pias1 selectively inhibits interferon-inducible genes and is important in innate immunity

PIAS1 selectively inhibits interferon-inducible genesand is important in innate immunity

Bin Liu1, Sheldon Mink2, Kelly A Wong3,4,5, Natalie Stein1, Crescent Getman1, Paul W Dempsey6,Hong Wu3,4,5 & Ke Shuai1,2,3

Interferon (IFN) activates the signal transducer and activator of transcription (STAT) pathway to regulate immune responses.

The protein inhibitor of activated STAT (PIAS) family has been suggested to negatively regulate STAT signaling. To understand

the physiological function of PIAS1, we generated Pias1–/– mice. Using PIAS1-deficient cells, we show that PIAS1 selectively

regulates a subset of IFN-c- or IFN-b-inducible genes by interfering with the recruitment of STAT1 to the gene promoter. The

antiviral activity of IFN-c or IFN-b was consistently enhanced by Pias1 disruption. Pias1–/– mice showed increased protection

against pathogenic infection. Our data indicate that PIAS1 is a physiologically important negative regulator of STAT1 and

suggest that PIAS1 is critical for the IFN-c- or IFN-b-mediated innate immune responses.

Both type I (interferon-a/b (IFN-a/b)) and type II (IFN-g) interferonshave antiviral, antiproliferative and immunoregulatory functions.Upon IFN-g stimulation, STAT1 becomes tyrosine phosphorylatedand translocates into the nucleus, where it binds to the IFN-gactivation sequence (GAS) to activate transcription. In the case ofIFN-a/b, both STAT1 and STAT2 are activated to form a transcrip-tional complex that binds to the IFN-a-stimulated responsive element(ISRE) for gene activation1–4.

The proper regulation of the cytokine-activated JAK-STAT pathwayis critical because abnormal JAK-STAT signaling is associated withcancer and immune disorders4. The JAK-STAT pathway is negativelyregulated at multiple steps by several groups of proteins4. The SOCS(suppressor of cytokine signaling) proteins are rapidly induced bycytokines and inhibit the JAK-STAT signaling through distinctmechanisms5,6. In the nucleus, the activity of STATs can be negativelyregulated by at least two molecular mechanisms: the dephosphoryla-tion of STATs by protein tyrosine phosphatases7 and the suppressionof STAT-mediated gene activation by the PIAS family of proteins.These negative regulators are important for controlling signalingstrength, kinetics and specificity of the JAK-STAT pathway4.

The mammalian PIAS protein family contains four members:PIAS1, PIAS3, PIASx and PIASy (ref. 4). PIAS1 was originally isolatedas a STAT1-interacting protein in a yeast two-hybrid screen8. Coim-munoprecipitation studies have indicated that PIAS1, PIAS3 andPIASx interact with STAT1, STAT3 and STAT4, respectively8–10. ThePIAS-STAT interaction is cytokine dependent. In reporter assays, PIASproteins inhibit STAT-mediated gene activation. In addition to PIAS1,PIASy also interacts with STAT1 (ref. 11). PIAS1 and PIASy inhibitSTAT1-dependent gene activation through distinct mechanisms.

Whereas PIAS1 can block the DNA-binding activity of STAT1, PIASyseems to act as a corepressor of STAT1. These studies suggest thatthere is specificity, as well as redundancy, in PIAS-mediated inhibitionof STAT signaling. However, the physiological function of mammalianPIAS proteins in the regulation of STAT signaling has not beendocumented.

To understand the physiological function of PIAS1, we generatedPias1–/– mice. Detailed gene activation analysis showed that PIAS1selectively regulates a subset of IFN-g- or IFN-b-responsive genes.Chromatin immunoprecipitation analysis showed enhanced recruit-ment of STAT1 to the endogenous promoter of a STAT1-dependentgene in Pias1–/– macrophages. Consistent with a negative regulatoryfunction for PIAS1 in interferon signaling, the removal of PIAS1resulted in enhanced immune responses to viral or microbial chal-lenges. Our results demonstrate an important regulatory function forPIAS1 in IFN-g- or IFN-b-mediated innate immune responses.

RESULTS

Generation of Pias1–/– mice

To study the biological function of PIAS1 in vivo, we generatedPias1–/– mice by homologous recombination in embryonic stemcells. Mouse Pias1 is composed of 14 exons, spanning 101.4 kilobases(kb) of the genomic DNA. A targeting construct was designed todelete exons 2–12 of Pias1 (Fig. 1a). Mice heterozygous for the Pias1-null mutation on a 129/Sv � C57BL/6 genetic background seemednormal and were intercrossed to obtain Pias1–/– offspring. Deletion ofPias1 was detected by Southern blot analysis (Fig. 1b). We confirmedthe absence of Pias1 mRNA from Pias1–/– mice by RNA blot analysisof total RNA isolated from the testes of wild-type and Pias1–/– mice

Published online 15 August 2004; doi:10.1038/ni1104

1Division of Hematology-Oncology, Department of Medicine; 2Department of Biological Chemistry; 3Molecular Biology Institute; 4Department of Molecular and MedicalPharmacology; 5Howard Hughes Medical Institute; 6Department of Microbiology, Immunology, and Molecular Genetics; University of California Los Angeles, Los Angeles,California, USA. Correspondence should be addressed to K.S. ([email protected]).

