Vol. 11, No. 7

MOLECULAR AND CELLULAR BIOLOGY, JUlY 1991, p. 3624-3632 0270-7306/91/073624-09$02.00/0 Copyright C 1991, American Society for Microbiology

Transcriptional Regulation by Fos and Jun In Vitro: Interaction among Multiple Activator and Regulatory Domains CORY ABATE, DANIEL LUK, AND TOM CURRAN* Department of Molecular Oncology & Virology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110

Received 20 February 1991/Accepted 10 April 1991

The proteins encoded by the proto-oncogenes c-fos and c-jun (Fos and Jun, respectively) form a heterodimeric complex that regulates transcription by interacting with the DNA-regulatory element known as the activator protein 1 (AP-1) binding site. Fos and Jun are members of a family of related transcription factors that dimerize via a leucine zipper structure and interact with DNA through a bipartite domain formed between regions of each protein that are rich in basic amino acids. Here we have defined other domains in the Fos-Jun heterodimer that contribute to transcriptional function in vitro. Although DNA-binding specificity is mediated by the leucine zipper and basic regions, Jun also contains a proline- and glutamine-rich region that functions as an ancillary DNA-binding domain but does not contribute directly to transcriptional activation. Transcriptional stimulation in vitro was associated with two regions in Fos and a single N-terminal activation domain in Jun. These activator regions were capable of operating independently; however, they appear to function cooperatively in the heterodimeric complex. The activity of these domains was modulated by inhibitory regions in Fos and Jun that repressed transcription in vitro. In the context of the heterodimer, the Jun activation domain was the major contributor to transcriptional stimulation and the inhibitory regions in Fos were the major contributors to transcriptional repression in vitro. Potentially, the inhibitory domains could serve a regulatory function in vivo. Thus, transcriptional regulation by the Fos-Jun heterodimer results from a complex integration of multiple activator and regulatory domains. A variety of processes have evolved in eukaryotic cells for coupling environmental cues to the selective regulation of gene expression. Cell surface stimuli evoke a series of intracellular signals that ultimately affect the levels or activities of proteins that bind to transcriptional control elements (for reviews, see references 33, 39, and 64). Many of these transcription factors interact with DNA in the form of dimeric complexes that employ a limited set of defined structural motifs. These conserved domains mediate interactions that generate an array of heterodimeric protein complexes formed from the products of related gene family members. In many cases, protein dimers may have distinct transcriptional properties although they exhibit similar DNA-binding specificities. This phenomenon is exemplified by the class of transcription factors that contain the leucine zipper motif. The fos and jun gene families encode leucine zipper-containing proteins that function in gene regulation by interacting cooperatively as heterodimeric complexes with the DNA-regulatory element known as the activator protein 1 (AP-1) binding site (reviewed in reference 18). Conservation among thefos and jun gene families is primarily restricted to the leucine zipper and an adjacent region that is rich in basic amino acids (14, 26, 38, 49, 66). The leucine zipper consists of a heptad repeat of leucines that align on one face of an oa-helix, forming a dimerization interface (30). Dimerization brings into appropriate juxtaposition adjacent basic regions present in both Fos and Jun that form a bimolecular DNA-binding domain (22, 29, 61). In contrast to Fos, Jun is capable of forming homodimeric complexes that can interact with DNA. However, the Fos-Jun heterodimer is more stable than the Jun homodimer, and this is reflected in the ability of Fos and Jun to interact cooperatively with *

Corresponding author.

