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Interleukin-12 (IL-12) promotes differentiation of T helper type 1(TH1) cells and augments production of interferon-γ (IFN-γ) throughactivation of the signal transducer and activator of transcription-4(STAT4)1–3. STAT proteins undergo receptor-mediated phosphoryla-tion on a conserved tyrosine to form SH2-phosphotyrosine–dependentSTAT dimers, which become active transcription factors, translocatingto the nucleus and interacting with one or more adjacent STAT recogni-tion sequences in DNA4–9. Recent studies have revealed additionalaspects of STAT regulation. Some nonphosphorylated STAT proteinsform active transcription factor complexes: for example, nonphospho-rylated STAT1 interacts with interferon regulatory factor-1 (IRF1), dri-ving basal expression of the low molecular mass polypeptide-2 (LMP2)gene10. Other nonphosphorylated STAT proteins move between thecytoplasm and nucleus before being phosphorylated11. Although gener-ally depicted as being monomeric6,12–15, some nonphosphorylatedSTAT proteins seem to reside in complexes of higher molecular weightthan would be predicted for a monomer16,17, which might reflect associ-ation with heterologous proteins18,19 or oligomeric forms of nonphos-phorylated STAT monomers20.

Crystallographic investigations have provided insights into the func-tional activities of certain STAT protein domains21,22. A structure of theSTAT1 core indicated that the SH2 domain undergoes important recip-rocal interactions with phosphotyrosine crucial for stabilizing activatedSTAT dimers bound to DNA21. The structure of the STAT4 N-domainwas interpreted as forming a dimer along a particular polar interface pro-posed to stabilize interaction between two STAT dimers bound to DNAto form STAT tetramers22. Mutation of an invariant tryptophan (W37)central to this interface in the STAT1 N-domain abolished cooperativeDNA binding by STAT1 and inhibited transcriptional responses22, sug-gesting that N-domain dimerization was required for STAT tetramer–meditated transcriptional activation. However, subsequent examination

of mutations affecting three other amino acid residues (Q36, T40 andE66) predicted to mediate interactions at the proposed interface leftSTAT4 tetramer formation intact, whereas the W37A mutation in STAT4prevented formation of both STAT4 dimers and tetramers because themutant was unable to undergo receptor-mediated tyrosine phosphoryla-tion23. The requirement of the STAT4 N-domain for STAT4 activation23

was recently confirmed for IL-12 signaling using STAT4-deficient trans-genic mice that express human full-length STAT4 or an N-terminal deletion mutant. Whereas the full-length STAT4 rescues IL-12responsiveness, the STAT4 N-terminally truncated mutant does notundergo phosphorylation and T lymphocytes expressing this mutant donot proliferate in response to IL-12 (ref. 24). Although these results donot exclude a role for the N-domain in STAT tetramer formation, theyindicate that the STAT4 N-domain may also have a role in receptor-induced phosphorylation of the nonphosphorylated STAT423.

Additional mutation analysis of the STAT1 N-domain was suggestiveof an alternative dimer interface to the one previously proposed25.Because these studies were done using STAT1 and not STAT4, the struc-tural aspects of STAT4 N-domain dimerization may require re-examina-tion to define mutations that selectively prevent STAT4 N-domaindimerization. The notion that the N-domain could mediate the forma-tion of STAT4 tetramers was based on results obtained using the W37Amutation22. However, this mutation is now known to globally disrupt theN-domain structure rather than to selectively prevent N-domain dimer-ization25. Therefore, a re-examination of the functional role of N-domain dimerization in STAT activation might be in order as well.

Here we show that the STAT4 N-domain caused assembly of nonphos-phorylated STAT4 dimers in vivo before receptor-driven activation. First,we identified N-domain mutations in the dimerization interface thatprevented N-domain dimerization and the assembly of nonphosphory-lated STAT4 dimers. Notably, these mutations also prevented subsequent

1Department of Pathology & Immunology, 2Howard Hughes Medical Institute and 3Department of Biochemistry and Molecular Biophysics, Washington UniversitySchool of Medicine, 660 S. Euclid Avenue, St. Louis, Missouri 63110, USA. Correspondence should be addressed to K.M.M. (murphy@immunology.wustl.edu).

Published online 4 January 2004; doi:10.1038/ni1032

N-domain–dependent nonphosphorylated STAT4dimers required for cytokine-driven activationNaruhisa Ota1,2, Tom J Brett1, Theresa L Murphy1, Daved H Fremont1,3 & Kenneth M Murphy1,2

The N-terminal protein interaction domain (N-domain) of the signal transducer and activator of transcription-4 (STAT4) is believed tostabilize interactions between two phosphorylated STAT4 dimers to form STAT4 tetramers. Here, we show that nonphosphorylatedSTAT4 dimers form in vivo before cytokine receptor–driven activation. Mutations in the N-domain dimerization interface abolishedassembly of nonphosphorylated STAT4 dimers and prevented STAT4 phosphorylation mediated by cytokine receptors. In addition, N-domain dimerization occurred for other STAT family members but was homotypic in character. This implies a conserved role for N-domain dimerization, which might include influencing interactions with cytokine receptors, favoring homodimer formation oraccelerating formation of the phosphorylated STAT dimer.

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STAT4 phosphorylation by cytokine receptors, indicating that the non-phosphorylated STAT4 dimers are required for normal receptor-drivenphosphorylation and activation. Finally, N-domain dimerization seemedto be a general phenomenon. N-domain dimerization was observedthroughout the STAT family, but was homotypic in character, which mayenhance the specificity of STAT signaling by favoring assembly of non-phosphorylated STAT homodimers rather than heterodimers. The pre-association of nonphosphorylated STAT dimers might allow the morerapid formation of the active dimers after activation involving anintramolecular rearrangement.