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using mouse Pias1 cDNA as a probe. The approximately 3-kb Pias1message was completely absent from Pias1–/– mice (Fig. 1c). Weconfirmed the absence of PIAS1 protein from Pias1–/– mice byimmunoblot analysis of whole-cell extracts prepared from Pias1+/+

and Pias1–/– bone marrow–derived macrophages (BMMs), spleen orthymus using antibody to PIAS1 (ref. 8; Fig. 1d). PIAS1 deficiency didnot cause a significant change in the expression of PIASy, PIAS3 orSTAT1 protein (Fig. 1d). In addition, the tyrosine phosphorylation ofSTAT1 at various time points after IFN-b or IFN-g treatment was notaltered in Pias1–/– splenocytes. In addition, the tyrosine phosphoryla-tion of STAT2 by IFN-b was also unaffected in Pias1–/– cells (Supple-mentary Fig. 1 online). We also obtained similar results in Pias1–/–

thymocytes, BMMs and primary fibroblasts (data not shown).Pias1–/– mice were produced at a frequency lower than the expected

mendelian ratio. At 3 weeks of age, Pias1–/– mice accounted for 12.9%of the offspring (Pias1+/+:Pias1+/–:Pias1–/– ¼ 33.3%:53.8%:12.9%,n ¼ 645). Genotyping of mice born on day 1 also showed a reducedmendelian ratio of Pias1–/– mice (Pias1+/+:Pias1+/–:Pias1–/– ¼ 34.4%:48.4%:17.2%, n ¼ 64). However, genotyping of embryos at embryonicday 17.5 showed a normal mendelian ratio (Pias1+/+:Pias1+/–:Pias1–/– ¼ 27%:51%:22%, n ¼ 33), suggesting a perinatallethality for Pias1–/– mice. The surviving PIAS1-deficient mice wererunts compared with their wild-type littermates, but these miceshowed no gross histological defects or premature death (data notshown). Pias1–/– males were approximately 40% smaller than theirwild-type counterparts, whereas Pias1–/– females were about 20–30%smaller than wild-type controls. Both males and females deficient forPIAS1 were fertile.

Specificity of PIAS1

To directly examine the function of PIAS1 in IFN-g– and IFN-b–mediated gene activation, we obtained BMMs from Pias1–/– mice andtheir wild-type littermates and treated them with IFN-g for varioustimes (0 h, 2 h, 4 h, 6 h and 8 h) or left them untreated. We isolatedtotal RNA from these cells and analyzed the induction of severalknown STAT1-target genes12–14 by IFN-g using quantitative real-timePCR (Q-PCR). The induction of Gbp1 (guanylate binding protein),Cxcl9 (CXC chemokine ligand 9) and Cxcl10 (CXC chemokine ligand10) by IFN-g was enhanced two- to fivefold in Pias1–/– cells comparedwith wild-type cells (Fig. 2a), whereas the expression of Irf1 (inter-feron regulatory factor 1), Nos2 (inducible nitric oxide synthase) and

Socs1 (suppressor of cytokine signaling 1) was not altered. We alsoanalyzed cells similarly after IFN-b treatment. Induction of Gbp1,Cxcl10, Cxcl9, Ifi203 (interferon-inducible 203 gene) and Ly6e wasincreased two- to threefold in Pias1–/– macrophages (Fig. 2b). Incontrast, induction of Irf7 or Ifi16 (also known as Ifi204) was notaffected in the absence of PIAS1 (Fig. 2b). We also examined thefunction of PIAS1 in IFN-g signaling in wild-type and Pias1–/– primaryembryonic fibroblasts. Similarly, PIAS1 deficiency resulted in enhancedinduction of Cxcl9 and Cxcl10, but not Irf1 (Fig. 2c). These resultssuggest that PIAS1 functions as an inhibitor of STAT1 and that PIAS1may regulate a specific subset of IFN-g- or IFN-b-inducible genes.

We next tested if the lack of PIAS1 affects STAT3-mediated geneactivation in response to interleukin 6 (IL-6) stimulation. We used asimilar Q-PCR analysis with RNA samples prepared from wild-typeand Pias1–/– cells, left untreated or treated with IL-6 for various times.The induction of STAT3 target genes Socs3 (ref. 15) and Junb16 by IL-6in BMMs or primary fibroblasts, respectively, was not affected in theabsence of PIAS1 (Fig. 2d). These data suggest that PIAS1 deficiencydoes not cause a global defect in IL-6 signaling.

To further characterize the specificity of PIAS1 in the negativeregulation of interferon-mediated gene activation, we used microarrayanalysis to evaluate gene expression profiles of wild-type and Pias1–/–

BMMs, left untreated or treated with IFN-g or IFN-b for 2 h or 4 h. Inthese conditions, about 650 genes were induced by either IFN-g orIFN-b by at least threefold. Among the interferon-inducible genes, 9%showed an induction of at least 1.3-fold in Pias1–/– macrophagescompared with wild-type controls (Fig. 2e and SupplementaryTable 1 online). The actual effect of PIAS1 on the induction ofthese genes was far greater than this by Q-PCR analysis (Fig. 2a,bversus Supplementary Table 1 online), indicating that microarrays areless sensitive than Q-PCR assays. Data from microarray analysissupport the conclusion that PIAS1 specifically regulates a subset ofinterferon-induced genes.

