinary approach to tions and phosphorylations on endogenous IFI16 in lymphocytes, provide evidence for a two-signal model for the function of IFI16 β in which we demonstrate an IFI16-mediated IFN- response. Impor- as a pathogenic DNA sensor. We define an evolutionarily con- tantly, the IFI16 NLS was acetylated in lymphocytes, as well as in served multipartite nuclear localization signal (NLS) required for macrophages. Mutagenesis and nuclear import assays showed that IFI16 sensing of HSV-1 viral DNA in the nucleus. We identify NLS NLS acetylations promote cytoplasmic localization by inhibiting acetylation as a molecular toggle of IFI16 localization and p300 as nuclear import. Additionally, broad-spectrum deacetylase inhibition a contributing acetyltransferase. Collectively, our results provide triggered accumulation of cytoplasmic IFI16, and we identify the critical insights into how IFI16 expands its range of surveillance acetyltransferase p300 as a regulator of IFI16 localization. Collec- against pathogenic DNA in a localization-dependent manner. tively, these studies establish acetylation as a molecular toggle of IFI16 distribution, providing a simple and elegant mechanism by Results which this versatile sensor detects pathogenic DNA in a localiza- IFI16 Has a Multipartite NLS. To study mechanisms regulating its tion-dependent manner. localization, we first searched for IFI16 NLS motifs. A putative bipartite NLS (residues 96–135) that included two lysine/arginine- proteomics | HIN200 protein | posttranslational modification | rich motifs, 96RKRKK100 (motif-1) and 128KRKK132 (motif-2), mass spectrometry | histone deacetylase was predicted (Fig. 1A and Fig. S1A). Indeed, a peptide including motif-2, 127QKRKKSTKEKA138, was shown to mediate nuclear he onset of mammalian innate immunity is marked by rec- import of a GST-peptide fusion (13). NLS motif-1 and motif-2 are Tognition of pathogen-associated molecular patterns by a rep- conserved among nuclear HIN200 proteins MNDA and IFIX, as fi ertoire of host sensors. Cellular localizations, target speci cities, well as in mammalian IFI16 homologs (Fig. 1 A and B and Fig. fi and downstream signaling pathways de ne their functions. The S1B). IFI16 also contains partial NLS motifs that we termed IFN-inducible HIN200 protein IFI16 has recently emerged as motif-3 and motif-4. Interestingly, in Bos taurus and Sus scrofa, a critical DNA sensor that stimulates innate immunity. IFI16 is motif-4 resembles a complete NLS motif and the murine coun- β required for IFN- production on dsDNA transfection and HSV- terpart can mediate nuclear localization (14). These partial motifs 1 infection (1). IFI16 binds dsDNA via its C-terminal HIN may be less active in primates because of amino acid substitutions. domains (1, 2) and associates with the endoplasmic reticulum Together, these observations indicate that a multipartite NLS is protein STING, triggering TBK1-dependent IFN-β induction (1). a common feature of nuclear HIN200 proteins. Additionally, DNA-stimulated IFI16 triggers inflammasome as- Although the high degree of conservation suggests critical func- sembly upon Kaposi’s sarcoma-associated herpes virus (KSHV) tions, the contributions of these motifs to nuclear import of full- infection, promoting secretion of proinflammatory cytokines (3). length IFI16 remain elusive. To determine their functions, we Interestingly, another HIN200 protein, AIM2, also initiates constructed deletions of motif-1 (residues 96–100), motif-2 (resi- DNA-dependent inflammasome assembly (4–7). dues 128–131), motif-3 (residues 134–136), and motif-4 (residues Although IFI16 targets and downstream pathways are starting to be defined, there are important unanswered questions regarding its cell type-dependent and dynamic subcellular localization. IFI16 is Author contributions: T.L. and I.M.C. designed research; T.L., B.A.D., and J.C. performed a predominantly nuclear protein (8) in lymphoid, epithelial, en- research; T.L., B.A.D., and I.M.C. analyzed data; and T.L. and I.M.C. wrote the paper. dothelial, and fibroblast tissues, as reviewed by Veeranki and The authors declare no conflict of interest. Choubey (9). However, its cytoplasmic localization has also been *This Direct Submission article had a prearranged editor. reported in macrophages (1), cells essential for DNA-induced in- 1To whom correspondence should be addressed. E-mail: [email protected]. nate immunity (10). Consistent with its dual subcellular localiza- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tion, recent reports suggest that IFI16 can sense pathogenic DNA 1073/pnas.1203447109/-/DCSupplemental.