RESULTSAn alternative STAT4 N-domain dimer interfaceBecause the N-domain dimerization interface initially proposed hasrecently been questioned on the basis of gel filtration and sedimenta-tion equilibrium analyses using STAT1 N-domain mutants25, andhas not been established experimentally for STAT4, we re-examinedthe proposed homodimeric assembly of the STAT4 N-domain basedon its crystal structure22 (PDB ID 1BGF). Given that there is onemolecule in the asymmetric unit of this structure, any potentialoligomeric assembly is necessarily created by crystallographic sym-metry. It has been observed that protein-protein interaction surfacesshow certain common characteristics, including large interfacialareas that are mostly hydrophobic and are complementary inshape26–28. We first evaluated each possible dimer assembly on thebasis of its buried solvent accessible surface area (buried ASAs; Fig. 1). Of the several possible dimers produced by crystal symmetry,only two potential homodimer assemblies show extensive interac-tion surfaces (>1,000 Å2 of buried ASA; Fig. 1a). Of these two, dimerB showed a more extensive interacting surface, which was highlyhydrophobic and had a higher degree of shape correlation (Sc) thandimer A, the assembly proposed previously22 (Fig. 1b). Thus, thiscomputational analysis indicated that dimer B is the most probablehomodimer assembly on the basis of crystallographic symmetry.Based on these observations, we tested whether dimer B representsthe physiological assembly of the STAT4 N-domain by introducingdestabilizing mutations at D19 and L78, residues that make contactspredicted to stabilize the dimer B assembly (Fig. 1).

To test whether these residues influence N-domain dimerization,we first generated soluble wild-type and mutant STAT4 N-domain

proteins (amino acids 1–121) and analyzed their ability to formhomodimers in solution (Fig. 2a,b). The wild-type STAT4 N-domainmigrated in gel filtration at an observed molecular mass consistentwith a dimer rather than monomer (Fig. 2a,b). N-domains with themutations T40A and E66A, located in the previously proposed dimerinterface22 (dimer A), also migrated as dimers, indicating that forSTAT4 that proposed interface may not, in fact, be relevant for physi-ologic dimer formation in solution. In contrast, soluble STAT4 N-domain proteins with mutations D19R or L78S migrated at a reducedmolecular mass consistent with monomers. These observations werefurther validated by dynamic light-scattering analysis (Fig. 2b). Thisanalysis calculates the particle size distribution (hydrodynamicradius) in solution, which is used to estimate the molecular weight.Wild-type and T40A and E66A N-domain mutant proteins showedhydrodynamic radii consistent with dimers, whereas D19R and L78Smutants seemed to be monomeric (Fig. 2b), consistent with resultsfrom gel filtration analysis. Notably, the STAT4 N-domain proteinharboring the W37A mutation was not sufficiently stable to be puri-fied for analysis, consistent with results for the same mutation in theSTAT1 N-domain reported previously25. Thus, hydrodynamic analy-sis confirmed that dimer B, but not dimer A, is the physiologic struc-ture that is relevant for N-domain dimerization.

Finally, we analyzed N-domain dimerization of wild-type and mutantSTAT4 in vivo using yeast two-hybrid analysis (Figs. 2c and 3). VariousSTAT4 N-domains were expressed both as LexA DNA-binding domain(LexA BD) fusion proteins (bait) in the yeast L40 strain29 and as Gal4activator domain (Gal4 AD) fusion proteins (prey) in the yeast AMR70strain. These yeast clones were mated and N-domain dimerization wasmeasured by analyzing colony growth on selective medium. Successfulbait-prey interactions in mated yeast cells allow them to grow on selectivemedium, which lacks tryptophan, leucine and histidine. The wild-typeSTAT4 N-domain showed bait-prey interactions consistent with N-domain dimerization (Fig. 2c). Similarly, T40A and E66A mutantsallowed a level of N-domain dimer formation similar to that of the wild-type STAT4 N-domain, as indicated by comparable colony growth under–Trp –Leu –His selection. Notably, the D19R and L78S STAT4 N-domainmutants showed no growth under –Trp –Leu –His selection, indicatinggreatly reduced dimer interaction in vivo. Thus, both in vitro and in vivoapproaches show that the STAT4 N-domain forms the dimer B, but notthe dimer A, assembly.

Figure 1 Analysis of STAT4 dimers produced by crystallographic symmetry to identify thephysiologic dimer. (a) Dimer A (produced by thefractional transformation –Y, –X, –Z+1/6 withtranslation 1, 1, 1) represents the dimer impliedpreviously22. Dimer B (produced by the fractionaltransformation X, X–Y, –Z+5/6 with translation 0,1, 0) represents an alternative interface recentlysuggested25. Highlighted residues were targetedfor mutational studies. Residues W37, T40, and E66 (magenta) are located in the dimer Ainterface, whereas residues D19 and L78 (cyan)are located in the dimer B interface. (b) Surfaceanalysis of the two dimers. According to thisanalysis, dimer B is a statistically bettermolecular interface (as compared to dimer A) and is more likely to represent a physiologicallyrelevant dimer.

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General and homotypic STAT N-domain dimerizationBecause STAT4 (Fig. 2) and STAT1 N-domains25 both show dimer for-mation, we wondered if other N-domains also possessed this property.The N-domain region of each STAT protein was expressed in the pFBL23and pGADT7 vectors to test each STAT N-domain as bait for each STATN-domain (prey) in a matrix of interactions by yeast mating (Fig. 3). TheN-domain of STAT4 formed dimers in this system as expected, andSTAT1 N-domain also dimerized with itself, consistent with a recentreport25. We also observed that the N-domains of the additional STATsshowed interactions as homodimers, so that interactions were observedprimarily only on the diagonal of the mating matrix (Fig. 3). For exam-ple, the N-domains of STAT5A and STAT5B differ at only 11 of the 130amino acid residues comprising the N-domain, yet still show selective,homotypic dimerization. For the STAT2 N-domain–LexA BD fusionprotein, we observed interaction with Gal4 AD itself (data not shown), sowe were unable to determine the specificity of STAT2 N-domain interac-tions. The largely diagonally restricted interactions indicate that N-domain dimerization is primarily homotypic in character within theSTAT family. Notably, N-domain dimerization occurred for STATs, such

as STAT6, that have not been strongly associated with tetramer forma-tion. Thus, the generality and homotypic character of N-domain dimer-ization may indicate other roles for N-domain dimerization in additionto stabilization of STAT tetramers.

