Acetylation modulates cellular distribution and DNAsensing ability of interferon-inducible protein IFI16Tuo 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 February27, 2012)

Detection of pathogenic nucleic acids is essential for mammalianinnate immunity. IFN-inducible protein IFI16 has emerged as a criticalsensor for detectingpathogenicDNA, stimulatingboth type I IFNandproinflammatory responses. Despite being predominantly nuclear,IFI16 can unexpectedly sense pathogenic DNA in both the cytoplasmand the nucleus. However, the mechanisms regulating its localiza-tion and sensing ability remain uncharacterized. Here, we proposea two-signal model for IFI16 sensing. We first identify an evolution-arily conserved multipartite nuclear localization signal (NLS). Next,using FISH and immunopurification, we demonstrate that IFI16detects HSV-1 DNA primarily in the nucleus, requiring a functionalNLS. Furthermore, we establish a localization-dependent IFN-β in-duction mediated by IFI16 in response to HSV-1 infection or viralDNA transfection. To identify mechanisms regulating the secondarycytoplasmic localization, we explored IFI16 posttranslation modifi-cations. Combinatorial MS analyses identified numerous acetyla-tions and phosphorylations on endogenous IFI16 in lymphocytes,in which we demonstrate an IFI16-mediated IFN-β response. Impor-tantly, the IFI16 NLS was acetylated in lymphocytes, as well as inmacrophages. Mutagenesis and nuclear import assays showed thatNLS acetylations promote cytoplasmic localization by inhibitingnuclear import. Additionally, broad-spectrum deacetylase inhibitiontriggered accumulation of cytoplasmic IFI16, and we identify theacetyltransferase p300 as a regulator of IFI16 localization. Collec-tively, these studies establish acetylation as a molecular toggle ofIFI16 distribution, providing a simple and elegant mechanism bywhich this versatile sensor detects pathogenic DNA in a localiza-tion-dependent manner.

proteomics | HIN200 protein | posttranslational modification |mass spectrometry | histone deacetylase

The onset of mammalian innate immunity is marked by rec-ognition of pathogen-associated molecular patterns by a rep-

ertoire of host sensors. Cellular localizations, target specificities,and downstream signaling pathways define their functions. TheIFN-inducible HIN200 protein IFI16 has recently emerged asa critical DNA sensor that stimulates innate immunity. IFI16 isrequired for IFN-β production on dsDNA transfection and HSV-1 infection (1). IFI16 binds dsDNA via its C-terminal HINdomains (1, 2) and associates with the endoplasmic reticulumprotein STING, triggering TBK1-dependent IFN-β induction (1).Additionally, DNA-stimulated IFI16 triggers inflammasome as-sembly upon Kaposi’s sarcoma-associated herpes virus (KSHV)infection, promoting secretion of proinflammatory cytokines (3).Interestingly, another HIN200 protein, AIM2, also initiatesDNA-dependent inflammasome assembly (4–7).Although IFI16 targets and downstreampathways are starting to

be defined, there are important unanswered questions regarding itscell type-dependent and dynamic subcellular localization. IFI16 isa predominantly nuclear protein (8) in lymphoid, epithelial, en-dothelial, and fibroblast tissues, as reviewed by Veeranki andChoubey (9). However, its cytoplasmic localization has also beenreported in macrophages (1), cells essential for DNA-induced in-nate immunity (10). Consistent with its dual subcellular localiza-tion, recent reports suggest that IFI16 can sense pathogenic DNA

in both the cytoplasm and the nucleus. Endogenous IFI16 wasshown to colocalize with transfected vaccinia virus (VACV)dsDNA in the cytoplasmof differentiatedTHP-1monocytes (1). Incontrast, nuclear IFI16 colocalized with KSHV DNA during earlyinfection, consequently activating inflammasome formation (3).These findings contradict the canonical notion that sensing ofpathogenic DNA is solely a cytoplasmic process, suggesting thatboth cytoplasmic and nuclear IFI16 may participate in viral DNAsurveillance. Hence, it is critical to understand the localization-dependentDNA sensing properties of IFI16. As reported for otherinnate sensors (e.g., Toll-like receptors), immune response may bedictated by the sensing context and cell type (11, 12). However,the precise molecular mechanisms regulating IFI16 localizationremain uncharacterized. Furthermore, the roles of subcellularlocalization in its DNA sensing function have not been assessed.Here, we used an integrative multidisciplinary approach to

