Proc. Nati. Acad. Sci. USAVol. 87, pp. 1032-1036, February 1990Biochemistry

Expression and purification of the leucine zipper and DNA-bindingdomains of Fos and Jun: Both Fos and Jun contact DNA directly

(oncogenes/transcription/protein complexes/gene regulation/Escherichia coli expression)

CORY ABATE, DANIEL LUK, REINER GENTZ*, FRANK J. RAUSCHER III, AND TOM CURRANtDepartment of Molecular Oncology and Virology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110

Communicated by Herbert Weissbach, November 6, 1989

ABSTRACT The protein products of the fos and junprotooncogenes interact cooperatively in the form of a het-erodimer with the activator protein 1 (AP-1) regulatory ele-ment. To characterize the properties of these proteins, we haveexpressed polypeptides comprised of the dimerization andDNA-binding domains of Fos and Jun in Escherichia coli. Themini-Fos (wbFos) and the mini-Jun (wbJun) proteins werepurified to apparent homogeneity by using a nickel affinitychromatography procedure. Purified wbFos and wbJun asso-ciated rapidly in vitro and interacted cooperatively with thehuman metallothionein H1A AP-1-binding site. However, effi-cient DNA binding of wbJun and wbFos-wbJun complexesrequired an additional activity present in nuclear extracts. Thisactivity was sensitive to alkylating agents and could be partiallymimicked by the presence of reducing and stabilizing agents.DNase I footprinting experiments demonstrated that Jun ho-modimeric complexes and Fos-Jun heterodimeric complexesinteracted with the same site on the human metallothionein H1Agene. Moreover, UV-crosslinking studies demonstrated thatFos and Jun contact DNA directly and that both proteinsinteracted equivalently with either strand of the AP-1-bindingsite.

A fundamental problem confronting many disciplines inmodern biology concerns the molecular mechanismswhereby long-term cellular phenotypic adaptations occur inresponse to environmental cues. Over the past several yearsa great deal of evidence has accumulated suggesting thattranscriptional regulatory proteins play a key role in couplingextracellular signals to gene expression in the nucleus byinteracting with cis-acting responsive elements in targetgenes. It has become apparent that several protooncogenes,the progenitors of retroviral oncogenes, are involved in theregulation ofgene expression. In particular, Fos and Jun, theprotein products of c-fos and c-jun, are rapidly and tran-siently induced by a diverse array of extracellular stimuli andhave been implicated in the transcriptional regulation oftarget genes in response to these stimuli (for review, see ref.1).

c-fos is the cellular gene from which the transforming genesof the FBJ (Finkel-Biskis-Jinkins) and FBR (Finkel-Biskis-Reilly) murine sarcoma viruses are derived (2, 3). Itsprotein product, Fos, forms a stable complex in the nucleuswith a cellular protein, originally termed p39 (4, 5). p39 hasnow been identified as Jun (6), the product ofthe cellular gene(c-jun) from which the oncogene (v-jun) is derived (7).Several concordant lines of research led to the identificationof Fos and Jun among the proteins that contribute to theDNA-binding activity associated with the mammalian tran-scription factor activator protein 1 (AP-1) (for review, see ref.1). The AP-1-binding site was first identified in the enhancer

regions of simian virus 40, human metallothionein IIA(hMTIIA), and several phorbol ester-responsive genes (8, 9).It is now apparent that similar nucleotide sequence motifs arepresent in negative and positive regulatory regions of severalgenes (see, e.g., refs. 10 and 11).

Studies carried out using proteins translated in vitro inrabbit reticulocyte lysates established that Fos and Jun forma heterodimeric complex that interacts with the AP-1-bindingsite (12). Although Fos alone does not bind to the hMTIIAAP-1 site, Jun can bind to this site as a homodimer with arelatively low affinity. The presence of Fos enhances thebinding of the Fos-Jun complex primarily by stabilizing theprotein-DNA interaction (12). Protein complex formation isrequired forDNA binding and occurs by a parallel interactionof the leucine-zipper domains thatjuxtaposes adjacent DNA-binding regions (13-17). The DNA-binding domain is com-prised of regions rich in basic amino acids and is contributedby Fos and Jun (14). Here we have used purified proteinssynthesized in Escherichia coli for a direct characterizationof the DNA-binding properties of Fos and Jun.