10558–10563 | PNAS | June 26, 2012 | vol. 109 | no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1203447109 Downloaded by guest on September 28, 2021 Fig. 1. Nuclear localization of IFI16 requires a multipartite NLS. (A) Sche- matic 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) Quanti- fication of relative nuclear abundances by an Operetta screen (mean ± SD of 3 biological replicates). MICROBIOLOGY 140–143) in full-length IFI16 C-terminally tagged with EGFP. Following transient expression in human osteosarcoma (U2OS) C Fig. 2. IFI16 sensing of viral DNA is localization-dependent. Cells were cells (Fig. 1 ), WT IFI16 localized predominantly to the nucleus. In infected with HSV-1 for the indicated times (A, B, and D) or transfected with contrast, both Δmotif-1 and -2 mutants had predominant cytoplas- a mixture of four HSV-1 DNA fragments (C) or VACV 70mer DNA (E)for3h. mic localizations, indicating that both motifs are nonredundant and (A) FISH assays demonstrate colocalization of HSV-1 genomic DNA with WT essential for nuclear localization. By comparison, Δmotif-3 and -4 IFI16 but not with NLS mutants at 2 hpi of HSV-1 infection in U2OS cells 63× remained mostly nuclear, with minor cytoplasmic localization. oil objective. (B) At 2 hpi, nuclear IFI16 binds more HSV-1 DNA than the NLS These subcellular distributions were quantified using a high- mutants in U2OS cells, as measured by co-IP of protein–DNA complexes and throughput screen (Fig. 1D). Although dynamic range was limited qPCR with four HSV-1 primer sets. DNA levels were normalized to isolated by out-of-plane signal, these data recapitulated the microscopy 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 results. Our results demonstrate that IFI16 has an evolutionarily 6 hpi) or VACV 70mer transfection in FlpIn293 cells was quantified by qPCR conserved multipartite NLS consisting of two essential motifs (1, 2) and normalized to corresponding mock treatments. Mean ± SD, n =3.*P < and two accessory motifs (3, 4). 0.05; **P < 0.01.

IFI16 Sensing of Viral DNA is Localization-Dependent. The distinct localization of the NLS mutants allowed us to test if IFI16 lo- deletion disrupts DNA binding, a mixture of four HSV-1 DNA calization influences its sensing of various pathogens. Herpesvi- fragments was transfected into the same U2OS cell lines. Sub- ruses deposit and replicate their dsDNA genome in the host sequent co-IP demonstrated that NLS mutants retained DNA- nucleus. Previous studies suggest that IFI16 can sense herpesvi- binding activity (Fig. 2C). In fact, their binding was consistently rus dsDNA in the cytoplasm (1) or nucleus (3). To determine if greater than that of nuclear WT IFI16, likely because transfected IFI16 localization is a critical determinant of sensing herpesvirus DNA first enters the cytoplasm and is more rapidly detected by DNA, we infected U2OS cell lines stably expressing WT IFI16- the cytoplasmic NLS mutants. Together, these data demonstrate EGFP or NLS deletion mutants (Δmotif-1 and Δmotif-2; Fig. S2) that WT IFI16 recognizes HSV-1 DNA primarily in the nucleus with HSV-1. Localization-dependent DNA sensing was assessed by FISH and protein-DNA coimmunopurification (co-IP). At 2 h and that detection of nuclear viral DNA requires a functional postinfection (hpi), prominent colocalization of IFI16 and HSV- NLS. To correlate this localization-dependent DNA binding with β 1 DNA was observed for WT IFI16-EGFP, but not for cyto- an immune response, we monitored expression of IFN- fol- plasmic NLS mutants (Fig. 2A). As reported for KSHV infection, lowing HSV-1 infections or DNA transfections. The U2OS cell a subset of WT IFI16 translocated into the cytoplasm (3). lines described above contain a considerable level of endogenous Consistent with the FISH data, co-IP assays showed a 10-fold IFI16, in addition to the EGFP-tagged IFI16. Therefore, increase in DNA binding level for WT IFI16-EGFP relative to FlpIn293 cell lines that express nuclear IFI16 or cytoplasmic NLS mutants (Fig. 2B). To exclude the possibility that NLS Δmotif-2 were constructed. EGFP-tagged IFI16 maintained the