The differential effect of PIAS1

PIAS1 can block the DNA-binding activity of STAT1 in vitro17. Tounderstand the molecular basis of PIAS1 specificity in the regulation ofinterferon-responsive genes, we tested the hypothesis that the intrinsicaffinity of STAT1 binding sequences present in STAT1-target genes maycontribute to the observed selective effect of PIAS1 in gene regulation.We used an electrophoretic mobility shift assay (EMSA) to examine the

Pias1+/+

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Figure 1 Generation of Pias1–/– mice.

(a) Restriction map of the genomic region

containing mouse Pias1; the 14 exons are

numbered. The target construct contains a

2.5-kb upstream EcoRI-EcoRI fragment, a

3.5-kb downstream EcoRI-BamHI fragment

and the PGKneopA cassette. After homologous

recombination, exons 2–12 of Pias1 are deleted.Probe, the C-terminal probe used for Southern

blot analysis. RI, EcoRI; RV, EcoRV; Bam,

BamHI. (b) Southern blot analysis of the tail

DNA from wild-type (+/+), heterozygous (+/–) or

Pias1–/– (–/–) mice. (c) Northern blot analysis

of the total RNA (10 mg/lane) isolated from the

testis of wild-type or Pias1–/– mice. The filter was

reprobed with the housekeeping gene Gapd to

show equal loading in each lane. (d) Immunoblot

analysis of whole-cell lysates from BMMs, spleen

or thymus (Thy) of wild-type or Pias1–/– mice.

The filter was probed with antibodies to various

proteins (right margin).

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relative STAT1 DNA binding affinity of several STAT1 target geneswhose promoters have been characterized before. We mixed a 32P-labeled oligonucleotide corresponding to the STAT1-binding elementin the promoter of Irf1 with recombinant, purified, tyrosine-phosphorylated STAT1 in the presence of various concentrations ofunlabeled GAS oligonucleotides from the promoters of Irf1 (ref. 18),Gbp1 (refs. 19,20), Cxcl9 (refs. 21,22) and Ly6e23. The DNA-bindingactivity of STAT1 was completely abolished by unlabeled Irf1 GAS at afivefold excess concentration, but not by Gbp1 or Cxcl9 or Ly6e GAS atthe same concentration (Fig. 3a). There was a reduction of about 80%in the DNA-binding activity of STAT1 on the Irf1 GAS in the presenceof a 20-fold excess of the Ly6e GAS, whereas there was no inhibition inthe presence of either Gbp1 or Cxcl9 GAS at the same concentration.These data indicate that STAT1 binds at least 20-fold more strongly tothe Irf1 GAS than to the Gbp1, Cxcl9 or Ly6e GAS. These data suggest

that there is a correlation between the STAT1 DNA-binding affinityand the responsiveness of STAT1 target genes toward PIAS1.

We next tested directly if PIAS1 differentially affects the binding ofSTAT1 toward a strong versus a weak DNA-binding site. We usedEMSA to examine the binding of STAT1 on a previously described,high-affinity STAT-binding element generated by mutating the cfospromoter (cfosM67)24 (a strong site) and on the Ly6e GAS (a weaksite) in the presence of various concentrations of bacterially purifiedPIAS1 (Fig. 3b). Quantitative analysis indicated that the DNA-bindingactivity of STAT1 was more sensitive to the inhibitory effect of PIAS1when binding to the weak Ly6e site than to the strong cfosM67 site(Fig. 3b). The relative affinity of the Ly6e site compared with thecfosM67 site was confirmed by competition analysis (Fig. 3c). Theseresults suggest that, at least in vitro, PIAS1 can inhibit the DNA-bindingactivity of STAT1 more efficiently on a weak site than on a strong site.

IFN-induced genes (100%)

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Figure 2 Analysis of gene activation by interferons in Pias1–/– and wild-type cells. (a) Gene activation in response to IFN-g (10 ng/ml), by Q-PCR analysis.

BMMs from Pias1–/– and wild-type littermates were left untreated or were treated with IFN-g (10 ng/ml) for various times (horizontal axes). Total RNA was

subjected to Q-PCR analyses using specific primers of STAT1-mediated genes (above graphs). Data is one representative of at least three independent

experiments. (b) Experiment as described in a, except cells were treated with IFN-b (500 U/ml). (c) Experiment as described in a, except primary embryonic

fibroblasts were used. Data are the average of results from three matched pairs of wild-type and Pias1–/– fibroblasts. (d) Experiment as described in a, except

BMMs and primary fibroblasts were stimulated with IL-6 (50 ng/ml) and were used to examine the induction of Socs3 and Junb, respectively. (e) Microarray

analyses of interferon-induced genes in wild-type and Pias1–/– macrophages. Interferon-induced genes are defined as genes induced by at least threefold byIFN-b or IFN-g for 2 h or 4 h in wild-type BMMs. PIAS1-affected genes are defined as genes hyperactivated by IFN-b or IFN-g in Pias1–/– macrophages by at

least 1.3-fold compared with that in wild-type BMMs.