provide evidence for a two-signal model for the function of IFI16as a pathogenic DNA sensor. We define an evolutionarily con-served multipartite nuclear localization signal (NLS) required forIFI16 sensing ofHSV-1 viralDNA in the nucleus.We identifyNLSacetylation as a molecular toggle of IFI16 localization and p300 asa contributing acetyltransferase. Collectively, our results providecritical insights into how IFI16 expands its range of surveillanceagainst pathogenic DNA in a localization-dependent manner.

ResultsIFI16 Has a Multipartite NLS. To study mechanisms regulating itslocalization, we first searched for IFI16 NLS motifs. A putativebipartite NLS (residues 96–135) that included two lysine/arginine-rich motifs, 96RKRKK100 (motif-1) and 128KRKK132 (motif-2),was predicted (Fig. 1A and Fig. S1A). Indeed, a peptide includingmotif-2, 127QKRKKSTKEKA138, was shown to mediate nuclearimport of a GST-peptide fusion (13). NLSmotif-1 andmotif-2 areconserved among nuclear HIN200 proteins MNDA and IFIX, aswell as in mammalian IFI16 homologs (Fig. 1 A and B and Fig.S1B). IFI16 also contains partial NLS motifs that we termedmotif-3 and motif-4. Interestingly, in Bos taurus and Sus scrofa,motif-4 resembles a complete NLS motif and the murine coun-terpart canmediate nuclear localization (14). These partial motifsmay be less active in primates because of amino acid substitutions.Together, these observations indicate that a multipartite NLS isa common feature of nuclear HIN200 proteins.Although the high degree of conservation suggests critical func-

tions, the contributions of these motifs to nuclear import of full-length IFI16 remain elusive. To determine their functions, weconstructed deletions of motif-1 (residues 96–100), motif-2 (resi-dues 128–131), motif-3 (residues 134–136), and motif-4 (residues

Author contributions: T.L. and I.M.C. designed research; T.L., B.A.D., and J.C. performedresearch; T.L., B.A.D., and I.M.C. analyzed data; and T.L. and I.M.C. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: icristea@princeton.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1203447109/-/DCSupplemental.

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140–143) in full-length IFI16 C-terminally tagged with EGFP.Following transient expression in human osteosarcoma (U2OS)cells (Fig. 1C),WT IFI16 localized predominantly to the nucleus. Incontrast, both Δmotif-1 and -2 mutants had predominant cytoplas-mic localizations, indicating that bothmotifs are nonredundant andessential for nuclear localization. By comparison, Δmotif-3 and -4remained mostly nuclear, with minor cytoplasmic localization.These subcellular distributions were quantified using a high-throughput screen (Fig. 1D). Although dynamic range was limitedby out-of-plane signal, these data recapitulated the microscopyresults. Our results demonstrate that IFI16 has an evolutionarilyconserved multipartite NLS consisting of two essential motifs (1, 2)and two accessory motifs (3, 4).

IFI16 Sensing of Viral DNA is Localization-Dependent. The distinctlocalization of the NLS mutants allowed us to test if IFI16 lo-calization influences its sensing of various pathogens. Herpesvi-ruses deposit and replicate their dsDNA genome in the hostnucleus. Previous studies suggest that IFI16 can sense herpesvi-rus dsDNA in the cytoplasm (1) or nucleus (3). To determine ifIFI16 localization is a critical determinant of sensing herpesvirusDNA, we infected U2OS cell lines stably expressing WT IFI16-EGFP or NLS deletion mutants (Δmotif-1 and Δmotif-2; Fig. S2)with HSV-1. Localization-dependent DNA sensing was assessedby FISH and protein-DNA coimmunopurification (co-IP). At 2 hpostinfection (hpi), prominent colocalization of IFI16 and HSV-1 DNA was observed for WT IFI16-EGFP, but not for cyto-plasmic NLS mutants (Fig. 2A). As reported for KSHV infection,a subset of WT IFI16 translocated into the cytoplasm (3).Consistent with the FISH data, co-IP assays showed a 10-foldincrease in DNA binding level for WT IFI16-EGFP relative toNLS mutants (Fig. 2B). To exclude the possibility that NLS