N-domain–mediated nonphosphorylated STAT4 dimers in vivoThe ability of N-domain to dimerize indicates that nonphosphorylatedSTAT4 might form dimers before receptor-induced phosphorylation. Totest this prediction directly, we coexpressed Myc-tagged STAT4 withhemagglutinin (HA)-tagged STAT4 in the human cell line U3A, whichlacks endogenous STAT4 expression23,30,31, and evaluated dimer forma-tion by immunoblot analysis (Fig. 4a). First, we used IFN-α treatment asa positive control to induce formation of phosphorylated STAT4 dimers.In this condition, immunoprecipitation (IP) using antibodies to Myc(anti-Myc) coprecipitated HA-tagged STAT4 (Fig. 4a, lane 1). However,even in the absence of IFN-α treatment, when STAT4 tyrosine phospho-rylation was undetectable, anti-Myc could still immunoprecipitate HA-tagged STAT4 (Fig. 4a, lane 2) and vice versa (Fig. 4a, lane 7), showing

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Figure 2 Mutational analysis of STAT4 N-domain dimerization. (a) Theoligomerization state of wild-type and mutant STAT4 N-domains was assessedby gel filtration chromatography. Elution profiles are shown for wild-typeSTAT4 N-domain (solid black line), and D19R (solid cyan line), T40A (solid magenta line), E66A (dashed magenta line) and L78S (dashed cyanline) mutants. Wild-type, T40A and E66A STAT4 N-domains have elutionprofiles indicative of dimers, whereas D19R and L78S STAT4 N-domainshave monomeric elution profiles. Similar results were obtained in threeindependent experiments. Arrows indicate the elution volumes and knownmolecular weights of the standards, ovalbumin (43.0 kDa), chymotrypsinogen(25.0 kDa) and ribonuclease A (13.7 kDa). (b) Hydrodynamic properties ofSTAT4 N-domain mutants measured by gel filtration and dynamic lightscattering. Purified wild-type or mutant STAT4 N-domains were analyzed foroligomerization by gel filtration chromatography and dynamic light scattering(see Methods). The calculated hydrodynamic radius of monomer (1.9 nm)and dimer (2.4 nm) STAT4 N-domains were determined from the crystalstructure22. These solution studies show that mutations at the dimer Binterface disrupt STAT4 N-domain dimerization, corroborating the results ofthe surface analysis. (c) Mutational analysis of STAT4 N-domain dimerizationby yeast two-hybrid assay. cDNAs encoding 1–130 amino acids of the wild-type or the indicated mutant STAT4 N-domains were each placed both inpFBL23, to produce a LexA BD fusion protein, and in pGADT7, to produce aGal4 AD fusion protein. After mating of the two strains, successful bait-preyinteractions allowed yeast growth on selective (–Trp –Leu –His) as well asnonselective (–Trp –Leu plate) medium. The T40A and E66A mutants showedgrowth on selective medium comparable with that of the wild type, consistentwith N-domain dimerization. The experiments using transformants with emptybait or prey vector showed less than 1.4% of wild-type growth.

Figure 3 Homotypic N-domain dimerization in the STAT family. L40 yeastexpressing the indicated STAT N-domain–LexA AD fusion protein were matedwith AMR70 yeast expressing the indicated STAT N-domain–Gal4 BD fusionprotein, grown on –Trp –Leu –His plates and examined for LacZ expression.The corresponding experiment using L40 with STAT2N.pFBL23 was notdone (n.d.), because His3 and LacZ expression were activated even whenthis transformant was mated with AMR70 with empty pGADT7. Data shownare representative of four independent experiments.

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that nonphosphorylated STAT4 dimers were present. Coimmuno-precipitation of Myc-tagged and HA-tagged STAT4 heterodimersrequired expression of both Myc-tagged and HA-tagged STAT4 (Fig. 4a,lanes 3, 4, 8 and 9), indicating that expression of both epitopes wasrequired to detect dimers by coimmunoprecipitation. Together, theseresults indicate that nonphosphorylated STAT4 forms a dimer beforeIFN-α treatment.

To test whether the N-domain is involved in the association of non-phosphorylated STAT4 dimers, we coexpressed Myc-tagged STAT4 andHA-tagged STAT4 harboring various N-domain mutations and analyzedtheir association by immunoblot analysis (Fig. 4b). Again, without IFN-α treatment, STAT4 tyrosine phosphorylation was undetectable, yetMyc-tagged and HA-tagged wild-type STAT4 protein as well as T40A andE66A STAT4 mutants could be coimmunoprecipitated (Fig. 4b, lanes 2, 4and 5). In contrast, neither the Myc-tagged and HA-tagged D19R STAT4mutant nor the L78S STAT4 mutant could be coimmunoprecipitated(Fig. 4b, lanes 3 and 6). To eliminate the possibility that the associationbetween Myc-tagged and HA-tagged STAT4 proteins was due to unde-tected phosphotyrosine–SH2 domain interactions, we mutated the con-served Y693 residue to phenylalanine in each of the constructs analyzed,

and re-examined their association in vivo as before (Fig. 4c). These Myc-tagged and HA-tagged STAT4 mutants could still be coimmunoprecipi-tated (Fig. 4c, lane 1). This association was abrogated by D19R and L78Smutations (Fig. 4c, lanes 2 and 5), but not by the T40A or E66A muta-tions (Fig. 4c, lanes 3 and 4), as before. In summary, these results directlydemonstrate the formation of nonphosphorylated STAT4 dimersthrough their N-domains in vivo before cytokine receptor activation.

N-domain dimerization and in vivo STAT4 activationNext we analyzed the functional effects of STAT4 N-domain mutationsusing IFN-α-induced tyrosine phosphorylation and DNA binding inhuman U3A cells23 (Fig. 5a). IFN-α treatment induced tyrosine phos-phorylation of wild-type STAT4 and the T40A and E66A STAT4mutants, consistent with earlier results23 (Fig. 5a, lanes 2, 5 and 6). Incontrast, IFN-α treatment did not induce tyrosine phosphorylation ineither the D19R or L78S STAT4 mutants (Fig. 5a, lanes 3 and 7).Additionally, for wild-type STAT4 and T40A and E66A STAT4 mutants,IFN-α treatment induced the formation of electrophoretic mobilityshift analysis (EMSA) complexes with the M67 probe containing a singlehigh-affinity consensus STAT binding site (Fig. 5b, lanes 2, 5 and 6).