Li et al. PNAS | June 26, 2012 | vol. 109 | no. 26 | 10559 Downloaded by guest on September 28, 2021 IFN stimulatory function upon DNA transfections (Fig. S3). The Posttranslational modification (PTM) is a possible mean for reg- 293 cells express low levels of endogenous IFI16 (Fig. S4) and ulating IFI16 subcellular localization (15). To identify PTMs are known to be poorly responsive to DNA transfections (1), within endogenous IFI16, we designed a targeted proteomics ap- thereby enabling us to study IFI16-mediated IFN response. proach, integrating cryogenic cell lysis, rapid immunoaffinity pu- Consistent with differential levels of DNA binding, nuclear WT rification, and MS (16) (Fig. 3C). Human CEM-T lymphoblast-like IFI16 mediated the highest IFN response following HSV-1 cells were selected for these analyses because they abundantly infections at 6 hpi (Fig. 2D), whereas cytoplasmic Δmotif-2 was express IFI16 (Fig. S4A); using shRNA knockdown, we demon- more responsive to transfected VACV 70mer DNA (Fig. 2E). strated that in lymphocytes, as in macrophages, endogenous IFI16 Interestingly, for both WT and Δmotif-2, there was no significant is required for the IFN-β response to VACV 70mer DNA (Fig. 3 A IFN-β induction at 2 hpi. In view of the prominent IFI16 DNA and B). Endogenous IFI16 was efficiently isolated (Fig. 3D), binding at 2 hpi in U2OS cells (Fig. 2 A and B), the lack of IFN-β digested in-gel or in-solution (17, 18) with trypsin or GluC, and induction at 2 hpi in 293 cells could reflect a difference in the analyzed by nano liquid chromatography (nLC) coupled MS/MS infection kinetics in different cell types. Additionally, the differ- with two complementary fragmentation techniques, collision-in- ences between 2 and 6 hpi may be attributable to the chronological duced dissociation (CID) and electron transfer dissociation order for DNA binding and the downstream outcome of IFN-β (ETD). An almost complete IFI16 sequence coverage was induction. Altogether, our results indicate that the IFI16-mediated obtained (>95%; Fig. S4B), leading to high-confidence identifi- IFN response to foreign DNA is indeed localization-dependent. cation of six phosphorylation and nine acetylation sites (Fig. 3E, Fig. S4 C and D, and Table S1). The majority of these PTMs have Endogenous IFI16 Is Acetylated Within NLS. Because cytoplasmic not been previously reported (Table S2). Thus, we present the NLS mutants can sense transfected DNA, the dynamic localization most comprehensive map of IFI16 phosphorylations and acetyla- of IFI16 can act to extend its range of DNA surveillance. tions to date (Fig. 4C). Notably, all identified phosphorylations (Fig. 4E, red pins) cluster within two predicted nonstructured regions of IFI16: the linker region (S95, S106, S153, S168, and S174) and the C terminus (S724). In contrast, lysine acetylations (Fig. 4E, green pins) were distributed within Pyrin (K45) or HIN (K214, K542, and K558) domains or between HIN domains (K444, K451, and K505). Importantly, we found that the two major NLS motifs (motif-1 and motif-2) each contain acetylations at K99 and K128, respectively (Fig. 4D). The K99 acetylation was also observed in endogenous IFI16 in THP-1 monocytes and ectopically expressed IFI16 in FlpIn293 cells (Fig. S5), indicating that NLS acetylation is a common event in multiple cell types. Because acetylation neutralizes the positive charge of the lysine, this modification may disrupt NLS binding to karyopherins of the nuclear translocation machinery (19), leading to cytoplasmic accumulation. Consistent with this hypothesis, the low levels of NLS acetylation present in CEM-T cells correlate with the minor fraction of IFI16 localized to the cytoplasm in these cells (Fig. S6A). Noteworthy, both lysine sites are highly conserved among IFI16 homologs and HIN200 family members, suggesting a common regulation of subcellular localization by acetylation.