MATERIAL AND METHODSConstruction and Purification ofwbfos and wbjun. A central

region of the c-fos (rat) gene (amino acids 116-211) wasreconstructed using a total of 16 oligonucleotides as indicatedin Fig. 1A. The sequence was compiled using the mostfrequently utilized codons present in a selection ofgenes thathad been successfully expressed at high levels in E. coli(R.G., unpublished data). The reconstructed fragment wasconfirmed by dideoxynucleotide sequencing and subclonedinto the pDS56-6xHis vector (18), as indicated in Fig. LA.This vector fuses six histidine residues plus another six aminoacids to the N terminus. The reconstructed fos gene isreferred to as "weebugfos," abbreviated wbfos. The DNA-binding and leucine-zipper domains ofjun were cloned intopDS56-6xHis using a Pst I-EcoRI fragment of pc-jun(rat)(12), as illustrated in Fig. 1B. This vector is referred to as"weebugjun," abbreviated wbjun. The recombinant proteinswere expressed and purified using a nickel-chelate affinityresin, as described (19, 20).

Protein Association and DNA-Binding Assays. Protein as-sociation, gel-shift assays, and UV-crosslinking assays wereperformed as described (6, 10, 12, 21). Nuclear extracts wereprepared from fresh rat liver by the method of Triezenberg etal. (22). Alkylation was performed by incubating cell extractsin the absence or presence of 10 mM methyl methanethio-sulfonate (Sigma) for 20 min at room temperature. Excessreagent was removed by gel filtration. For some samples,alkylation was reversed by the addition of excess dithiothrei-

Abbreviations: hMTIIA, human metallothionein I1A; AP-1, activatorprotein 1; DTT, dithiothreitol; BSA, bovine serum albumin; NP40,Nonidet P-40.*Present address: F. Hoffmann La Roche Inc., Basel, Switzerland.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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A pDS56AT GA GATCS CAT CAC CAT CAC CAT CAC TCC GGT CGT GCG CAB TCC ATC GGT CGT 60TACXTTC ABCTM AC TG OTA YAG GMC CCAGCA CGC GTCA6G TAG CCA GCAM 6a H H NH H 13 6 R A l8 I e R

CGC GGT AM GT6jGAA CM CTG TCC CCG GM GAG GAA GAG AM CGTJCGC ATC CGC CGT GM 120 69GCG CCA m CM CTr 6Tr GP AMG GGC CTT CTC CTr CTC m GA GC6 TAG GCP GCA CTTR 6 K V E Q L 8 P E E E E K R R I R R E

46 # v

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IWCGTAA AM ATM GCG GCA CG AMI TC CGT A M CCGTCGT GM CTMC GM ACC CTG 180GCA TTG m TAC CGC CGT CGC m ACG GCA I AC GCAGCA CT GAC TGG CTG ToGGR N K M A A A K C R I R R R E L T D T -

C4 GCG GM ACC GAC CAM CTG GM GM GM AM TCC GCGICTG CM ACC GM ATC GCG AM 240GI CGCCTTGG cT TCGmCTCTG CI 6*T ArMGCGC GTTTGG CTr TAG CGC TTGC A E T D a A E D E Kl A U T E I A N

CTG CTG AM GM AM GM MCTG GATe C ATC CTG GCG GCA CAC CGT CCM I(, TIC AM 300GAC GM m CIT lOTT CI lIC GMe CTC Am TM GM CGC CIT GTIIICA GC CIC MG m1

L K E K E K * E F I L A A H R P A C KMEmdM

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FIG. 1. Construction of wbFos and wbJun and purification of the recombinant proteins. (A) The nucleotide sequence of wbfos and predictedamino acid sequence of wbFos are illustrated. The nucleotides and amino acids contributed by the pDS56 vector are indicated by shading. Theextent of the individual synthetic oligonucleotides is indicated with bars in the nucleotide sequence. The leucines of the leucine zipper arehighlighted. The positions ofBamHI, Pst I, and HindIll sites used for the constructions are indicated. The single-letter amino acid code is used.(B) Schematic illustration of the domains of Fos and Jun expressed from the Pn25 promoter in the pDS56-6xHis vector. The vector providesan initiator methionine codon (M) and fuses six histidine (H6) residues to the N terminus. RBS is the ribosome binding site. (C) wbFos (lane2) and wbJun (lane 3) were purified by affinity chromatography on nickel-chelate columns. Approximately 1 Ag of purified proteins was resolvedon a 15% polyacrylamide/SDS gel and stained with silver nitrate. Lane 1 contains molecular mass standards labeled in kDa.

tol (DTI) (20 mM) and DTT was subsequently removed bygel filtration. For DNase footprinting, a 100-base-pair frag-ment (nucleotides -55 to -154) of the hMTIIA promoter wasamplified by the polymerase chain reaction. Convenientrestriction sites were generated using oligonucleotides thatadded BamHI and HindI11 restriction sites to the 5' and 3'ends ofthe fragment. The amplified fragment was end-labeledand the sequence was confirmed. DNase footprinting assayswere performed according to the method of Jones et al. (23).