Figure 5 Formation of nonphosphorylated STAT4 dimers is required forSTAT4 activation and function. (a) U3A cells stably expressing either wild-type STAT4 (WT) or the indicated STAT4 mutants, or infected with GFP-RV(Empty), were either treated (+) with IFN-α or left untreated (–), and celllysates were analyzed for phosphotyrosine and STAT4 protein (see Methods).(b) Nuclear extracts were prepared from cells treated as described in a andanalyzed by EMSA for STAT4 DNA binding activity using the M67 and GASprobes as previously described23. The Eα probe indicates similar amount ofprotein loading and quality of nuclear extracts between samples. (c) U3Acells stably infected with retroviruses expressing either wild-type STAT4 (WT)or the indicated STAT4 mutants, or infected with GFP-RV (Empty), wereeither treated (+) with pervanadate or left untreated (–) for 5 min. Celllysates were immunoprecipitated (IP) with anti-STAT4 and immunoblotted(IB) for phosphotyrosine (P-Tyr) and STAT4 as above. Data are representativeof three independent experiments.

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Figure 4 Formation of nonphosphorylated STAT4 dimers is mediated by N-domains. (a) Coimmunoprecipitation of C-terminal Myc-tagged STAT4 and HA-tagged STAT4. U3A cells were infected (+) with retrovirus expressing Myc-tagged STAT4 (STAT4-Myc) or HA-tagged STAT4 (STAT4-HA) or left uninfected (–),and treated (+) with IFN-α or left untreated (–) (see Methods). Cell lysates were immunoprecipitated (IP) with anti-Myc (c-Myc) or anti-HA (HA) as indicated.Precipitates were analyzed by immunoblotting (IB) for phosphotyrosine (P-Tyr), HA epitope (HA), Myc epitope (c-Myc) or murine STAT4 (STAT4) as indicated.(b) Full-length wild-type (WT), D19R, T40A, E66A or L78S STAT4 were stably expressed by retrovirus with a Myc epitope tag (STAT4-Myc) or an HA epitopetag (STAT4-HA) as indicated and analyzed as described in a. (c) Wild-type or mutant full-length STAT4 with Y693F mutations and with a Myc epitope tag(STAT4Y693F-Myc) or an HA epitope tag (STAT4Y693F-HA), as indicated, were analyzed as in a.

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STAT binding of this site was expected to be unaffected by N-domain–mediated tetramerization. Complexes were also observed with the GASprobe derived from the murine IL-2Rα enhancer containing two adja-cent STAT complexes to interact with STAT4 selectively as tetramers23

(Fig. 5b, lanes 2, 5 and 6). In contrast, D19R and L78S STAT4 mutantsdid not generate EMSA complexes with either probe (Fig. 5b, lanes 3and 7), consistent with the nonphosphorylated state of these mutants. Inall cases, the W37A mutation eliminated phosphorylation and EMSAbinding, as previously described23. Thus, formation of nonphosphory-lated STAT4 dimers, mediated by the N-domain, was required for IFN-αreceptor–mediated STAT4 phosphorylation.

These results show that mutations preventing N-domain dimeriza-tion also prevent STAT4 phosphorylation under physiologic cytokinestimulation. Conceivably, however, these mutations might simply dis-rupt the entire STAT structure rather than selectively altering the pro-posed dimer interface, and so might make STAT4 phosphorylationimpossible in all circumstances. Our gel filtration analysis provides evi-dence against such a global disruption, because the isolated N-domains,except for W37A, behaved as soluble proteins of the predicted mono-meric size. To test directly whether these STAT4 mutants could undergophosphorylation, we repeated the experiments above using non-physiologic pervanadate treatment to inhibit phosphatases (Fig. 5c). Asexpected, pervanadate treatment induced tyrosine phosphorylation ofwild-type STAT4 (Fig. 5c, lane 2). The N-domain mutants that wereunable to undergo cytokine-induced STAT4 phosphorylation could bephosphorylated after pervanadate treatment (Fig. 5c, lanes 3 and 7).Only the W37A mutation inhibited STAT4 phosphorylation after per-vanadate treatment (Fig. 5c, lane 4), indicating that previously reportedfunctional results for the W37A mutation may be due to structural dis-ruption22. Pervanadate treatment, however, did not generate fully func-tional STAT activation as assessed by EMSA analysis, even for wild-typeSTAT4 (see Supplementary Fig. 1 online). Conceivably, pervanadatemay interfere with a number of processes required for full STAT4 activa-tion, but nonetheless this result prevents further analysis of artificiallyphosphorylated STAT4 mutants. Thus, all mutants, except W37A,showed the ability to be phosphorylated, implying that these mutationsdid not alter the global structure of STAT4.

STAT4 is also activated in TH1 cells by IL-12 receptor signaling, andinduction of IFN-γby IL-12 and IL-18 is strongly dependent on STAT4activation32–34, providing a sensitive in vivo assay for STAT4 activation.We therefore expressed wild-type and mutant STAT4 in primary STAT4-deficient T cells and analyzed effects on IL-12- and IL-18-induced IFN-γproduction during IL-12-induced TH1 development (Fig. 6). STAT4-deficient T cells infected with empty retroviral vector (GFP-RV) did notinduce IFN-γ in response to IL-12 and IL-18 stimulation, as expected.Expression of wild-type STAT4 and the T40A and E66A STAT4 mutantsin STAT4-deficient T cells restored IL-12- and IL-18-induced IFN-γ

production, whereas the D19R or L78S STAT4 did not (Fig. 6a). As acontrol, treatment with phorbol 12-myristate 13-acetate (PMA) andionomycin induced IFN-γ production (>44% positive staining) in allcells, consistent with the fact that induction of IFN-γby this treatment isnot STAT4 dependent34. This control also shows that the defect in cellsexpressing the D19R and L78S STAT4 mutants was specific to cytokinesignaling and not a general defect in IFN-γ gene inducibility. Weobtained similar results when the developing TH1 cells were maintainedin culture for several weeks to obtain strongly polarized cytokine pro-duction with higher IFN-γ production (Fig. 6b). Again, infection byempty retroviral vector generated cells unable to produce IFN-γ inresponse to IL-12 and IL-18 signaling, whereas expression of wild-typeSTAT4 and the T40A or E66A STAT4 mutants, but not the D19R or L78SSTAT4 mutants, restored IFN-γ induction (Fig. 6b). Thus, formation ofnonphosphorylated STAT4 dimers, mediated by the N-domain, wasrequired for IL-12- and IL-18-induced IFN-γproduction by TH1 cells.