Acetylation Within NLS Inhibits Nuclear Import. To evaluate the impact of PTMs on NLS function, we assessed subcellular localizations of IFI16-EGFP mutants for K99 and K128 within the NLS, as well as adjacent S95, S106, and S153. Lysines were mutated to arginine (R) or glutamine (Q) to mimic the nonacetylated or acetylated state, respectively, and to nonfunctional alanine (A). Serines were mutated to nonphosphorylated A or phosphomimic aspartate (D). Localizations of these EGFP-tagged mutants were assayed by transient transfection in U2OS cells in a high- throughput screen (∼1,500 cells per sample; Fig. 4B). For both K99 and K128 sites, the relative nuclear abundances of Q and A mutants were significantly reduced compared with the R mutants, indicating that acetylation at these sites interferes with nuclear localization. Although both Q and A mutations disrupt the positive charges, Q mutants were more defective than A mutants, because the bulky side chain of Q may be less tolerated by the importin binding site. S95, S106, and S153 mutations did not significantly reduce nuclear localization, suggesting these phosphorylations have only minor roles in IFI16 localization. Consistently, confocal Fig. 3. Endogenous IFI16 is acetylated within the NLS and mediates a type I microscopy revealed a predominant cytoplasmic localization for IFN response in lymphocytes. shRNA-mediated knockdown of IFI16 (A) acetyl-mimic mutants and partial rescue of nuclear localization for compromises IFN-β expression in response to VACV 70mer transfection in A CEM-T lymphocytes (B). (C) Combinatorial mass spectrometric approach to R mutants (Fig. 4 ). These results indicate K99 and K128 are identify PTMs on endogenous IFI16 from CEM-T cells. (D) Coomassie-stained critical sites that have an impact on IFI16 localization. SDS/PAGE shows efficient IFI16 isolation; dotted lines indicate IFI16 isoforms. Next, we tested if the reduced nuclear localization of acetyl- (E) Map of IFI16 phosphorylations (red pins) and acetylations (green pins). (F) mimics resulted from inhibited nuclear import. An in vitro nuclear Identification of NLS acetylations (ac) using ETD and CID MS/MS. import assay (Fig. S7A) was performed using synthetic acetylated

10560 | www.pnas.org/cgi/doi/10.1073/pnas.1203447109 Li et al. Downloaded by guest on September 28, 2021 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.

and unmodified motif-1 peptides (Fig. S7B). Equivalent amounts the ability to acetylate K99 in vitro (Fig. 4F and Fig. S6E). These of the synthetic peptides were covalently conjugated to GST- results establish a role for p300 in regulating IFI16 localization. EGFP cargos (Fig. S7C), and the resulting peptidyl-protein con- jugates were incubated with isolated HeLaS3 nuclei in the pres- Discussion ence of cytosolic extract and ATP. As observed by fluorescence Localization-Dependent DNA Sensing. Mammalian cells use nucleic microscopy and flow cytometry (Fig. 4C), motif-1 showed signifi- acid sensing mechanisms for detecting intracellular pathogens in MICROBIOLOGY cant nuclear import activity. Nuclear import of motif-1 was less multiple cellular compartments. Cytoplasmic sensors, such as prominent than that of a strong monopartite NLS (SV40 large RIG-I- or Nod-like receptors and AIM2, patrol the cytosolic space, T antigen), consistent with motif-1 being part of a multipartite whereas membrane-bound Toll-like receptors guard endosomal NLS. Importantly, the nuclear import activity of acetylated motif-1 compartments. Accumulating evidence indicates that activation of peptide was drastically reduced, indicating that K99 acetylation these pattern-recognition receptors requires not only nucleic acids can inhibit nuclear import. of appropriate chemical nature but their relevant localization, p300 Regulates the Cytoplasmic Localization of Newly Synthesized suggesting a two-signal model for innate immunity (22). IFI16. Because acetylation affected IFI16 nuclear import, we pre- Here, we assessed the role of cellular localization in mediating dicted that inhibition of deacetylase activity would block nuclear the DNA sensing function of the IFN-inducible protein IFI16, an import, promoting cytoplasmic accumulation of IFI16. To monitor emerging DNA sensor. The results from FISH, viral DNA binding, the nuclear import of newly synthesized IFI16, we induced the and IFN-β expression assays demonstrated that nuclear IFI16 lo- expression of IFI16-EGFP by tetracycline in the FlpIn cell line. calization is indeed essential for sensing HSV-1 DNA in the nu- Although treatment with the sirtuin inhibitor nicotinamide did not cleus. Our result is consistent with the observation that nuclear affect IFI16 nuclear import (Fig. S6C), treatment with the broad- IFI16 detects KSHV DNA to elicit an inflammatory response (3). spectrum histone deacetylase (HDAC) inhibitor trichostatin A led Moreover, we previously showed that IFI16 binds the genomic to significant accumulation of cytoplasmic IFI16-EGFP (Fig. 4D) DNA of human cytomegalovirus (HCMV) (23). Although this B in a dose-dependent manner (Fig. S6 ). Cytoplasmic localization interaction was required for transcriptional activation of the im- was not an artifact of compromised nuclear integrity, because mediate early promoter, it is possible that IFI16 may also detect nuclear marker p84 was not redistributed. These data suggest that HCMV DNA in the nucleus to elicit innate immunity. Because HDACs regulate IFI16 localization, further supporting acetylation these viruses represent the three subfamilies of herpesviruses, α as a critical determinant of IFI16 cellular distribution. We also (HSV-1), β (HCMV), and γ (KSHV), we envisage that IFI16 could observed that K99 in motif-1 resembles a p300 acetylation motif, with a positively charged residue at position −3 (20), and that be a common nuclear DNA sensor for herpesviruses (Fig. 5). a p300 binding motif (21) is upstream of motif-1 (Fig. S6D). To test Why is DNA sensing critical in the nucleus? Herpesviruses if p300 acetyltransferase regulates IFI16 localization, we tran- replicate their dsDNA genomes in host nuclei and are known to siently overexpressed p300 or P/CAF (P300/CBP-associated fac- evade host immunity (24). After cell entry, the viral genome is tor) in the IFI16-inducible cell line and assessed IFI16 localization protected in the cytoplasm by the capsid before nuclear deposition. (Fig. 4F). Although untransfected and P/CAF-transfected cells did Indeed, our data show that HSV-1 DNA efficiently escapes the not alter IFI16 localization, cells transfected with p300 displayed surveillance of cytoplasmic IFI16 mutants (Fig. 2B). Consequently, a striking IFI16 cytoplasmic accumulation of IFI16-EGFP. Addi- the host nucleus represents the last line of defense and a critical tionally, we demonstrated that the purified p300 HAT domain has stage for detection of herpes virus DNA.

Li et al. PNAS | June 26, 2012 | vol. 109 | no. 26 | 10561 Downloaded by guest on September 28, 2021 The nucleus was previously thought to be a “forbidden” zone expand its range of DNA surveillance. The presence of cytoplasmic for sensing, because an accurate mean of discriminating viral from IFI16 in macrophages and lymphocytes is in accordance with their host DNA in a single compartment seems challenging, although specialized functions in eliciting systemic host immunity as a rapid models have been hypothesized (25). Our results indicated that response to viral DNA. Thus, promoting cytoplasmic localization upon HSV-1 infection, the diffused nuclear localization of IFI16 of a DNA sensor, such as IFI16, may maximize immune system (Fig. S2) was drastically altered, becoming concentrated within sensitivity to DNA viruses. From a regulatory perspective, NLS viral DNA-containing nuclear bodies (Fig. 2A). This localization acetylation provides a simple and elegant mechanism for fine- change may suggest a higher affinity of IFI16 for viral DNA than tuning IFI16 distribution for its DNA sensing function or other for host chromosomes, an observation worthy of future in- localization-dependent activities. vestigation. In summary, our results lend significant support to Based on these findings, various IFI16 distributions may be establishing IFI16 as a nuclear DNA sensor and contribute pre- achieved by differential acetyltransferase and deacetylase activities viously undescribed evidence for localization-dependent sensing regulating NLS acetylation. Indeed, we show that p300 over- of pathogenic nucleic acids. expression or HDAC inhibition triggers cytoplasmic accumulation of IFI16. It is tempting to hypothesize that the diverse IFI16 Redefining the IFI16 NLS. Although a bipartite NLS encompassing localizations observed in different cell types are modulated by cell region 127–145 was previously considered as the IFI16 NLS, an type-specific activity levels of regulatory enzymes. At present, the extensive characterization of NLS motifs was lacking before this number of NLS acetylations regulating cellular localization remains study. To assess the localization-dependent DNA sensing functions surprisingly low, because, to our knowledge, less than a dozen of IFI16, we first predicted and experimentally confirmed its com- acetylated NLSs have been reported. The actual frequency of these plete NLS motifs, identifying an evolutionarily conserved multi- phenomena may be underestimated. Recent large-scale proteomic