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Enhanced promoter occupancy of STAT1

To further understand the molecular basis of PIAS1 specificity inregulating interferon-responsive genes, we analyzed the recruitment ofSTAT1 to the promoters of STAT1 target genes by chromatin immu-noprecipitation assay. The promoter of Gbp1, a known STAT1 targetgene whose expression was increased in Pias1–/– macrophages (Fig. 2),has two IFN-g-responsive subregions to which STAT1 binds25

(Fig. 4a). The proximal subregion contains an ISRE sequence, whereasthe distal subregion contains a GAS and an ISRE sequence (Fig. 4a).We prepared cell extracts from wild-type and Pias1–/– macrophages,left them untreated or treated with IFN-g for 20 min or 1 h, thensubjected them to chromatin immunoprecipitation analysis withantibodies to STAT1 or IgG (control). We quantified bound DNAby Q-PCR using specific primers (Fig. 4a). The binding of STAT1 toeither the proximal or the distal region of Gbp1 promoter wasincreased in Pias1–/– macrophages compared with wild-type cells(Fig. 4a). Irf1 is a STAT1 target gene, but its induction is not affectedin the absence of PIAS1 (Fig. 2). The promoter of Irf1 contains aGAS sequence to which STAT1 binds18. We did similar chromatinimmunoprecipitation analysis with the Irf1 promoter. The binding ofSTAT1 to the Irf1 promoter was similar in wild-type and Pias1–/–

macrophages (Fig. 4b). These results suggest that the ability of PIAS1to influence the promoter occupancy of STAT1 in response tointerferons contributes to the specificity of PIAS1 in the regulationof STAT1 target genes.

Enhanced interferon-mediated antiviral responses

We examined whether PIAS1, which regulates a specific subset ofinterferon-induced genes, is important in regulating the interferon-activated, STAT1-dependent innate immune responses to viral infec-tion26. This was suggested because some of the PIAS1-sensitive genes,such as Gbp1, are believed to have antiviral function27. To directlyexamine if PIAS1 is involved in interferon-dependent innate immu-nity, we challenged BMMs from wild-type and Pias1–/– mice primedwith IFN-b (5 U/ml or 50 U/ml) with mouse g-herpes virus-68(MHV-68) for various time periods28. We examined the viral replica-tion by immunoblot analyses using antibodies to the viral capsidprotein M9, the viral tegument protein ORF45 or the viral capsidprotein ORF26 (ref. 29). The expression of viral proteins was inhibitedin Pias1–/– macrophages compared with wild-type cells (Fig. 5a).Notably, this inhibitory effect was more profound in cells primed witha higher dose of IFN-b, indicating that the enhanced antiviral responsein Pias1–/– cells is interferon dependent. Quantitative plaque assaysindicate that at 72 h after infection there was about 90% inhibition ofviral production in Pias1–/– macrophages compared with that of wild-type cells primed with 50 U/ml of IFN-b (Fig. 5b).

We also examined the cellular response to viral infection byanalyzing the induction of antiviral genes. We did Q-PCR analysisof RNA samples from wild-type and Pias1–/– macrophages infectedwith MHV-68 for various times. MHV-68 infection induced theexpression of Gbp1 and Cxcl10 in wild-type macrophages, and there

Ly6e Ly6eLy6e

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a bFigure 4 Chromatin immunoprecipitation assays

of STAT1 target gene promoters. (a) Enhanced

DNA-binding activity of STAT1 to the endogenous

Gbp1 promoter in response to IFN-g in Pias1–/–

BMMs. Chromatin immunoprecipitation (ChIp)

assays of BMMs from Pias1–/– and wild-type

littermates. Cells were left untreated or were

treated with IFN-g (10 ng/ml) for 20 min or 1 h,

and cell extracts were analyzed by chromatin

immunoprecipitation assay with antibody to

STAT1. Rabbit IgG was used as a negative control.

STAT1-bound DNA was quantified by Q-PCR(primers, above graphs) and normalized with the

input DNA. (b) Experiment as described in a

except that primers specific for the Irf1 promoter

were used. Data are from one representative of

two independent experiments.

Figure 3 The differential affect of PIAS1 on the DNA-binding activity of STAT1 to GAS elements.

(a) EMSAs. 32P-labeled STAT1-binding site from the Irf1 promoter was incubated with bacterially

purified, tyrosine-phosphorylated STAT1, with or without various concentrations of unlabeled GAS

oligonucleotides from the Irf1, Gbp1, Cxcl9 or Ly6e promoter. (b) PIAS1 differentially affects the

DNA-binding activity of STAT1 to GAS. EMSAs with purified phosphorylated STAT1 and 32P-labeled

cfosM67 or Ly6e probe, in the presence or absence of various concentrations of purified GST-PIAS1

protein. Data were quantified and are plotted as the percentage binding relative to STAT1 alone.