In vivo STAT1 phosphorylation without N-domain dimerizationAs shown above, N-domain–mediated STAT4 dimerization was requiredfor STAT4 activation and function. We wondered if other N-domainsalso possess this property. Because M28A and L78A mutations have beenreported to disrupt the STAT1 N-domain dimerization25, we analyzedwild-type STAT1 and M28A, L78A and N-domain–truncated STAT1mutants for the ability to undergo phosphorylation in U3A cells (seeSupplementary Fig. 2 online). The loss of the N-domain still allowedIFN-α- or IFN-γ-induced phosphorylation of STAT1 (SupplementaryFig. 2), although this STAT1 is not functional35. M28A and L78A muta-tions did not affect the ability of STAT1 to undergo IFN-α- or IFN-γ–induced phosphorylation (Supplementary Fig. 2), indicating thatSTAT1 may be phosphorylated by a different system from STAT4.

Figure 6 N-domain dimerization–dependent STAT4 activation in vivo. (a) STAT4-deficient primary DO11.10 T cells were activated in vitro (seeMethods) and infected as described46 with retrovirus expressing wild-typeSTAT4 (WT) or the indicated STAT4 mutant, or with empty retrovirus (Empty).T cells were stimulated on day 3 after infection either with IL-12 and IL-18 orwith PMA and ionomycin and analyzed on day 4 for intracellular production of IFN-γ as previously described34. Panels show events gated on live GFP+ Tcells. Numbers in the upper right quadrants are percentages of IFN-γ positivecells. Data are representative of three independent experiments. (b) Cellsdescribed in a were purified by fluorescence-activated cell sorting for GFPexpression, and were restimulated on a weekly basis with antigen under TH1-inducing conditions as described34 to generate long-term TH1 lines. Theselines were restimulated as indicated and IFN-γ production was analyzed asdescribed in a. Data are presented as in a.

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DISCUSSIONThe STAT4 N-domain was previously proposed to stabilize interactionsbetween two activated STAT dimers, to form tetramers binding to clus-tered nonconsensus STAT elements in cytokine-responsive genes22. Thismodel was very attractive because STAT4 forms tetramers on clusteredSTAT elements in the IFN-γgene first intron7, STAT5 forms tetramers onthe CD25 promoter-enhancer region36,37 and such recognition of non-consensus STAT elements might provide a means for selective target-gene activation. However, subsequent experiments initially did notsupport this hypothesis23, suggesting rather that the STAT4 N-domainparticipated in receptor-mediated phosphorylation, and it was laterdetermined that the original mutational analysis22 was based on anincorrect interpretation of the physiologic dimer interface25. In addition,functional data supporting STAT tetramer formation were based on theW37A mutations in STAT1, rather than STAT4 (ref. 22), and subsequentwork analyzing STAT5 tetramers also used the W37A mutation36,37,which is now recognized to disrupt the N-domain structure25.

Our purpose here was to analyze the biological role of N-domaindimerization in STAT4 activity. Because this required us to identify spe-cific mutations that would selectively interrupt N-domain dimerization,rather than globally disrupting the N-domain, we started this study byanalyzing the dimerization of soluble N-domain proteins in solution invitro and in a yeast two-hybrid system. These approaches allowed us toidentify and confirm an alternative interface from the one previouslyproposed22 and to produce mutations that selectively interrupt N-domain dimerization without global disruption of the domain. Next, weselected two systems for functional analyses, IFN-α activation of STAT4in human U3A cells38 and IL-12-induced STAT4-dependent responses inmouse T cells32. Analyzing the functional consequences of these muta-tions showed that STAT4 dimerization via the N-domain occurs in vivobefore cytokine signaling and that these nonphosphorylated STAT4dimers are the preferred receptor-kinase substrate and are required forcytokine-mediated STAT4 activation. Finally, we found that N-domaindimerization is shared by most other STAT family members.

Previously, STAT activation was generally thought to involve tyrosinephosphorylation by the activated receptor–JAK kinase complex of cyto-plasmic STAT monomers. After phosphorylation, STAT monomerswould eventually associate with other appropriately phosphorylatedSTAT monomers to form reciprocal SH2-phosphotyrosine interactionsstabilizing the activated STAT dimer (see Supplementary Fig. 3 online).In that model, the formation of appropriate STAT dimers would bedelayed by the process of diffusion-limited association between phos-phorylated monomers, and would also be in competition with nonpro-ductive unions between heterologous STAT species and with abortiveunions between phosphorylated STAT monomers and nonphosphory-lated monomers. By contrast, our results suggest a model in which STATmonomers are preassembled into nonphosphorylated dimers via the N-domain, a process that can occur in the entire time leading up tocytokine receptor triggering (Supplementary Fig. 3 online). In ourmodel, formation of activated STAT dimers is enhanced because the twopreassociated STAT monomers are presented to the receptor–JAK kinasecomplex simultaneously, favoring synchronized phosphorylation of thetwo STAT monomers and allowing formation of the active STAT dimerby a simple intramolecular rearrangement. Additionally, in our model,cytokine receptors that concurrently activate more than one STATspecies39 could avoid generating mixed STAT dimers which mightdegrade or complicate signaling specificity. For example, IL-12 inducesphosphorylation of STAT1, STAT3 and STAT4 (refs. 1,40). The prefer-ence for homotypic N-domain dimerization would favor the preassoci-ation primarily of nonphosphorylated STAT1 homodimers, STAT3homodimers and STAT4 homodimers, which upon phosphorylation

and intramolecular rearrangement would primarily favor activatedSTAT homodimers.