the AP-1 binding site (42a, 48). The many heterodimeric complexes that can be formed among the Fos and Jun family members have similar DNA-binding properties (15, 26, 42a, 49, 66). However, there are some indications that these protein dimers have different effects on target gene transcription (13, 54). In the majority of cell types, the levels of expression of fos,jun, and related genes are relatively low. However, their expression can be induced transiently by a plethora of extracellular signals, including those associated with mitogenesis, differentiation, and neuronal excitation (reviewed in references 17 and 41). Although the AP-1 site was originally identified as a phorbol ester-responsive element (5, 31, 32), it is present in many genes that are responsive to other signalling events (18). In addition, Fos and Jun can interact with a related DNA sequence termed the cyclic AMP (cAMP)-responsive element (CRE) (21, 37, 48, 51). Moreover, Jun and Fos can form heterodimeric complexes with certain members of the CRE/ATF families that interact preferentially with CRE sites (8, 24, 37). The CRE site is also a target for second messengers other than cAMP, including calcium (55). Thus, AP-1 and CRE sites can be viewed as general responsive elements that act at the distal end of complex signal transduction pathways. Many inducible and resident leucine zipper dimers are capable of interacting with these DNA sequences. Therefore, mechanisms must exist that promote the selective interaction of protein dimers with specific target genes in each cell type. Some potential regulatory mechanisms have already been uncovered. For example, the availability of distinct subsets of leucine zipper dimers varies at different times, in different cell types, and in response to particular signals (14, 55, 56). In addition, a novel reduction-oxidation (redox) mechanism has been identified, involving a conserved cysteine residue in the basic region of Fos and Jun. This redox mechanism 3624

VOL . 1 l,

1991

TRANSCRIPTIONAL REGULATION BY Fos AND Jun IN VITRO

regulates DNA-binding activity in vitro (4) and potentially could provide a regulatory role in vivo, since a nuclear redox factor has been identified that promotes Fos-Jun DNAbinding activity (la, 3). Recent studies indicate that the transcriptional activity of Fos and Jun is influenced by competitive and cooperative interactions with other transcription factors that bind to overlapping or adjacent DNA sequences (19, 27, 36, 45, 52, 53, 62, 65). There are also some indications that direct interactions occur between leucine zipper dimers and steroid receptors (19, 27, 65). In addition to these regulatory features, it is likely that the specificity of the Fos and Jun family members involves regions of the protein that are less well conserved than the leucine zipper and basic regions. We have previously established that transcriptional stimulation by the Fos-Jun heterodimer in vitro involves activation domains present in both proteins (2). Here we have examined the transcriptional activities of a series of truncated Fos and Jun polypeptides in the context of homo- or heterodimeric complexes with a sensitive and quantitative in vitro assay. This analysis has delineated the domains in Fos and Jun that mediate transcriptional regulation in vitro. The results suggest that the transcriptional activity of the FosJun heterodimer involves the integration of several positive and negative domains. MATERIALS AND METHODS Expression and purification of Fos and Jun deletions in Escherichia coli. Full-length Fos and Jun and truncated polypeptides containing Fos amino acids 116 to 211 and Jun amino acids 224 to 334 were synthesized as histidine fusion proteins in E. coli as described before (la, 3). A series of additional truncated genes were constructed from full-length fos and jun by polymerase chain reaction with oligonucleotides that corresponded to the 5' and 3' ends of the coding sequence to be amplified. The 5' oligonucleotides contained an SphI site (including the initiator methionine) and six histidine codons, and the 3' oligonucleotides contained a HindIII site immediately after the stop codon. Polymerase chain reactions were performed according to the specifications of the manufacturer (Perkin-Elmer-Cetus). The products were gel purified, digested with SphI and HindIII, and cloned into the complementary sites of the vector pdS56 (la). The sequences were confirmed by dideoxynucleotide sequencing. The hexahistidine fusion proteins were synthesized in E. coli and purified from cell lysates by nickel affinity chromatography in the presence of 6 M guanidineHCI as described previously (la, 3). The purified proteins were renatured by extensive dialysis against 25 mM sodium phosphate, pH 7.5, containing 1 mM dithiothreitol and 5%

glycerol. Protein association and DNA-binding assays. Proteins were incubated for 15 min at 37°C in binding buffer (10 mM Tris-HCl [pH 7.9],5 mM MgCl2, 50 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol, 5% glycerol, 5% sucrose) to allow dimer formation. DNase I footprinting of protein dimers was performed with a 100-bp fragment of the human metallothioneinIIA (HMTIIA) promoter that contained the AP-1 site (la, 3). Gel retardation assays were performed with a 25-bp oligonucleotide that contained the HMTIIA AP-1 site as described before (3, 48). For methylation interference, the 100-bp HMTIIA fragment was partially methylated by using dimethylsulfate, as per the manufacturer's (New England Nuclear) instructions. Modified DNA (100,000 cpm) was incubated with protein dimers (0.2 ,uM) as for gel retardation