deletion disrupts DNA binding, a mixture of four HSV-1 DNAfragments was transfected into the same U2OS cell lines. Sub-sequent co-IP demonstrated that NLS mutants retained DNA-binding activity (Fig. 2C). In fact, their binding was consistentlygreater than that of nuclear WT IFI16, likely because transfectedDNA first enters the cytoplasm and is more rapidly detected bythe cytoplasmic NLS mutants. Together, these data demonstratethat WT IFI16 recognizes HSV-1 DNA primarily in the nucleusand that detection of nuclear viral DNA requires a functionalNLS. To correlate this localization-dependent DNA binding withan immune response, we monitored expression of IFN-β fol-lowing HSV-1 infections or DNA transfections. The U2OS celllines described above contain a considerable level of endogenousIFI16, in addition to the EGFP-tagged IFI16. Therefore,FlpIn293 cell lines that express nuclear IFI16 or cytoplasmicΔmotif-2 were constructed. EGFP-tagged IFI16 maintained the

Fig. 1. Nuclear localization of IFI16 requires a multipartite NLS. (A) Sche-matic of IFI16 and alignment of the multipartite NLS of IFI16, IFIX, andMNDA. (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 isstained with DAPI, and the cytoplasm is stained with CellMask. (D) Quanti-fication of relative nuclear abundances by an Operetta screen (mean ± SD of3 biological replicates).

Fig. 2. IFI16 sensing of viral DNA is localization-dependent. Cells wereinfected with HSV-1 for the indicated times (A, B, and D) or transfected witha 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 WTIFI16 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 NLSmutants in U2OS cells, as measured by co-IP of protein–DNA complexes andqPCR with four HSV-1 primer sets. DNA levels were normalized to isolatedprotein levels. (C) Cytoplasmic NLS mutants bind more transfected HSV-1DNA fragments. (D and E) IFN-β expression following HSV-1 infection (2 and6 hpi) or VACV 70mer transfection in FlpIn293 cells was quantified by qPCRand normalized to corresponding mock treatments. Mean ± SD, n = 3. *P <0.05; **P < 0.01.

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IFN stimulatory function upon DNA transfections (Fig. S3). The293 cells express low levels of endogenous IFI16 (Fig. S4) andare known to be poorly responsive to DNA transfections (1),thereby enabling us to study IFI16-mediated IFN response.Consistent with differential levels of DNA binding, nuclear WTIFI16 mediated the highest IFN response following HSV-1infections at 6 hpi (Fig. 2D), whereas cytoplasmic Δmotif-2 wasmore responsive to transfected VACV 70mer DNA (Fig. 2E).Interestingly, for both WT and Δmotif-2, there was no significantIFN-β induction at 2 hpi. In view of the prominent IFI16 DNAbinding at 2 hpi in U2OS cells (Fig. 2 A and B), the lack of IFN-βinduction at 2 hpi in 293 cells could reflect a difference in theinfection kinetics in different cell types. Additionally, the differ-ences between 2 and 6 hpi may be attributable to the chronologicalorder for DNA binding and the downstream outcome of IFN-βinduction. Altogether, our results indicate that the IFI16-mediatedIFN response to foreign DNA is indeed localization-dependent.

Endogenous IFI16 Is Acetylated Within NLS. Because cytoplasmicNLSmutants can sense transfectedDNA, the dynamic localizationof IFI16 can act to extend its range of DNA surveillance.