Our results are strictly applicable to STAT4 at the moment. In the caseof STAT4, we directly demonstrated the formation of N-domain–dependent nonphosphorylated homodimers, which were required forreceptor-induced tyrosine phosphorylation of STAT4 and downstreamtranscriptional activation. Previous STAT4 phosphorylation that wasindependent of a functional N-domain may have resulted from in vitrokinase treatment22 or from unknown kinase activities in insect cells36,each of which may bypass normal physiologic barriers. In addition, notall STAT species may require N-domain–mediated dimerization forreceptor-driven phosphorylation. We showed that loss of the STAT1 N-domain still allows IFN-α- or IFN-γ-induced phosphorylation of STAT1,although this STAT1 was not functional35. N-domain mutations thatwere reported recently to disrupt the STAT1 N-domain dimerization25

do not effect on the ability of STAT1 to undergo IFN-γ- or IFN-α-induced phosphorylation, indicating that STAT1 may be phosphorylatedby a different system from STAT4. In summary, our data provide an addi-tional role for the N-domain in STAT4 activation, in which N-domaindimerization participates in the formation of nonphosphorylateddimeric precursors, which, in the case of STAT4, is required for cytokine-mediated STAT activation and responses.

METHODSCytokines, antibodies and reagents. Recombinant (r) human IFN-α A was pur-chased from Serotec. Murine and human rIFN-γand STAT3 cDNA were gifts fromR.D. Schreiber (Washington University School of Medicine). Human rIL-2 wasprovided by Takeda. Murine rIL-12 was from Genetics Institute. Murine rIL-18was purchased from Medical and Biological Laboratories. Yeast strains L40 andAMR70 were gift from A. Shaw (Washington University School of Medicine).pFBL23 was a gift from J. Camonis (Paris, France). Monoclonal antibody specificfor the Myc tag and polyclonal antisera specific for murine STAT1 and STAT4 andfor the HA tag were purchased from Santa Cruz Biotechnology. Peroxidase-conjugated RC20 antibody to phosphotyrosine was purchased from TransductionLaboratories. Peroxidase-conjugated goat anti-rabbit was purchased from JacksonImmunoResearch Laboratories. TriColor-conjugated anti–murine CD4 andFITC-conjugated anti–murine CD62L were purchased from Caltag. APC-conjugated anti–murine IFN-γwas purchased from BD Biosciences. Monoclonalanti-CD3 (500A2), anti-CD28 (37.51) and anti-IL-4 (11B11) were as described34.

Surface analysis. Solvent accessible surface areas (ASAs) were calculated with theprogram NACCESS41 using a 1.4-Å probe sphere. Buried ASA was calculated asthe sum of the ASA for the isolated components (two STAT4 N-domainmonomers) minus that of the dimer. The buried ASAs were further analyzed bythe percentage that is contributed by nonpolar (all but N or O) and polar surfaces(N and O). Shape correlation (Sc) values were calculated with the program SC27

using the default parameters.

Plasmid construction and protein preparation of STAT4 N-domain mutations.The plasmid pTYB1 was modified for expression of wild-type and mutant STAT4N-domains (1–121 amino acids) using the IMPACT-CN system (New EnglandBiolabs) as follows. Wild-type STAT4 N-domain was amplified by PCR usingSTAT4 cDNA23 as template and the primers STAT4-TYB forward (F) and STAT4-TYB reverse (R). Sequences for all oligonucleotide primers are provided inSupplementary Table 1 online. The PCR product was digested with NdeI, treatedwith T4 polynucleotide kinase, ligated to the pTYB1 vector prepared by digestionwith SapI, blunted with Vent polymerase, digested with NdeI, and treated withcalf intestinal phosphatase, to produce STAT4N.pTYB1. STAT4N.pTYB1 wasused as template to generate other mutant STAT4 N-domains using QuickChange Site Directed Mutagenesis (Stratagene) with primers D19R (F), D19R(R), L78S (F) and L78S (R). To generate W37A, T40A and E66A mutations inSTAT4N.pTYB1, previously described primers were used23. Expression andpurification of wild-type and mutant STAT4 N-domain proteins were doneaccording to the instructions of the manufacturer of the IMPACT-CN system(New England Biolabs).

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Gel filtration analysis. A Superdex 75 gel filtration column (16/60; AmershamBiosciences) used at a flow rate of 1.0 ml/min was calibrated using albumin (67.0 kDa, elution volume 55.1 ml), ovalbumin (43.0 kDa, 61.3 ml), chy-motrypsinogen A (25.0 kDa, 71.3 ml) and ribonuclease A (13.7 kDa, 80.9 ml)(Amersham Biosciences). In all cases the column buffer consisted of 20 mMHEPES (pH 7.6), 150 mM NaCl and 5 mM DTT. The molecular masses of thewild-type and mutant STAT4 N-domains were calculated from their respectiveelution volumes by reference to a calibration plot. Confidence intervals reported inFigure 2b reflect the standard deviation from the mean elution volume for threereplicate runs.

Dynamic light-scattering analysis. Dynamic light-scattering (DLS) experimentswere carried out using a DynaPro MSX Molecular Sizing Instrument (ProteinSolutions) and data were analyzed with the Dynamics software package (V6.2).Measurements were conducted at 20 °C at a sample concentration of 1 mg/ml in abuffer containing 20 mM HEPES (pH 7.6), 150 mM NaCl and 5 mM DTT. Thesolutions were prefiltered through a 0.02-µm membrane filter (Anodisc 13;Whatman) into a 12-ml sample cuvette for analysis. The values were frommonomodal fits and are an average of 20 measurements.