3625

assays. The free DNA was resolved from protein-DNA complexes by polyacrylamide gel electrophoresis. The DNA in the entire gel was transferred electrophoretically onto DEAE (NA45) paper (Schleicher & Schuell) overnight at 100 mA with 0.5x TBE. The DNA bands were identified by autoradiography and eluted from the DEAE paper in 1 M NaCl-20 mM Tris-Cl (pH 8)-0.1 mM EDTA for 1 h at 55°C. The DNA was concentrated by ethanol precipitation and cleaved with piperidine, and the DNA fragments were resolved on a 10% acrylamide-6 M urea sequencing gel. In vitro transcription assays. Nuclear extracts were prepared from HeLa cells by the procedure of Dignam et al. (20) as modified by Briggs et al. (11). In vitro transcription reactions were performed by a modification of the procedure described previously (2). Briefly, endogenous AP-1 binding activity was depleted from HeLa nuclear extracts by incubation with an oligonucleotide containing the AP-1 site (0.025 ,uM final concentration in a 25-,ul reaction mix) for 10 min at 30°C. Transcription reactions were performed with a linearized DNA template (10 ,ug/ml) containing six tandem AP-1 sites or six mutated AP-1 sites (2). Protein dimers (0.2 ,uM) were incubated with template DNA for 10 min at 30°C in buffer containing 40 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.6), 5 mM MgCl2, 70 mM potassium chloride, 1 mM dithiothreitol, 12% glycerol, 1% polyvinyl alcohol, and 200 U of RNasin (Promega) (final concentrations in 25-,ul reaction mix). Subsequently, the oligo-depleted HeLa nuclear extract (50 ,ug) was added, and transcription complexes were formed by incubation for 5 min at 30°C. RNA synthesis was initiated by the addition of a nucleotide mix containing 0.5 mM each GTP, ATP, and CTP and 0.01 mM [32P]UTP (1 p.Ci), and reactions proceeded for 40 min at 30°C. The radioactive RNA products were extracted with phenol-chloroform, precipitated with ethanol, and resolved on 6% acrylamide-7 M urea gels. The radioactive bands were visualized by autoradiography and quantitated by densitometric scanning. The transcription reactions were performed at least five times, and data presented are representative of these experiments.

RESULTS Expression and purification of truncated Fos and Jun polypeptides. Previously, we demonstrated that purified Fos and Jun stimulated AP-1-dependent transcription in HeLa cell extracts and that transcriptional activity was influenced by both members of the heterodimeric complex (2). To define the regions responsible for transcriptional regulation, a series of truncated Fos and Jun polypeptides were synthesized in and purified from E. coli (Fig. 1A and B). Each of the Fos and Jun proteins contained the leucine zipper and basic regions (Fos amino acids 139 to 211 and Jun amino acids 240 to 334) and lacked portions of the N or C terminus (Fig. 1A and B). These polypeptides were expressed as histidine fusion proteins and were purified from E. coli cell lysates by nickel affinity chromatography (la, 3). The proteins obtained after one pass over the nickel column were approximately 90 to 95% pure (Fig. 1C). Jun contains an ancillary DNA-binding domain. DNase I footprinting analysis revealed that the truncated Fos and Jun polypeptides protected the same region of the HMTIIA promoter as did the full-length proteins (Fig. 2A and B). When assayed at equimolar concentrations, both Jun homodimeric and Fos-Jun heterodimeric complexes interacted with the AP-1 site with the same specificity (Fig. 2A and B). However, the footprints obtained with Jun homodimers

3626

MOL. CELL. BIOL.

ABATE ET AL.

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lacking amino acids 186 to 240 (Junl86-240) were weaker than those obtained with full-length Jun. In addition, methylation interference analysis indicated that full-length and truncated Fos and Jun contacted the same guanine nucleotides in the AP-1 site (data not shown). In gel retardation assays, heterodimers containing the various Fos and Jun polypeptides exhibited similar apparent affinities for the AP-1 site (Fig. 3A and B). In contrast, the apparent affinities of Jun homodimers for the AP-1 site varied significantly (Fig. 3B). For example, Jun224-334 and Jun240-334 exhibited very low levels of gel shift activity compared with equimolar