Posttranslational modification (PTM) is a possible mean for reg-ulating IFI16 subcellular localization (15). To identify PTMswithin endogenous IFI16, we designed a targeted proteomics ap-proach, integrating cryogenic cell lysis, rapid immunoaffinity pu-rification, andMS (16) (Fig. 3C). HumanCEM-T lymphoblast-likecells were selected for these analyses because they abundantlyexpress IFI16 (Fig. S4A); using shRNA knockdown, we demon-strated that in lymphocytes, as in macrophages, endogenous IFI16is required for the IFN-β response to VACV 70merDNA (Fig. 3Aand B). Endogenous IFI16 was efficiently isolated (Fig. 3D),digested in-gel or in-solution (17, 18) with trypsin or GluC, andanalyzed by nano liquid chromatography (nLC) coupled MS/MSwith two complementary fragmentation techniques, collision-in-duced dissociation (CID) and electron transfer dissociation(ETD). An almost complete IFI16 sequence coverage wasobtained (>95%; Fig. S4B), leading to high-confidence identifi-cation of six phosphorylation and nine acetylation sites (Fig. 3E,Fig. S4 C and D, and Table S1). The majority of these PTMs havenot been previously reported (Table S2). Thus, we present themost comprehensive map of IFI16 phosphorylations and acetyla-tions to date (Fig. 4C).Notably, all identified phosphorylations (Fig. 4E, red pins) cluster

within two predicted nonstructured regions of IFI16: the linker re-gion (S95, S106, S153, S168, and S174) and the C terminus (S724).In contrast, lysine acetylations (Fig. 4E, green pins) were distributedwithin Pyrin (K45) or HIN (K214, K542, and K558) domains orbetween HIN domains (K444, K451, and K505). Importantly, wefound that the two major NLS motifs (motif-1 and motif-2) eachcontain 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 FlpIn293cells (Fig. S5), indicating that NLS acetylation is a common eventin multiple cell types. Because acetylation neutralizes the positivecharge of the lysine, this modification may disrupt NLS binding tokaryopherins of the nuclear translocation machinery (19), leadingto cytoplasmic accumulation. Consistent with this hypothesis, thelow levels ofNLSacetylation present inCEM-T cells correlatewiththeminor fraction of IFI16 localized to the cytoplasm in these cells(Fig. S6A). Noteworthy, both lysine sites are highly conservedamong IFI16 homologs and HIN200 family members, suggestinga common regulation of subcellular localization by acetylation.

Acetylation Within NLS Inhibits Nuclear Import. To evaluate theimpact of PTMs on NLS function, we assessed subcellular local-izations of IFI16-EGFPmutants forK99 andK128within theNLS,as well as adjacent S95, S106, and S153. Lysines were mutated toarginine (R) or glutamine (Q) to mimic the nonacetylated oracetylated state, respectively, and to nonfunctional alanine (A).Serines were mutated to nonphosphorylated A or phosphomimicaspartate (D). Localizations of these EGFP-tagged mutantswere assayed by transient transfection in U2OS cells in a high-throughput screen (∼1,500 cells per sample; Fig. 4B). For bothK99and K128 sites, the relative nuclear abundances of Q and Amutants were significantly reduced compared with the R mutants,indicating that acetylation at these sites interferes with nuclearlocalization. Although bothQ andAmutations disrupt the positivecharges, Q mutants were more defective than A mutants, becausethe bulky side chain of Q may be less tolerated by the importinbinding site. S95, S106, and S153 mutations did not significantlyreduce nuclear localization, suggesting these phosphorylationshave only minor roles in IFI16 localization. Consistently, confocalmicroscopy revealed a predominant cytoplasmic localization foracetyl-mimic mutants and partial rescue of nuclear localization forR mutants (Fig. 4A). These results indicate K99 and K128 arecritical sites that have an impact on IFI16 localization.Next, we tested if the reduced nuclear localization of acetyl-

mimics resulted from inhibited nuclear import. An in vitro nuclearimport assay (Fig. S7A) was performed using synthetic acetylated

Fig. 3. Endogenous IFI16 is acetylated within the NLS and mediates a type IIFN response in lymphocytes. shRNA-mediated knockdown of IFI16 (A)compromises IFN-β expression in response to VACV 70mer transfection inCEM-T lymphocytes (B). (C) Combinatorial mass spectrometric approach toidentify PTMs on endogenous IFI16 from CEM-T cells. (D) Coomassie-stainedSDS/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.