Plasmid constructions for yeast two-hybrid analysis. Wild-type N-domains ofeach murine STAT were placed into the pFBL23 vector29 to generate an N-termi-nal fusion of the N-domain fused to LexA DNA binding domain (LexA BD) asfollows. For each STAT, the N-domain coding region was amplified using primersSTAT1-FBL (F), STAT1-FBL (R), STAT2-FBL (F), STAT2-FBL (R), STAT3-FBL(F), STAT3-FBL (R), STAT4-FBL (F), STAT4-FBL (R), STAT5A-FBL (F),STAT5A-FBL (R), STAT5B-FBL (F), STAT5B-FBL (R), STAT6-FBL (F) andSTAT6-FBL (R). PCR reactions were carried out using the corresponding STATcDNA template23. For STAT1, STAT4 and STAT6, PCR products were digestedwith XhoI and SalI and ligated into SalI-digested pFBL23 (ref. 29). The STAT2PCR product was digested with MfeI and BamHI and ligated into EcoRI- andBamHI-digested pFBL23. The STAT3 PCR product was digested with EcoRI andBamHI and ligated into EcoRI- and BamHI-digested pFBL23. The STAT5A PCRproduct was digested with EcoRI and BglII and ligated into EcoRI- and BamHI-digested pFBL23. The STAT5B PCR product was digested with EcoRI and ligatedinto EcoRI-digested pFBL23.

Wild-type N-domains of each murine STAT member were placed into thepGADT7 vector (BD Biosciences Clontech) to generate Gal4 activator domain(Gal4 AD) fusion proteins as follows. For each STAT, the N-domain coding regionwas amplified using primers STAT1-GAD (F), STAT1-GAD (R), STAT4-GAD (F),STAT4-GAD (R), STAT6-GAD (F) and STAT6-GAD (R). For STAT2, STAT3,STAT5A and STAT5B, the same primers were used as for cloning into pFBL23. TheSTAT1, STAT4 and STAT6 PCR products were digested with EcoRI and BamHIand ligated into EcoRI- and BamHI-digested pGADT7. The remaining STAT PCRproducts were digested as described for the corresponding STAT fragment in thepFBL23 subcloning and cloned into pGADT7 digested with EcoRI or EcoRI plusBamHI. The STAT N-domain region subcloned in these vectors consisted ofamino acids 1–130 for STAT1, STAT3, STAT4, STAT5A and STAT5B, amino acids1–129 for STAT2 and amino acids 1–125 for STAT6.

To generate STAT4 N-domain mutations for yeast two-hybrid analysis, QuickChange Site Directed Mutagenesis was carried out on both the STAT4N.pFBL23and STAT4N.pGADT7 plasmids using the primer pairs R87E (F) and R87E (R).D19R and L78S were made with the oligonucleotide primers described above, andW37A, T40A and E66A mutations with primers described previously23.

Yeast two-hybrid analysis. STAT4 N-domain–LexA BD fusion proteins encodedin pFBL23 and STAT4 N-domain–Gal4 AD fusion proteins encoded in GADT7were transformed into yeast strains L40 and AMR70, respectively, and cultured in5 ml of –Trp and –Leu media, respectively, at 30 °C overnight. In Figure 3, yeaststrains L40 and AMR70 were transformed with STAT N.pFBL23 or STATN.pGADT7 plasmids, respectively. The mating procedures and LacZ assay weretaken from the manufacturer’s manual (BD Biosciences). The correspondingexperiment using L40 with STAT2N.pFBL23 was not done, because His3 and LacZexpression were activated even when this transformant was mated with AMR70bearing empty pGADT7.

Constructs for retroviral expression in U3A cells. We had previously clonedwild-type murine STAT4 cDNA into the retroviral vector GFP-RV and introduced

the W37A, T40A and E66A mutations into the STAT4 N-domain as described23.Additional D19R, L78S and R87E mutations were introduced using Quick ChangeSite Directed Mutagenesis using the D19R (F), D19R (R), L78S (F), L78S (R),R87E (F) and R87E (R) primers described above. An epitope from the influenza AHA was placed at the C terminus of STAT4 in GFP-RV to produce STAT4-HA.GFP-RV by Quick Change Site Directed Mutagenesis with primers ST4-HA(F) and ST4-HA (R). We had previously placed the STAT4 cDNA into an alternatebicistronic retroviral vector, CD4-RV30, that uses a truncated human CD4 recep-tor as a marker. We placed an epitope from the c-Myc gene42, EQKLISEEDL,derived from residues 408–432 of human c-Myc, at the C-terminus of STAT4 inCD4-RV to produce STAT4-Myc.CD4-RV, by Quick Change Site DirectedMutagenesis with primers ST4-Myc (F) and ST4-Myc (R). Mutations in theSTAT4 N-domain in these retroviral constructs were introduced by Quick ChangeSite Directed Mutagenesis using primers described above for D19R, T40A, E66Aand L78S. Virus was prepared from these constructs and used to infect U3A cells asdescribed30. Finally, we mutated the conserved STAT4 tyrosine Tyr693 to pheny-lalanine in all wild-type and mutant STAT4 constructs in both GFP-RV and theCD4-RV vectors by Quick Change Site Directed Mutagenesis using primers ST4-Y693F (F) and ST4-Y693F (R).

For the construction of STAT1.GFP-RV, the STAT1 cDNA30 was amplified byPCR using STAT1.pBSSK as the template and the oligonucleotides STAT1-FBL (F)and STAT1 (R). PCR products were phosphorylated with T4 polynucleotide kinaseand then ligated to the GFP-RV43 vector prepared by digestion with BglII, bluntedwith Vent polymerase and treated with calf intestinal phosphatase. STAT1.GFP-RVwas used as template to generate mutant STAT1.GFP-RV using Quick Change SiteDirected Mutagenesis with the oligonucleotides M28A (F), M28A (R), L78A (F)and L78A (R). An N-domain–truncated STAT1 (∆N STAT1) was designed to initi-ate from a methionine followed by residue Q132 of native STAT1. ∆N STAT1 cDNAwas amplified by PCR reaction using STAT1.pBSSK as template and the oligonu-cleotides STAT1C (F) and STAT1 (R). PCR products were phosphorylated with T4polynucleotide kinase and then ligated to the GFP-RV vector prepared by digestionwith BglII, blunted with Vent polymerase and treated with calf intestinal phos-phatase. All constructs were verified by sequencing.