RESULTS AND DISCUSSIONExpression and Purification of wbFos and wbJun. Previous

mutagenesis studies demonstrated that the dimerization andDNA-binding domains of Fos are contained within aminoacids 116-211 (14) and that those of Jun are contained withinthe C-terminal region, amino acids 206-315 (24). Thesedomains of Fos and Jun were expressed in E. coli as fusionproteins containing six histidine residues at the N termini, asillustrated in Fig. 1B. Histidine fusion proteins can be rapidlypurified by affinity chromatography using a nickel-chelatecolumn (19, 20). Previous attempts to express Fos at highlevels in E. coli were not successful (25), in part, because ofthe prevalence ofcodons infrequently utilized in E. coli in thec-fos sequence (26). Thus, the mini-fos gene (wbfos), con-taining the sequence coding for Fos amino acids 116-211, wasreconstructed by ligation of synthetic oligonucleotides con-taining codons frequently used in E. coli (Fig. 1A). Afterinduction by isopropyl 8-D-thiogalactopyranoside, thewbFos polypeptide constituted approximately 30%o oftotal E.coli protein and had an apparent molecular mass of 15 kDa onSDS/polyacrylamide gels (Fig. 1C). For expression of theappropriate region of Jun in E. coli, a DNA fragment,obtained by digestion of pc-jun(rat) (12) with Pst I and EcoRI,was cloned into the expression vector pDS56 (18) (Fig. 1B).

The level of expression of the mini-Jun protein (wbJun)approached 5% of total E. coli protein content and it had anapparent molecular mass of 19 kDa (Fig. 1C). Both wbFosand wbJun were solubilized in the presence of 6 M guanidinehydrochloride and were purified by affinity chromatographyon a nickel-chelate adsorbent in the presence ofguanidine, asdescribed (20). The proteins were solubilized by extensivedialysis against 25mM Mes (pH 6.0). The purity ofthe wbFosand wbJun preparations was estimated to be at least 95%(Fig. 1C).

Dimerization and DNA-Binding Properties of PurifiedwbFos and wbJun. Coimmunoprecipitation assays, per-formed using radiolabeled wbFos and wbJun expressed in E.coli, demonstrated that these proteins associated efficientlyin vitro (Fig. 2A). These data are in agreement with ourprevious finding that in vitro-translated Fos and Jun associaterapidly after mixing (12, 14) and are in contrast with otherreports that suggested cotranslation is required for het-erodimer formation (15). In fact, the Fos-Jun complex hasbeen shown to form after translocation of the newly synthe-sized proteins to the nucleus (4).

Full-length Fos and Jun translated in reticulocyte lysatesinteract cooperatively with the AP-1-binding site in gel-retardation assays (12). Similar results were obtained withwbFos and wbJun translated in vitro (Fig. 2B). These dataconfirmed the specificity of the truncated wbFos and wbJunproteins for the AP-1-binding site and suggested that thehexa-histidine addition at the N termini did not interfere withthe protein-DNA interaction. In contrast to the results ob-tained with the in vitro-translated proteins, purified wbFosand wbJun (5 ,g/ml) exhibited no DNA-binding activity ingel-shift assays (Fig. 2B). However, DNA binding was de-tected when the protein concentration was increased to 400Ag/ml. This protein concentration is much higher than thatobtained by in vitro translation (less than 1 ,ug/ml). To

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presence of [35S]methionine. Extracts were immunoprecipitated with anti-Fos or anti-Jun antibodies as indicated, the products were resolvedon a 15% polyacrylamide/SDS gel, and an autoradiograph is shown. Lane Std contains '4C-methylated molecular mass standards (Amersham).(B) The DNA-binding properties of wbFos and wbJun were assessed by gel-retardation assays. wbFos and wbJun, either translated inreticulocyte lysates or purified from E. coli, were incubated together at 37rC for 30 min. Purified proteins were assayed alone or in the presence

of a reticulocyte lysate (retic; 5 ILI), liver nuclear extract (liver nucleus; 5 jig), or liver whole cell extract (30 ,ug), as indicated. 32P-labeledoligonucleotides containing an AP-1 site were added in the presence of poly(dl dC). Protein-DNA complexes were resolved from unboundoligonucleotides on low-ionic-strength polyacrylamide gels (6.5%). NA, no addition.