(c) Experiment as described in a, except that 32P-labeled cfosM67 probe was used. Data are one

representative of three independent experiments.


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was increased induction of Gbp1 and Cxcl10 in Pias1–/– macrophages(Fig. 5c). We conclude that the removal of PIAS1 results in increasedantiviral responses. These results are consistent with the idea thatPIAS1 functions as a negative regulator of interferon signaling.

To confirm that the interferon-activated JAK-STAT pathway iscritical for the antiviral response against MHV-68 infection, weused wild-type and Jak1–/– murine embryonic fibroblasts and leftthem untreated or treated them with IFN-b, followed by MHV-68infection for various times. We examined viral replication by immuno-blot analysis using antibodies to viral proteins ORF26 and M9 (Fig. 5d)or by the measurement of viral titers (Fig. 5e). Consistent with theresults obtained in BMMs (Fig. 5a,b), viral replication was inhibited inthe presence of IFN-b in wild-type fibroblasts. In contrast, viralproduction was increased in JAK1-deficient cells. In addition, IFN-bfailed to inhibit viral replication in Jak1–/– cells. We also obtained similar

results in STAT1-deficient human fibroblasts (data not shown). Theseresults confirm that the interferon-activated JAK-STAT pathway has acritical function in the antiviral response to MHV-68 infection.

To further examine the function of PIAS1 in the antiviral response,we infected wild-type and Pias1–/– primary embryonic fibroblasts withvesicular stomatitis virus (VSV), a single-stranded RNA virus knownto be highly sensitive to STAT1 signaling26, in the presence of variousconcentrations of IFN-g. Again, the interferon-mediated antiviralresponse was increased in Pias1–/– cells (Fig. 5f).

Increased antiviral and antimicrobial responses

To examine further the physiological function of PIAS1 in interferonsignaling, we challenged Pias1–/– mice and the matched wild-typecontrols with VSV. Stat1–/– mice are hypersensitive to VSV infec-tion30,31. In accordance with the idea of a negative regulatory function

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Figure 5 Enhanced antiviral responses in Pias1–/– cells. (a) BMMs from Pias1–/– mice and their wild-type littermates were primed with IFN-b(5 U/ml, left; or 50 U/ml, right) for 16 h, then challenged with MHV-68 at multiplicity of infection of 5. Protein extracts from cells collected at various

times after infection were analyzed by immunoblot. Data are from one representative of five independent pairs of BMMs. (b) Plaque assay. Supernatants

from BMMs in a pretreated with IFN-b (50 U/ml) after infection with MHV-68 were analyzed by plaque assay to determine viral titers. (c) Wild-type

and Pias1–/– macrophages were infected with MHV-68 at a multiplicity of infection of 5. Antiviral gene induction was determined by Q-PCR. Data are from

one representative of three independent pairs of BMMs. (d) Experiment as described in a, except wild-type and Jak1–/– mouse embryonic fibroblasts were

infected with MHV-68 at a multiplicity of infection of 0.1. (e) The viral titers in the medium of cells infected with MHV-68 for 48 h from d were determined.

Unt, untreated. (f) Wild-type and Pias1–/– primary embryonic fibroblasts pretreated with various doses of IFN-g for 24 h were infected with VSV at a

multiplicity of infection of 1. Cell viability was determined 24 h after infection.

P < 0.001

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5 × 106 CFU2 × 106 CFUa b c

Figure 6 Enhanced antiviral and antibacterial activity in Pias1–/– mice. (a) Age- and gender-matched wild-type and Pias1–/– mice (n ¼ 5) were challenged

intravenously with 5 � 107 PFU of VSV and were monitored for survival for 6 d. (b) Age- and gender-matched wild-type and Pias1–/– mice (n ¼ 7) were

intraperitoneally infected with 2 � 106 CFU of L. monocytogenes and were monitored for survival. P value was determined by paired t-test. (c) Experiment

as described in b except that 5 � 106 CFU of L. monocytogenes was administered and nine pairs of mice were used.


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for PIAS1 in STAT1 signaling, all Pias1–/– mice survived challenge with5 � 107 plaque-forming units (PFU) of VSV, whereas 60% of wild-type mice succumbed to the same dose of VSV challenge in the sameconditions (Fig. 6a).

To examine the function of PIAS1 in microbial infection, wechallenged Pias1–/– and the matched wild-type control mice withListeria monocytogenes at various concentrations. When infected with2 � 106 colony-forming units (CFU) of bacteria, 30% of wild-type micedied 4 d after infection, whereas Pias1–/– mice all survived (Fig. 6b). Inaddition, all wild-type mice succumbed to infection with 5 � 106 CFUof bacteria after 96 h, whereas 22% of Pias1–/– mice survived (Fig. 6c).These results support the idea of a negative regulatory function forPIAS1 in innate immunity against pathogenic infection.