partite structure. The newly identified motif-1 was equally critical studies have generated databases of protein acetylations (27, 28), for nuclear localization as the reported motif-2, whereas motif-3 highlighting acetylation as a more widespread modification than and motif-4 were less important, albeit indispensable for full func- initially thought. By analyzing these databases, we noticed several tion. Motif-1 and motif-2 comprise a consensus bipartite NLS acetylations within putative NLS motifs of other proteins, such as – Ac Ac structure (K/R)(K/R)X10 12(K/R)3/5 (19), with a spacer (27 aa) M phase phosphoprotein 8 (K AKAGK LK) and nucleolar fi – Ac Ac signi cantly longer than the canonical length (10 12 aa). Despite protein 5 (KAK KAK IKVK). Considering that NLS acetylation – their scarcity, long spacers (16 37 aa) have been documented and could exist in low stoichiometry or within low-abundance proteins, supported by genetic (26) and structural (19) evidence. Thus, the it is likely that more sites will be identified through targeted fi newly de ned IFI16 NLS, along with its counterparts in mammalian approaches. Among the limited existing examples, the impact of homologs, showcase additional examples of multipartite NLSs. acetylation on NLS function seems versatile, either promoting (29–31) or preventing (32, 33) nuclear import. Together with our Acetylation as a Regulator of IFI16 Subcellular Localization. To ex- findings for IFI16 acetylation, these studies exemplify diverse plore possible mechanisms underlying the unexplained cytoplasmic mechanisms by which acetylation can modulate NLS function. IFI16 localization, we constructed a comprehensive map of acety- In contrast to acetylation, phosphorylation is commonly repor- lations and phosphorylations in endogenous IFI16 using combi- ted as a modulator of NLS function. IFI16 was reported to be natorial MS. Our results revealed two critical acetylation sites that phosphorylated at unknown sites (8, 34, 35), and few PTM sites negatively regulate NLS function, indicating that impeded nuclear were identified via global whole-cell studies (Table S1). Addi- import could be a source for cytoplasmic IFI16. Thus, these ace- tionally, an IFI16 peptide carrying the phospho-mimetic S132D tylations can serve as a toggle to control the destination of newly could moderately compromise nuclear import in vitro, although synthesized IFI16 (Fig. 5). Importantly, the IFI16 NLS acetylation this phosphorylation was not reported to exist in vivo (13). Our exists in various cell types, including CEM-T lymphocytes and study identified both known and previously undescribed phos- differentiated THP-1 monocytes. Because we demonstrated that phorylations on endogenous IFI16, and indicated that the NLS IFI16 sensing ability is localization-dependent, this toggle can phosphorylation sites S95, S106, and S153 have little impact on localization. These PTMs may play other functional or structural roles, or they may participate in crosstalk with other PTMs. In summary, we report that IFI16 is a DNA sensor that possesses multiple acetylation and phosphorylation sites, as well as an evolutionarily conserved multipartite NLS. We demonstrate that sensing of pathogenic DNA by IFI16 is consistent with a two-signal model of innate immunity, depending on localization of both the sensor and pathogenic DNA target. We determine that IFI16 cellular distribution and sensing functions are modulated by a combination of genetically encoded (NLS) and posttranslational (acetylation) mechanisms, and we identify p300 as an enzyme in- volved in IFI16 regulation. These observations reveal a simple and elegant acetylation-dependent mechanism that fine-tunes the range of IFI16 surveillance activity to the benefit of host innate immunity. Materials and Methods Complete materials and methods are given in SI Materials and Methods.A brief description is provided below.

Fig. 5. Working model for the localization-dependent sensing activity of Cell Culture and Construction of Stable Cell Lines. CEM-T, U2OS, Flp-In T-REx IFI16. Detection of herpes viral DNA occurs in the nucleus, and detection of HEK293, and HeLaS3 cells were cultured using standard procedures. U2OS cell transfected DNA or cytoplasmic viral DNA occurs in the cytoplasm. A mul- lines stably expressing IFI16-EGFP or mutants were generated by plasmid tipartite NLS is required for nuclear import. NLS acetylation impedes nuclear transfection, G418 selection, and ﬂow cytometry sorting. Flp-In T-REx import of newly synthesized IFI16 and is regulated by HDACs and p300. HEK293-inducible cell lines were constructed according to the manufacturer’s Ac, lysine acetylation. instructions (Life Technologies).