determine whether factors present in the reticulocyte lysatecontributed to DNA binding, purified wbFos and wbJun (2pkg/ml) were assayed for DNA-binding activity in the pres-ence of the reticulocyte lysate (Fig. 2B). In this case, thewbFos-wbJun complex exhibited a high degree of AP-1DNA-binding activity. In addition to reticulocyte lysate, avariety of eukaryotic cell extracts, such as rat liver extract,were able to stimulate the DNA-binding activity of bothwbJun and the wbFos-wbJun complex (Fig. 2B). The highestlevel of activity were associated with the nuclear fraction ofcell extracts (Fig. 2B). In contrast to the effect on DNAbinding, the ability of wbFos and wbJun to form het-erodimeric complexes was not influenced by the presence ofreticulocyte lysate or cell extracts (data not shown). Thus,cell extracts contain a factor(s) that increases the DNA-binding activity of the Fos-Jun complex without promotingor stabilizing heterodimer formation.The biochemical properties of the cellular factor were

characterized further using dialyzed rat liver nuclear extractsas a source of activity. The activity was sensitive to prote-olysis and heat treatment suggesting that it is proteinaceous(data not shown). Additionally, the activity was abolished bytreatment of liver nuclear extracts with alkylating agents,which block sulfhydryl groups, and was restored by reversalofalkylation (Fig. 3A). This suggests that the cellular factor(s)contains free sulfhydryl groups that are essential for promot-ing the DNA-binding activity of wbFos and wbJun. By usinghigher concentrations of wbFos and wbJun, the requirementfor the cellular factor was overcome by the inclusion ofelevated levels of reducing agents such as DTT (5 mM) in thepresence of stabilizing agents such as bovine serum albumin(BSA; 1 mg/ml), or Nonidet P-40 (NP40; 0.1%) (Fig. 3B).None of these agents was effective on its own (Fig. 3B).However, stimulation of Jun homodimer DNA-binding ac-tivity was observed only with the nuclear factor and was notseen with DTT plus BSA. Interestingly, preparations ofAP-1DNA-binding activity, purified from mammalian cells byoligonucleotide affinity chromatography, exhibited the sameenhancement of gel-shift activity in the presence of eithernuclear extracts or DTT plus stabilizing agents (Fig. 3C).Thus, the requirement for a reducing environment and sta-bilizing agents in the DNA-binding assay is a general propertyof the mixture of AP-1-binding proteins and is not a specific

feature of polypeptides expressed in E. coli. A critical levelof DTT was required for these effects (5 mM) as all DNA-binding assays were performed in the presence of 1 mM DTT.It is not likely that the reducing agents are required todissociate aberrant protein aggregates since the wbFos andwbJun proteins migrated primarily as monomers on nonre-ducing SDS gels (data not shown). Additionally, wbFos andwbJun that were renatured in the presence of high levels ofreducing agents (20 mM DTT) exhibited a similar enhance-ment ofDNA binding in the presence of cell extracts or DTTplus NP40. The effect does not appear to be a stable modi-fication since proteins that were incubated with nuclearextracts and then repurified still required the addition ofeither nuclear extract or DTT plus NP40 for efficient DNAbinding (data not shown). Although the physiological signif-icance of these observations is not clear at present, it ispossible that the reducing and stabilizing agents mimic theactivity of a factor present in cell nuclei that modulates theDNA-binding activity of Fos-Jun complexes in vivo. Inter-esting, both Fos and Jun contain a cysteine residue in theDNA-binding region that is highly conserved in c-fos andc-jun-related genes (17). The nature of the nuclear activity isunclear at present; however, several experimental ap-proaches have suggested that it is not a kinase or a phospha-tase.The relative affinities of the wbFos and wbJun protein

complexes for the hMTIIA AP-1 site were determined usingoptimal DNA-binding conditions-i.e., in the presence of ratliver extract (Fig. 3D). Both wbJun and wbFos-wbJun com-plexes exhibited a high degree ofDNA-binding activity whenassayed under these conditions. However, the wbFos-wbJuncomplex had an 8-fold higher apparent affinity. wbFos alonedid not exhibit DNA binding even at the highest proteinconcentration tested (0.29 gM). Thus, similar to the full-length proteins translated in vitro (12), wbFos and wbJuninteract cooperatively with the AP-1-binding site.wbJun and wbFos-wbJun Protein Complexes Interact with

the Same DNA Region and Both wbFos and wbJun ContactDNA Directly. The interaction of wbJun and wbFos-wbJunprotein complexes with DNA was examined by DNase Iprotection assays using a region ofthe hMTIIA enhancer (Fig.4A). Although wbFos alone did not interact with this DNAfragment, both wbJun and wbFos-wbJun complexes pro-