Lipopolysaccharide-induced endotoxic shock

The interferon-activated JAK-STAT pathway is involved in lipopoly-saccharide (LPS)–induced endotoxic shock. Mice deficient in IFN-greceptor or STAT1 show resistance to LPS-induced toxicity25,32,33,whereas mice lacking SOCS1, an inhibitor of the JAK-STAT pathway,are hypersensitive to LPS-induced endotoxic shock34,35. The observedphenotypes of Pias1–/– mice to pathogenic infection suggest thatPIAS1 may be involved in the LPS response. We examined theresponse of Pias1–/– mice toward LPS-induced septic shock. Wechallenged Pias1–/– mice and age- and gender-matched wild-typecontrols with a sublethal dose of LPS and monitored the responsive-ness of these mice (Fig. 7). Consistent with a negative regulatoryfunction of PIAS1 in interferon signaling, Pias1–/– mice were hyper-sensitive to LPS-induced endotoxic shock.

Protein sumoylation and p53 regulation

PIAS1 has a SUMO (small ubiquitin-related modifier) E3 ligaseactivity, and PIAS1 regulates the activity of p53 through sumoyla-tion36–38. To examine the physiological function of PIAS1 in theregulation of p53 signaling, we analyzed g-irradiation-induced p53-dependent apoptosis in wild-type and Pias1–/– thymocytes by annexinV assay. Apoptosis induced by g-irradiation at various doses was notaltered in the absence of PIAS1 (Fig. 8a), suggesting that PIAS1 is notessential in regulating the apoptotic activity of p53.

The PIAS homologs SIZ1 and SIZ2 in yeast function as SUMOE3 ligases, and the deletion of SIZ1 and SIZ2 results in anoverall reduction in protein sumoylation39. We examined the functionof PIAS1 in regulating protein sumoylation. We found no defectin basal SUMO3 or SUMO1 protein modification in wild-typeand Pias1–/– thymocytes (Fig. 8b and data not shown). Cellularstress signals can induce the conjugation of SUMO3 to manyproteins40. We noted increased protein SUMO3 conjugation inthymocytes under osmotic shock induced by sorbitol (Fig. 8b).However, the induction of SUMO3 modification of proteins bysorbitol treatment in Pias1–/– thymocytes was not altered (Fig. 8b).We also obtained similar results in primary fibroblasts (datanot shown). Thus, PIAS1 deficiency does not have a substantialdefect in global protein sumoylation either constitutively or uponsorbitol treatment.

DISCUSSION

Studies in cultured cells have suggested that PIAS1 can regulate thetranscriptional activity of STAT1 and other transcription factorsincluding p53, androgen receptor and SMAD4. To understand thephysiological function of PIAS1, we generated Pias1–/– mice. Ourresults have demonstrated that PIAS1 is a physiological negativeregulator of STAT1 and that PIAS1 interferes with the recruitmentof STAT1 to the promoters of endogenous genes. Detailed geneactivation and microarray studies showed an unexpected specificityof PIAS1 in the regulation of IFN-g- or IFN-b-mediated geneactivation. Functional studies suggested that PIAS1 is important ininterferon-mediated innate immunity to pathogenic infection.

The JAK-STAT pathway can be negatively regulated by severalgroups of proteins, including the SOCS family of proteins and proteintyrosine phosphatases4–6. These negative regulators act by ‘switchingoff’ the overall cytokine responses. In contrast, PIAS1 shows specificityin the regulation of interferon signaling. Our results suggest thatinterferon-responsive genes can be negatively regulated in subgroupsby gene-specific modulators such as PIAS1. The ability to regulatecytokine-responsive genes in subgroups may provide additional flex-ibility in the modulation of specific biological functions of cytokines.

How is the specificity of PIAS1 in the negative regulation ofinterferon-responsive genes achieved? Our results indicate that thedifferential effect of PIAS1 on the binding of STAT1 to the promotersof STAT1 target genes contributes to the observed PIAS1 specificity.This conclusion is supported by the finding that removal of PIAS1enhanced the binding of STAT1 to the promoter of Gbp1 (PIAS1-sensitive gene) but not to that of Irf1 (PIAS1-insensitive gene). Weexamined how PIAS1 can differentially affect the binding of STAT1 togene promoters. Our results suggest that the DNA-binding affinity ofSTAT1-binding sites present in the promoters of STAT1 target genescan largely influence the PIAS1 effect. PIAS1 has a more profoundeffect on genes containing weak STAT1-binding sites (for example,Gbp1, Cxcl9 and Ly6e) than genes containing a strong STAT1-bindingsite (for example, Irf1). EMSA studies showed that PIAS1 inhibits theDNA-binding activity of STAT1 more efficiently on a weak binding

P < 0.001

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0 24 48 72 96Time (h)

+/+–/–

Figure 7 Pias1–/– mice are hypersensitive to LPS-induced endotoxic shock.

Age- and gender-matched wild-type and Pias1–/– mice (n ¼ 6) wereintraperitoneally injected with 20 mg/g body weight of LPS from Escherichia

coli serotype O55:B5 and were monitored for survival. P value was

determined by paired t-test.

Anti-SUMO3

83

175

kDa Sumoylatedproteins

Sorbitol (min): 60301006030100

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Pias1+/+

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%)

a b

Figure 8 PIAS1 in p53 signaling and protein sumoylation. (a) Annexin V

apoptosis assays of thymocytes freshly isolated from Pias1–/– and wild-typelittermates (n ¼ 5), left untreated or treated with various doses of

g-irradiation (IR). (b) Protein extracts of thymocytes from wild-type (+/+) or

Pias1–/– (–/–) mice (n ¼ 3) left untreated or treated with sorbitol (0.3 M),

analyzed by immunoblot with antibody to SUMO3.