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and unmodified motif-1 peptides (Fig. S7B). Equivalent amountsof the synthetic peptides were covalently conjugated to GST-EGFP cargos (Fig. S7C), and the resulting peptidyl-protein con-jugates were incubated with isolated HeLaS3 nuclei in the pres-ence of cytosolic extract and ATP. As observed by fluorescencemicroscopy and flow cytometry (Fig. 4C), motif-1 showed signifi-cant nuclear import activity. Nuclear import of motif-1 was lessprominent than that of a strong monopartite NLS (SV40 largeT antigen), consistent with motif-1 being part of a multipartiteNLS. Importantly, the nuclear import activity of acetylatedmotif-1peptide was drastically reduced, indicating that K99 acetylationcan inhibit nuclear import.

p300 Regulates the Cytoplasmic Localization of Newly SynthesizedIFI16. Because acetylation affected IFI16 nuclear import, we pre-dicted that inhibition of deacetylase activity would block nuclearimport, promoting cytoplasmic accumulation of IFI16. Tomonitorthe nuclear import of newly synthesized IFI16, we induced theexpression of IFI16-EGFP by tetracycline in the FlpIn cell line.Although treatment with the sirtuin inhibitor nicotinamide did notaffect IFI16 nuclear import (Fig. S6C), treatment with the broad-spectrum histone deacetylase (HDAC) inhibitor trichostatin A ledto significant accumulation of cytoplasmic IFI16-EGFP (Fig. 4D)in a dose-dependent manner (Fig. S6B). Cytoplasmic localizationwas not an artifact of compromised nuclear integrity, becausenuclear marker p84 was not redistributed. These data suggest thatHDACs regulate IFI16 localization, further supporting acetylationas a critical determinant of IFI16 cellular distribution. We alsoobserved that K99 in motif-1 resembles a p300 acetylation motif,with a positively charged residue at position −3 (20), and thata p300 bindingmotif (21) is upstreamofmotif-1 (Fig. S6D). To testif p300 acetyltransferase regulates IFI16 localization, we tran-siently overexpressed p300 or P/CAF (P300/CBP-associated fac-tor) in the IFI16-inducible cell line and assessed IFI16 localization(Fig. 4F). Although untransfected and P/CAF-transfected cells didnot alter IFI16 localization, cells transfected with p300 displayeda striking IFI16 cytoplasmic accumulation of IFI16-EGFP. Addi-tionally, we demonstrated that the purified p300 HAT domain has

the ability to acetylate K99 in vitro (Fig. 4F and Fig. S6E). Theseresults establish a role for p300 in regulating IFI16 localization.

DiscussionLocalization-Dependent DNA Sensing. Mammalian cells use nucleicacid sensing mechanisms for detecting intracellular pathogens inmultiple cellular compartments. Cytoplasmic sensors, such asRIG-I- orNod-like receptors andAIM2, patrol the cytosolic space,whereas membrane-bound Toll-like receptors guard endosomalcompartments. Accumulating evidence indicates that activation ofthese pattern-recognition receptors requires not only nucleic acidsof appropriate chemical nature but their relevant localization,suggesting a two-signal model for innate immunity (22).Here, we assessed the role of cellular localization in mediating