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FIG. 4. (A) DNase footprintingof wbFos and wbJun. A 100-base-pair fragment of the hMTIIA pro-moter (nucleotides -154 to -54)was digested with BamHI or Hind-III and end-labeled. Protein-DNAcomplexes were digested withDNase (0.04 mg/ml) and the result-ing DNA fragments were resolvedon 10%o polyacrylamide/6 M ureagels. (B) UV-crosslinking analysis.An oligonucleotide containing thehMTIIA AP-1 site was labeled onboth strands by filling-in with re-verse transcriptase (Top). Alterna-tively, individual strands (Middleand Bottom) were labeled with T4polynucleotide kinase and annealedwith the nonlabeled complementarystrand. wbFos and wbJun were in-cubated at 37°C with DTT (10 mM)and NP40 (0.1%) prior to addition ofthe 32P-labeled oligonucleotides.Protein-DNA complexes were ex-posed to UV irradiation for 10 minon ice. Proteins were immunopre-cipitated with either anti-Fos or an-ti-Jun antibodies and the productswere resolved on a 15% polyacryl-amide/SDS gel. Denatured com-plexes (lanes D) were boiled in 1%SDS and 10%6 (vol/vol) 2-mercapto-ethanol prior to immunoprecipita-tion. N, native extracts; NA, noaddition.

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tected a 23-base-pair region that contained the AP-1-bindingsite on both coding and noncoding strands. Thus, bothhomomeric and heteromeric protein complexes interactedwith the same region of DNA. To determine whether bothproteins contact DNA directly, UV-crosslinking analysis wasperformed (Fig. 4B). UV treatment resulted in transfer of32P-labeled oligonucleotides to both wbFos and wbJun (Fig.4B). This demonstrates that both proteins contact DNA. Inaddition, both wbFos and wbJun were efficiently crosslinkedto the hMTIIA AP-1 site when individual strands of theoligonucleotide were labeled (Fig. 4B), suggesting that nei-ther wbFos nor wbJun exhibits strand specificity for inter-action with DNA. Although wbJun was labeled with the32P-labeled hMTIIA AP-1 site in the absence of wbFos,wbFos did not incorporate substantial amounts of label in theabsence ofwbJun. Thus, Fos contacts DNA directly but onlyin the form of a heterodimeric protein complex with Jun. Incombination, these data suggest that the observed cooper-ativity of the interaction of the Fos-Jun complex with thehMTIIA AP-1 site reflects an enhanced stability of the het-erodimeric protein complex relative to the Jun homomericcomplex, rather than an alteration in the specificity of theDNA-protein interaction.

SIGNIFICANCEFos and Jun are among the many proteins that interact withthe AP-1-binding site (1). The leucine-zipper and basic-regionmotifs are highly conserved among the c-fos and c-jun genefamilies, which suggests that multiple protein complexesformed between members ofthese gene families interact withDNA in the same way as the Fos-Jun complex (17). Theavailability of purified Fos and Jun proteins comprised ofthese highly conserved domains will facilitate a detailedcharacterization of this model. Indeed, by using purifiedproteins, we have established that homodimeric Jun andheterodimeric Fos-Jun complexes interact with the sameregion ofDNA and that both proteins contact DNA directly.Additionally, we have identified an activity present in nuclearextracts that enhances the DNA-binding properties of Junhomodimeric and Fos-Jun heterodimeric complexes. Al-though the nature and significance of this activity is unclearat present, it is conceivable that it modulates the affinity andspecificity of Fos-Jun complexes for the AP-1-binding site invivo.The AP-1-binding site, originally identified as a phorbol

12-myristate 13-acetate-response element (8, 9) is present innumerous genes, many ofwhich are not induced by treatmentwith this agent (11, 21). Moreover, AP-1 DNA-binding ac-tivity is increased by treatment of cells with a wide range ofstimulatory agents in addition to phorbol 12-myristate 13-acetate, including serum and nerve growth factor (21), cal-cium ionophore (11), and excitation of neurons in vivo (27).There is tremendous variability in the nucleotide sequence ofAP-1-binding sites and Fos and Jun have also been shown to

interact with the cAMP-responsive element (12). The mech-anism whereby specific AP-1 protein complexes recognizetarget gene regulatory elements in the nucleus is likely to behighly complex. Unraveling this complexity will be greatlyfacilitated by the availability of purified components such asFos and Jun.

We thank E. Gagne and R. Roeder for helpful discussions and forthe gift of oligonucleotide affinity-purified AP-1 from HeLa cells. Wethank J. I. Morgan, H. Weissbach, and P. Kim for useful commentson the manuscript and for discussions.

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