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site than on a strong binding site. In addition to the contribution ofthe intrinsic DNA-binding affinity of the STAT1-binding site, theoverall binding of STAT1 to an endogenous gene promoter may alsobe affected by other factors, such as the influence of non-STATtranscription factors on the promoter, which may provide an addi-tional level of regulation on PIAS1 specificity. Finally, PIASy can alsointeract with STAT1 and repress STAT1-dependent transcription11.Thus, a redundant function of other PIAS proteins may also con-tribute to the observed specificity of PIAS1 in the regulation ofinterferon-responsive genes.

Because PIAS1 seems to affect only a subset of interferon-responsivegenes, one important issue is the biological importance of suchspecific regulation. interferons are critical for host defense againstviral infection. Our results indicate that cells lacking PIAS1 showenhanced antiviral responses, including reduced viral replication andenhanced antiviral gene expression. There was a more profoundantiviral effect in Pias1–/– cells primed with higher doses of interferons.Therefore, although other signaling events may also contribute toantiviral responses, these results demonstrate that the enhancedantiviral phenotype in Pias1–/– cells is interferon dependent. Consis-tent with a negative regulatory function of PIAS1 in interferonsignaling, Pias1–/– mice show increased protection against viral infec-tion. Thus, PIAS1 may be a valuable target for the design oftherapeutic drugs that can specifically enhance the antiviral activityof interferons. In addition, Pias1–/– mice showed enhanced antimi-crobial activity and increased sensitivity toward LPS-induced endo-toxic shock. Thus, PIAS1 is an important regulator of innate immuneresponses.

Biochemical studies indicate that PIAS1 has a SUMO E3 ligaseactivity, which has been suggested to regulate several transcriptionfactors, such as p53 (refs. 36-38). Our data suggest that PIAS1deficiency has no substantial effect on the p53-mediated apoptosis.In addition, the lack of PIAS1 does not affect global protein sumoyla-tion either constitutively or after stress treatment. It remains to bedetermined whether the lack of PIAS1 effect on the regulation of thesesignaling events may result from redundant functions of other PIASproteins. The function of PIAS1 SUMO ligase activity in the regula-tion of STAT1 signaling has been studied in vitro or in overexpressionconditions, but the results are controversial41,42. The physiologicalgene targets of PIAS1 in interferon signaling identified here should bevaluable for further studies of the potential function of the PIAS1-mediated protein sumoylation in the regulation of STAT1. Thephysiological importance of the SUMO E3 ligase activity of PIAS1in cellular signaling remains to be clarified.

The experiments reported here focused on the characterization ofthe immunologic phenotypes of Pias1–/– mice. Pias1–/– mice wererunts and showed a partial perinatal lethality. These phenotypes maybe caused by the dysregulation of STAT1 signaling or, alternatively, bythe abnormal regulation of other transcription factors in the absenceof PIAS1. Further studies are needed to understand the molecularbasis for these phenotypes.

METHODSGeneration of Pias1–/– mice. Mouse Pias1 is composed of 14 exons, over about

101.4 kb of genomic DNA. The translational start codon (ATG) is located in the

first exon. To disrupt Pias1, we used a targeting vector containing 2.5-kb

upstream and 3.5-kb downstream flanking sequences of Pias1, as well as the

PGKneopA cassette for positive selection (Fig. 1a). The targeting construct was

electroporated into mouse embryonic stem cells derived from the 129 strain,

followed by G418 selection (300 mg/ml). We obtained two clones containing the

disrupted allele. Individual clones of targeted embryonic stem cells were

injected into C57BL/6 blastocysts and implanted into pseudopregnant females.

Chimeric mice were bred to obtain germline transmission. Heterozygous mice

seemed normal and were bred to obtain Pias1–/– mice. The proper deletion of

Pias1 was detected by Southern blot analysis, in which genomic DNA isolated

from the tails of the mice were subjected to EcoRV digestion, followed by

electrophoresis and hybridization with an external probe from the 3¢ flanking

region of Pias1. The wild-type allele produces an 11-kb fragment, whereas a

9-kb band indicates the Pias1-null allele (Fig. 1b). Animal experiments were

done with the approval of the UCLA Animal Research Committee.

Isolation of BMMs. BMMs were differentiated from marrow cells from 4- to

8-week-old Pias1–/– mice and their wild-type littermates as described43. BMMs

were maintained in 1� DMEM containing 10% fetal bovine serum, 1%

penicillin/streptomycin and 30% L929 conditioned medium containing

macrophage colony-stimulating factor for 7 d before they were used for

various experiments.

Microarray analysis. Microarray analysis essentially followed the manufac-

turer’s instructions (Affymetrix). BMMs from wild-type or Pias1–/– littermates

were left untreated or were treated with IFN-b (500 U/ml) or IFN-g (10 ng/ml)

for 2 h or 4 h. Total RNA was prepared with RNA-STAT60 (Tel-Test) and

purified with the RNeasy kit (Qiagen). Double-stranded complementary DNA

was synthesized from 20 mg total RNA according to Affymetrix methodology

and purified with Phase Lock Gels (Eppendorf). Biotin-labeled RNA was

synthesized with the BioArray High Yield RNA Transcript Labeling Kit (Enzo).