the DNA sensing function of the IFN-inducible protein IFI16, anemerging DNA sensor. The results from FISH, viral DNAbinding,and IFN-β expression assays demonstrated that nuclear IFI16 lo-calization is indeed essential for sensing HSV-1 DNA in the nu-cleus. Our result is consistent with the observation that nuclearIFI16 detects KSHV DNA to elicit an inflammatory response (3).Moreover, we previously showed that IFI16 binds the genomicDNA of human cytomegalovirus (HCMV) (23). Although thisinteraction was required for transcriptional activation of the im-mediate early promoter, it is possible that IFI16 may also detectHCMV DNA in the nucleus to elicit innate immunity. Becausethese viruses represent the three subfamilies of herpesviruses, α(HSV-1), β (HCMV), and γ (KSHV), we envisage that IFI16 couldbe a common nuclear DNA sensor for herpesviruses (Fig. 5).Why is DNA sensing critical in the nucleus? Herpesviruses

replicate their dsDNA genomes in host nuclei and are known toevade host immunity (24). After cell entry, the viral genome isprotected in the cytoplasm by the capsid before nuclear deposition.Indeed, our data show that HSV-1 DNA efficiently escapes thesurveillance of cytoplasmic IFI16mutants (Fig. 2B). Consequently,the host nucleus represents the last line of defense and a criticalstage for detection of herpes virus DNA.

Fig. 4. IFI16 NLS acetylation prevents nuclear import. (A) Confocal microscopy of K99 and K128 mutants 40× objective. (B) Relative nuclear abundance ofIFI16 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 flowcytometry 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.

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The nucleus was previously thought to be a “forbidden” zonefor sensing, because an accurate mean of discriminating viral fromhost DNA in a single compartment seems challenging, althoughmodels have been hypothesized (25). Our results indicated thatupon HSV-1 infection, the diffused nuclear localization of IFI16(Fig. S2) was drastically altered, becoming concentrated withinviral DNA-containing nuclear bodies (Fig. 2A). This localizationchange may suggest a higher affinity of IFI16 for viral DNA thanfor host chromosomes, an observation worthy of future in-vestigation. In summary, our results lend significant support toestablishing IFI16 as a nuclear DNA sensor and contribute pre-viously undescribed evidence for localization-dependent sensingof pathogenic nucleic acids.

Redefining the IFI16 NLS. Although a bipartite NLS encompassingregion 127–145 was previously considered as the IFI16 NLS, anextensive characterization of NLS motifs was lacking before thisstudy. To assess the localization-dependent DNA sensing functionsof IFI16, we first predicted and experimentally confirmed its com-plete NLS motifs, identifying an evolutionarily conserved multi-partite structure. The newly identified motif-1 was equally criticalfor nuclear localization as the reported motif-2, whereas motif-3and motif-4 were less important, albeit indispensable for full func-tion. Motif-1 and motif-2 comprise a consensus bipartite NLSstructure (K/R)(K/R)X10–12(K/R)3/5 (19), with a spacer (27 aa)significantly longer than the canonical length (10–12 aa). Despitetheir scarcity, long spacers (16–37 aa) have been documented andsupported by genetic (26) and structural (19) evidence. Thus, thenewly defined IFI16NLS, alongwith its counterparts inmammalianhomologs, showcase additional examples of multipartite NLSs.

Acetylation as a Regulator of IFI16 Subcellular Localization. To ex-plore possiblemechanisms underlying the unexplained cytoplasmicIFI16 localization, we constructed a comprehensive map of acety-lations and phosphorylations in endogenous IFI16 using combi-natorial MS. Our results revealed two critical acetylation sites thatnegatively regulate NLS function, indicating that impeded nuclearimport could be a source for cytoplasmic IFI16. Thus, these ace-tylations can serve as a toggle to control the destination of newlysynthesized IFI16 (Fig. 5). Importantly, the IFI16 NLS acetylationexists in various cell types, including CEM-T lymphocytes anddifferentiated THP-1 monocytes. Because we demonstrated thatIFI16 sensing ability is localization-dependent, this toggle can