10562 | www.pnas.org/cgi/doi/10.1073/pnas.1203447109 Li et al. Downloaded by guest on September 28, 2021 Fluorescence Imaging and Operetta Screen. Cells were stained with anti-GFP MS. Peptides were separated by reverse phase liquid chromatography on an (laboratory of I.M.C.) and anti-p84 (Abcam) antibodies, and visualized on Ultimate 3000 nanoRSLC system (ThermoFisher Scientific) coupled online to a Zeiss LSM 510 (Carl Zeiss MicroImaging) or Leica TCS SP5 confocal micro- an LTQ Orbitrap Velos ETD mass spectrometer (ThermoFisher Scientific). MS scope (Leica Microsystems). Relative nuclear abundances of IFI16-EGFP and and data-dependent MS/MS scans were acquired sequentially. Peptide fi mutants in U2OS cells were quanti ed on an Operetta system (PerkinElmer). fragmentation used CID or ETD. MS/MS data were searched by SEQUEST in Proteome Discoverer (ThermoFisher Scientific) against a human protein da- FISH. U2OS cell lines for IFI16-EGFP or NLS mutants were infected with HSV-1 tabase (SwissProt) and common contaminants, plus reversed sequences (strain 17+) at multiplicity of infection of 5. At 2 hpi, cells were immunostained (21,569 entries). SEQUEST results were refined by X!Tandem in Scaffold for GFP. HSV-1 genome was stained by FISH, and FISH probes were nick- (Proteome Software). Peptide probabilities were calculated by Percolator in translated using pBAC HSV-1 with Cy3-dCTP labeling (PerkinElmer). Proteome Discover and PeptideProphet in Scaffold. PTM probabilities for site localization were scored using SLoMo (36). Protein–DNA Complex Co-IP. HSV-1–infected or DNA-transfected U2OS cells were cross-linked with 1% paraformaldehyde and lysed in buffer by sonication. IFI16 protein–DNA complexes were affinity-purified on magnetic beads and re- Nuclear Import and in Vitro Acetylation Assays. IFI16 motif-1 peptides were verse cross-linked. Two-thirds of the sample was digested with proteinase K. DNA synthesized as unmodified or acetylated. For nuclear import assay, GST-GFP was purified and quantified by quantitative PCR (qPCR) with four HSV-1 primers proteins were conjugated to NLS peptides and incubated with digitonin-per- (Table S3). One-third of the sample was digested with DNase I. Isolated IFI16 was meabilized HeLaS3 nuclei (SI Materials and Methods). Nuclear import was quantified by Western blot and densitometry to normalize the qPCR data. assessed by microscopy and flow cytometry. For acetylation assay, unmodified motif-1 peptide was incubated with the recombinant catalytic domain of p300 Isolation of Endogenous IFI16 and Enzymatic Digestion. CEM-T cells were acetyltransferase (Enzo Life Sciences) and analyzed on a MALDI LTQ Orbitrap harvested, cryogenically disrupted, and lysed in buffer: 20 mM K-Hepes (pH mass spectrometer (ThermoFisher Scientific) (37). 7.4), 0.1 M KoAc, 2 mM MgCl2, 0.1% Tween-20, 1 μM ZnCl2,1μM CaCl2, 0.6% μ Triton X-100, 0.2 M NaCl, and 10 g/mL DNase I, plus protease and phos- ACKNOWLEDGMENTS. We thank T. Greco, J. Wang (laboratory of I.M.C.), phatase inhibitor mixtures. IFI16 was affinity-purified on M-270 epoxy C. DeCoste, and J. Goodhouse (Core Facilities, Princeton University) for technical magnetic beads (Life Technologies) conjugated with anti-IFI16 antibodies support and L. Runnels (University of Medicine and Dentistry of New Jersey- (1:1 wt/wt of 50004 and 55328; Abcam) at 4 °C for 1 h. For in-gel digestion, Robert Wood Johnson Medical School), B. Sodeic (Hannover Medical School), IFI16 was resolved by SDS/PAGE, excised, and digested with trypsin (Prom- and J. Flint (Princeton University) for sharing reagents. This work was supported ega) or endoproteinase Glu-C (Roche). Peptides were extracted in 1% formic by National Institutes of Health National Institute on Drug Abuse Grant acid (FA) and 0.5% FA/0.5% acetonitrile. For in-solution digestion, we used DP1DA026192 and Human Frontier Science Program Organization Award an optimized filter-aided sample preparation method (18). RGY0079/2009-C (to I.M.C.).

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