FIG. 1. Expression of truncated Fos and Jun polypeptides and transcriptional stimulation in vitro. (A and B) Diagram of Fos and Jun polypeptides and transcriptional activity. Truncated Fos (F) and Jun (J) proteins contained the amino acids indicated. All of the Fos and Jun constructs contained the leucine zipper region (LLL) and basic amino acids (+ + +). The N terminus of these in Fos contained a region rich in prolines (P) and a region rich in acidic residues (--- ); the C terminus contained an acidic and proline-rich region (P). N-terminal of the leucine zipper in Jun is a proline- and glycine-rich region (P-G) and a proline- and glutamine-rich region (P-Q). The transcriptional activities of Fos and Jun polypeptides were determined as described in the legends to Fig. 5 and 6. The fold transcriptional stimulation, determined by densitometric scanning of autoradiograms, was normalized to basal transcriptional activity. The results represent the mean of five independent experiments, with standard deviations indicated. (C) The Fos and Jun polypeptides were expressed in E. coli as hexahistidine fusion proteins and purified by nickel affinity chromatography. The purified proteins (2 ,Lg) were resolved on a 13.5% polyacrylamide-sodium dodecyl sulfate gel and visualized by staining with Coomassie brilliant blue. Markers correspond to molecular mass standards (Bio-Rad) in kilodaltons (phosphorylase B, 92.5 kDa; bovine serum albumin, 68 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 20 kDa; lysozyme, 14 kDa).

amounts of full-length Jun, Jun9O-334, Junl86-334, and Junl98-334 (Fig. 3B). This effect was observed over a range of protein concentrations (data not shown), indicating that the apparent relative affinities of Jun224-334 and Jun240-334 homodimers were lower than those of other Jun proteins. These data demonstrate that a region of Jun contained within

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TRANSCRIPTIONAL REGULATION BY Fos AND Jun IN VITRO

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amino acids 190 to 224 promotes a high-affinity interaction of homodimers with DNA. Interestingly, this domain contains a high content of proline and glutamine residues, which together comprise 50%o of the total amino acid content of the region.

Although these Jun polypeptides exhibited marked differin DNA-binding affinity, the full-length and truncated proteins contacted the same bases of the AP-1 site in methylation interference assays (data not shown). Thus, the proline- and glutamine-rich region functions as an ancillary DNA-binding domain to stabilize the interaction of Jun homodimers with DNA indirectly. This result is in contrast with the findings of a previous study, in which the affinities of truncated Jun proteins for DNA were reported to be equivalent, as measured by DNase I footprinting assays (10). However, DNase I footprinting analysis does not readily distinguish the affinity differences among homodimers formed with the truncated Jun polypeptides (Fig. 2B). The effect of the proline-glutamine domain on the DNA-binding activity of Jun homodimers was only evident in gel shift assays (compare Fig. 2B and 3B). While it is possible that this effect represents a peculiarity of the gel shift assay, we believe that it reflects the greater sensitivity of the gel shift assay to differences in stabilities of protein-DNA complexes. Both Jun and Fos contain activation domains. HeLa cell extracts provide an excellent source of mammalian basic transcription factors for in vitro transcription assays (20). However, these extracts also contain high levels of endogeences

nous AP-1 activity (Fig. 4, lane 1). Indeed, AP-1 was first purified from HeLa nuclear extracts (32). Previously, endogenous AP-1 activity was depleted from HeLa extracts by oligonucleotide affinity chromatography (2, 10). However, this procedure also resulted in a general reduction in transcriptional activity, which limited the levels of stimulation obtained following the addition of Fos and Jun (1). To circumvent this problem, we depleted HeLa nuclear extracts of endogenous AP-1 by incubation with an oligonucleotide containing the AP-1 binding site (Fig. 4, lane 2). This procedure efficiently and specifically reduced endogenous AP-1 transcriptional activity (Fig. 4, compare lanes 1 and 2) but did not influence the transcription of a template containing mutated AP-1 sites (Fig. 4, compare lanes 6 and 7). The addition of Jun or Fos-Jun complexes resulted in transcriptional activation of a template containing six AP-1 sites (Fig. 4, lanes 4 and 5) but did not affect the transcription of a template containing six mutated AP-1 sites (Fig. 4, lanes 9 and 10). In the absence of Jun, no transcriptional stimulation was obtained with any of the Fos polypeptides (Fig. 4, lane 3; Fig. 5A). This was expected, since Fos does not bind to DNA on its own (48). Furthermore, this result indicates that there were no Jun-related proteins available to cooperate with Fos in the depleted HeLa extracts. A subset of heterodimers formed between the Fos polypeptides and Jun224334 exhibited transcriptional activity (Fig. 1A and 5A). Since Jun224-334 does not stimulate transcription in the absence of