expand its range of DNA surveillance. The presence of cytoplasmicIFI16 in macrophages and lymphocytes is in accordance with theirspecialized functions in eliciting systemic host immunity as a rapidresponse to viral DNA. Thus, promoting cytoplasmic localizationof a DNA sensor, such as IFI16, may maximize immune systemsensitivity to DNA viruses. From a regulatory perspective, NLSacetylation provides a simple and elegant mechanism for fine-tuning IFI16 distribution for its DNA sensing function or otherlocalization-dependent activities.Based on these findings, various IFI16 distributions may be

achieved by differential acetyltransferase and deacetylase activitiesregulating NLS acetylation. Indeed, we show that p300 over-expression or HDAC inhibition triggers cytoplasmic accumulationof IFI16. It is tempting to hypothesize that the diverse IFI16localizations observed in different cell types are modulated by celltype-specific activity levels of regulatory enzymes. At present, thenumber ofNLS acetylations regulating cellular localization remainssurprisingly low, because, to our knowledge, less than a dozenacetylated NLSs have been reported. The actual frequency of thesephenomena may be underestimated. Recent large-scale proteomicstudies have generated databases of protein acetylations (27, 28),highlighting acetylation as a more widespread modification thaninitially thought. By analyzing these databases, we noticed severalacetylations within putative NLS motifs of other proteins, such asM phase phosphoprotein 8 (KAcAKAGKAcLK) and nucleolarprotein 5 (KAKAcKAKAcIKVK). Considering thatNLS acetylationcould exist in low stoichiometry or within low-abundance proteins,it is likely that more sites will be identified through targetedapproaches. Among the limited existing examples, the impact ofacetylation on NLS function seems versatile, either promoting(29–31) or preventing (32, 33) nuclear import. Together with ourfindings for IFI16 acetylation, these studies exemplify diversemechanisms by which acetylation can modulate NLS function.In contrast to acetylation, phosphorylation is commonly repor-

ted as a modulator of NLS function. IFI16 was reported to bephosphorylated at unknown sites (8, 34, 35), and few PTM siteswere identified via global whole-cell studies (Table S1). Addi-tionally, an IFI16 peptide carrying the phospho-mimetic S132Dcould moderately compromise nuclear import in vitro, althoughthis phosphorylation was not reported to exist in vivo (13). Ourstudy identified both known and previously undescribed phos-phorylations on endogenous IFI16, and indicated that the NLSphosphorylation sites S95, S106, and S153 have little impact onlocalization. These PTMs may play other functional or structuralroles, 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 evo-lutionarily conserved multipartite NLS. We demonstrate thatsensing of pathogenic DNA by IFI16 is consistent with a two-signalmodel of innate immunity, depending on localization of boththe sensor and pathogenic DNA target. We determine that IFI16cellular distribution and sensing functions are modulated bya 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 andelegant acetylation-dependentmechanism that fine-tunes the rangeof IFI16 surveillance activity to the benefit of host innate immunity.

Materials and MethodsComplete materials and methods are given in SI Materials and Methods. Abrief description is provided below.

Cell Culture and Construction of Stable Cell Lines. CEM-T, U2OS, Flp-In T-RExHEK293, and HeLaS3 cells were cultured using standard procedures. U2OS celllines stably expressing IFI16-EGFP or mutants were generated by plasmidtransfection, G418 selection, and flow cytometry sorting. Flp-In T-RExHEK293-inducible cell lines were constructed according to themanufacturer’sinstructions (Life Technologies).

Fig. 5. Working model for the localization-dependent sensing activity ofIFI16. Detection of herpes viral DNA occurs in the nucleus, and detection oftransfected DNA or cytoplasmic viral DNA occurs in the cytoplasm. A mul-tipartite NLS is required for nuclear import. NLS acetylation impedes nuclearimport of newly synthesized IFI16 and is regulated by HDACs and p300.Ac, lysine acetylation.

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Fluorescence Imaging and Operetta Screen. Cells were stained with anti-GFP(laboratory of I.M.C.) and anti-p84 (Abcam) antibodies, and visualized ona Zeiss LSM 510 (Carl Zeiss MicroImaging) or Leica TCS SP5 confocal micro-scope (Leica Microsystems). Relative nuclear abundances of IFI16-EGFP andmutants in U2OS cells were quantified on an Operetta system (PerkinElmer).