Samples were cleaned, fragmented and hybridized to mouse genome

(MGU74Av2) Genechips (Affymetrix) as instructed. GeneChips were stained

with phycoerythrin-streptavidin (Molecular Probes) and were scanned with a

GeneChip scanner (Affymetrix).

Q-PCR. Q-PCR was done as described28. First-strand complementary DNA was

produced by reverse transcription of 1–3 mg total RNA using superscript II

(Invitrogen). Q-PCR was carried out using the iCycler thermocycler (BioRad)

in a final volume of 25 ml containing Taq polymerase, 1� Taq buffer

(Stratagene), 125 mM dNTP, SYBR Green I (Molecular Probes) and fluoroscein

(BioRad). Amplification conditions were as follows: 95 1C for 3 min; 40 cycles

of 95 1C for 30 s, 60 1C for 30 s and 72 1C for 30 s. Actin was used to

standardize the levels of cDNA. The primer sequences used in Q-PCR assays are

in Supplementary Table 2 online.

EMSA. EMSA was done as described8. The sequences of the oligonucleotides

used are as follows, with the core GAS sequences underlined: Irf1, 5¢-GATCGTGATTTCCCCGAAATGACG-3¢; Gbp1, 5¢-CATGAGTTTCATAT-

TACTCTAAATC-3¢; Cxcl9, 5¢-GATCCTTACTATAAACTCCCCGTTTATGT-

GAAATGGA-3¢; Ly6e, 5¢-CATGTTATGCATATTCCTGTAAGTG-3¢; and

cfosM67, 5¢-GATCCATTTCCCGTAAATC-3¢.

Chromatin immunoprecipitation assay. Chromatin immunoprecipitation

assays used the ChIP Assay Kit (Upstate Biotech) as instructed by the

manufacturer. In all, 1 � 107 wild-type or Pias1–/– BMMs were left untreated

or were treated with IFN-g (10 ng/ml) for 20 min or 1 h. Cell extracts were

prepared and chromatin was sheared by sonication (10 s at 30% of the

maximum strength, for a total of six times). Chromatin immunoprecipitation

assays used antibody to STAT1 or rabbit IgG as a negative control. Bound DNA

was quantified by Q-PCR and was normalized with the input DNA. The primer

sequences used are in Supplementary Table 2 online.

Viral infection and plaque assays. MHV-68 was obtained from American Type

Culture Collection (VR1465). MHV-68 viral infection was done as described44.

Supernatants from infected cells were used to measure viral titers by plaque

assays. Monolayers of BHK-21 cells (ATCC CCL-10) were used for plaque

assays, which were overlaid with 1% methylcellulose (Sigma) for 5 d. The cells

were then fixed and stained with 0.2% crystal violet in 20% ethanol, and

plaques were counted to determine the titers. VSV (Indiana strain) was

obtained from D. Nayak (University of California at Los Angeles, Los Angeles,

California). Primary embryonic fibroblasts were plated in triplicate into 96-well

tissue culture plates at a density of 1 � 104 cells per well. Cells were pretreated

with various doses of IFN-g for 24 h. Cells were then washed once with PBS


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and infected with VSV. Cell viability was determined 24 h after infection by

crystal violet staining followed by spectroscopy at 540 nm as described30. For

infection of mice with VSV, Pias1–/– and age- and gender-matched wild-type

mice were challenged with 5 � 107 PFU of VSV by means of tail vein injection

and were monitored for survival every 24 h for 6 d.

Apoptosis assays. Thymocytes freshly isolated from Pias1–/– and wild-type

littermates were left untreated or were treated with various doses of g-

irradiation, then were cultured in RPMI medium plus 10% FBS and 1%

penicillin and streptomycin. Apoptosis was determined 16 h after treatment

with an annexin V apoptosis detection kit as instructed by the manufacturer

(R&D Systems).

Accession numbers. Microarray data were submitted to the National Center

for Biotechnology Information Gene Expression Omnibus (accession number

GSE1552; http://www.ncbi.nlm.nih.gov/geo/).

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSWe thank R. Sun for antibodies to MHV-68 viral proteins; R. Schreiber forJak1–/– cells; and J. Gao and B. Nguyen for technical assistance. Supported bythe National Institutes of Health (K.S. and H.W.), American Cancer Society(K.S.) Howard Hughes Medical Institute (H.W.), Leukemia & LymphomaSociety (B.L.), National Institutes of Health–National Cancer Institute (traininggrant 5T32 CA009056 to S.M.), University of California at Los Angeles,(Sprague Jr. Fellowship to K.A.W.) and US Public Health Service (NationalResearch Service award, GM07185 to K.A.W.).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 10 May; accepted 13 July 2004

Published online at http://www.nature.com/natureimmunology/

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pias1 selectively inhibits interferon-inducible genes and is important in innate immunity

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