Expression of Myc-tagged and HA-tagged STAT4 proteins in U3A fibroblast.Phoenix-Amphotropic packaging cell lines were transfected with retroviral vec-tors expressing Myc-tagged STAT4 and STAT4 mutants and HA-tagged STAT4and STAT4 mutants by calcium phosphate precipitation as described43, and U3Acells were infected as described30. Infected U3A cells were purified by cell sortingfor expression of the GFP and the hCD4 surface markers as described30. Sortedcells were expanded; they were >90% pure and stably expressed the retroviralmarkers. Monolayers of fibroblasts were incubated with 500 U/ml of human IFN-α A at 37 °C for 25 min. Whole-cell lysates were prepared by incubating cells inlysis buffer containing 20 mM HEPES (pH 7.6), 150 mM NaCl and 2.0% Tween 20at 4 °C for 1 h. Immunoprecipitation was carried out with mouse monoclonalanti-Myc or rabbit polyclonal anti-HA and protein G–Sepharose (AmershamBiosciences). Immunoprecipitates were resolved by denaturing SDS-PAGE andwere transferred to nitrocellulose. Phosphotyrosine-containing proteins, HA-tagged protein, Myc-tagged protein and STAT4 protein were detected by blottingwith peroxidase-conjugated RC20, rabbit polyclonal anti-HA, mouse monoclonalanti-Myc or rabbit polyclonal anti-STAT4, respectively.

Analysis of tyrosine-phosphorylated wild-type and mutant STAT proteins.Phoenix-Amphotropic packaging cell lines were transfected with retroviral vec-tors expressing full length STAT4 and STAT4 mutants as described43. Monolayersof fibroblasts were incubated with 500 U/ml of human IFN-α A at 37 °C for 25 min. Whole-cell lysates were prepared by incubating cells in RIPA buffer (150 mM NaCl, 1.0% Nonidet P-40 deoxycholate, 0.1% SDS and 50 mM Tris-Cl(pH 8.0)) at 4 °C for 1 h, and STAT4 molecules were precipitated with rabbit poly-clonal anti-STAT4 and protein G–Sepharose. Phosphotyrosine-containing pro-teins and STAT4 protein were detected as above. A 20× stock of pervanadatesolution was prepared 5 min before use by diluting 105 µl of 1 M Na3VO4 and 33µl of 30% H2O2 to 5 ml in distilled water. Pervanadate treatment was done at 37 °Cfor 5 min (for IP experiments) or 25 min (for EMSA experiments). For analysis ofSTAT1 proteins (Supplementary Fig. 2 online), Phoenix-Amphotropic packagingcell lines were transfected with retroviral vectors as described above. Monolayersof fibroblasts were incubated with 1,000 U/ml of human IFN-α A or human

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IFN-γat 37 °C for 25 min. Immunoprecipitation was carried out with rabbit poly-clonal anti-STAT1 and protein G–Sepharose. Phosphotyrosine-containing pro-teins and STAT1 protein were detected as described above.

Electrophoretic mobility shift analysis. EMSA was carried out as described previ-ously23. The following DNA probes were used in this study: M67SIE1, GTCGACATTTCCCGTAAATCGTCGA; GAS c+n23, GTGCAGTTTCTTCTG\AGAAGTACCAGACATTTCTGATAAGAGAG; Eα Y-box23, TCGACATTTTTCTGATTGGTTAAAAGTC.

Intracellular cytokine staining of T cells. We previously used GFP-RV to expressSTAT4 in cells23,44, but higher STAT4 levels were reported using the vectorSTAT4.pBMN-I-GFP45. Thus, for primary T cell infections, we usedSTAT4.pBMN-I-GFP for all STAT4 expression and introduced all mutations of theN-domain by Quick Change Site Directed Mutagenesis using primers describedabove for D19R, T40A, E66A and L78S. Naive STAT4-deficient DO11.10 T cellswere purified by cell sorting for CD4 and CD62L expression using FITC-conjugated monoclonal anti–murine CD62L and TriColor-conjugated mono-clonal anti–murine CD4, stimulated with 5 µg/ml of plate-bound anti-CD3 and 2 µg/ml of monoclonal anti-CD28 under TH1-inducing conditions (50 U/ml ofhuman rIL-2, 10 U/ml of murine rIL-12, 100 U/ml of murine rIFN-γand 10 µg/mlof anti-IL-4), infected on day 1 and day 2 after stimulation as described43, washedon day 3 and restimulated either with 10 U/ml of murine IL-12 and 1 ng/ml ofmurine IL-18 for 18 h or with 50 ng/ml of PMA and 1 µM of ionomycin for 5 h. 1 µg/ml of brefeldin A (Epicentre) was supplied for the final 3 h. Cells were fixedwith 2% formaldehyde in PBS and treated with FACS buffer (3% FBS in PBS) con-taining 0.05% saponin, incubated with either APC-conjugated monoclonal anti-IFN-γ or isotype control and analyzed by flow cytometry (FACSCalibur, BDBiosciences). These same conditions were used to derive cells by weekly restimula-tion with 0.3 µM of OVA(323–339) peptide and irradiated BALB/c splenocytes(2,000 rad) under TH1-inducing conditions (50 U/ml of human rIL-2, 1,000 bac-teria/ml of heat-killed Listeria monocytogenes, 100 U/ml of murine rIFN-γ and 10 µg/ml of anti-IL-4; Fig. 6b). Cell lines were washed on day 3 after their last stim-ulation and restimulated either with 10 U/ml of murine IL-12 and 1 ng/ml ofmurine IL-18 for 18 h or with 50 ng/ml of PMA and 1 µM of ionomycin for 5 h.For both restimulations, 1 µg/ml of brefeldin A was supplied for the final 3 h.Intracellular staining for IFN-γwas done as described above.

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

ACKNOWLEDGMENTSThe authors thank G.R. Stark (Lerner Research Institute, Cleveland, Ohio, USA) forproviding the U3A cells, and J.D. Farrar (University of Texas Southwestern MedicalCenter, Dallas, Texas, USA) for helpful discussions.

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

Received 16 August; accepted 24 November 2003Published online at http://www.nature.com/natureimmunology.com/

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