FISH. U2OS cell lines for IFI16-EGFP or NLS mutants were infected with HSV-1(strain 17+) at multiplicity of infection of 5. At 2 hpi, cells were immunostainedfor GFP. HSV-1 genome was stained by FISH, and FISH probes were nick-translated using pBAC HSV-1 with Cy3-dCTP labeling (PerkinElmer).

Protein–DNA Complex Co-IP. HSV-1–infected or DNA-transfected U2OS cellswere cross-linked with 1% paraformaldehyde and lysed in buffer by sonication.IFI16 protein–DNA complexes were affinity-purified on magnetic beads and re-verse cross-linked. Two-thirds of the samplewasdigestedwithproteinaseK.DNAwas purified and quantified by quantitative PCR (qPCR) with four HSV-1 primers(Table S3). One-third of the sample was digested with DNase I. Isolated IFI16wasquantified by Western blot and densitometry to normalize the qPCR data.

Isolation of Endogenous IFI16 and Enzymatic Digestion. CEM-T cells wereharvested, cryogenically disrupted, and lysed in buffer: 20 mM K-Hepes (pH7.4), 0.1 M KoAc, 2 mM MgCl2, 0.1% Tween-20, 1 μM ZnCl2, 1 μM CaCl2, 0.6%Triton X-100, 0.2 M NaCl, and 10 μg/mL DNase I, plus protease and phos-phatase inhibitor mixtures. IFI16 was affinity-purified on M-270 epoxymagnetic beads (Life Technologies) conjugated with anti-IFI16 antibodies(1:1 wt/wt of 50004 and 55328; Abcam) at 4 °C for 1 h. For in-gel digestion,IFI16 was resolved by SDS/PAGE, excised, and digested with trypsin (Prom-ega) or endoproteinase Glu-C (Roche). Peptides were extracted in 1% formicacid (FA) and 0.5% FA/0.5% acetonitrile. For in-solution digestion, we usedan optimized filter-aided sample preparation method (18).

MS. Peptides were separated by reverse phase liquid chromatography on anUltimate 3000 nanoRSLC system (ThermoFisher Scientific) coupled online toan LTQ Orbitrap Velos ETD mass spectrometer (ThermoFisher Scientific). MSand data-dependent MS/MS scans were acquired sequentially. Peptidefragmentation used CID or ETD. MS/MS data were searched by SEQUEST inProteome Discoverer (ThermoFisher Scientific) against a human protein da-tabase (SwissProt) and common contaminants, plus reversed sequences(21,569 entries). SEQUEST results were refined by X!Tandem in Scaffold(Proteome Software). Peptide probabilities were calculated by Percolator inProteome Discover and PeptideProphet in Scaffold. PTM probabilities for sitelocalization were scored using SLoMo (36).

Nuclear Import and in Vitro Acetylation Assays. IFI16 motif-1 peptides weresynthesized as unmodified or acetylated. For nuclear import assay, GST-GFPproteins were conjugated to NLS peptides and incubated with digitonin-per-meabilized HeLaS3 nuclei (SI Materials and Methods). Nuclear import wasassessed by microscopy and flow cytometry. For acetylation assay, unmodifiedmotif-1 peptidewas incubatedwith the recombinant catalytic domain of p300acetyltransferase (Enzo Life Sciences) and analyzed on a MALDI LTQ Orbitrapmass spectrometer (ThermoFisher Scientific) (37).

ACKNOWLEDGMENTS. We thank T. Greco, J. Wang (laboratory of I.M.C.),C. DeCoste, and J. Goodhouse (Core Facilities, PrincetonUniversity) for technicalsupport and L. Runnels (University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School), B. Sodeic (Hannover Medical School),and J. Flint (Princeton University) for sharing reagents. This workwas supportedby National Institutes of Health National Institute on Drug Abuse GrantDP1DA026192 and Human Frontier Science Program Organization AwardRGY0079/2009-C (to I.M.C.).

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