3628

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tides containing only one of these domains were less active, e.g., Fosl39-380 (8-fold) and Fosll6-211 (5-fold), and a truncated protein that did not contain either activator domain was essentially inactive (2-fold) (Fig. 1A and 5A). In contrast to the situation with Fos, Jun was active by itself in vitro. Transcriptional stimulation by the various Jun homodimers required an N-terminal domain corresponding to amino acids 90 to 186 (Fig. 6). Whereas Jun and Jun9O-334 were active in transcriptional stimulation (8-fold and 22-fold, respectively), Jun polypeptides truncated beyond amino acid 186 did not produce significant levels of transcriptional

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FIG. 5. Transcriptional stimulation by Fos polypeptides. (A and B) The Fos polypeptides were assayed alone or in the presence of Jun224-334, Jun9O-334, or full-length Jun (J) at saturating protein levels (0.2 ,uM). The DNA template (10 ,ug/ml) contained six AP-1 sites, and HeLa nuclear extracts (50 ,ug) were preincubated with an AP-1 oligonucleotide. The radiolabeled RNA products were resolved on 6% acrylamide-7 M urea gels and visualized by autoradiography. The arrow indicates the specific RNA products. NA, no addition.

TRANSCRIPTIONAL REGULATION BY Fos AND Jun IN VITRO

VOL. 11, 1991 +

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activity (Fig. 1B and 6), although they did interact with DNA (Fig. 2B and 3B). These data demonstrate that the prolineand glutamine-rich region of Jun does not function in transcriptional stimulation, although it does act as an ancillary DNA-binding domain. This is in contrast with a prior study, which indicated that the activation domain of Jun in vitro corresponded to amino acids 215 to 255 (10). However, the proline-glutamine-rich domain (amino acids 215 to 255) does not stimulate transcription in transfection assays (6, 7, 26, 28), whereas the N-terminal region defined here as an activation domain functions in vivo as well as in vitro. The transcriptional activity of Jun9O-334 (22-fold) was significantly greater than that of full-length Jun (8-fold). Thus, the N terminus of Jun inhibits the transcriptional activity of the homodimer in vitro (10); however, it also contributes to DNA binding of Jun homodimers. It is noteworthy that in all cases, transcriptional stimulation by the Jun activation domain was significantly greater than that obtained with Fos activator regions (compare Fig. 1A and B). The presence of Fos had quite a significant effect on the ability of Jun polypeptides to stimulate transcription. In part, this was a consequence of the increased DNA-binding affinity of Fos-Jun heterodimers. A truncated Fos protein (Fosl39-211), containing the minimal DNA-binding domain but lacking an activation domain (Fig. 1A and SA), enhanced the ability of full-length Jun and Jun9O-334 to stimulate transcription from 8- to 18-fold and from 22- to 30-fold, respectively (Fig. 1B and 6). However, even in the presence of Fosl39-211, the Jun proteins truncated beyond amino acid 186 were inactive in transcription assays (Fig. 1B and 6). These heterodimers exhibited high levels of DNA-binding activity (Fig. 2B and 3B), underscoring the point that the proline-glutamine-rich region of Jun is not an activator region. The activation domain of Jun lies between amino acids 90 and 186 and has a high content of prolines (9o), a net negative charge (-5), and an unusually high glycine content (16%). Both acidic and proline-rich regions have been associated with transcriptional activation domains (39), while glycine-rich regions have not yet been reported. Thus, this region has some characteristics of previously described activation domains, although the significance of the prevalent amino acids is not yet clear. In some of the transcription assays, a degree of variability in the level of stimulation was observed (see, e.g., Fig. 6, lane 5). However, the enhancement was calculated from five independent assays and was

3629

relatively consistent, as indicated by the standard deviations (Fig. 1B). Fos contains domains that repress transcription in vitro. The transcriptional activity of full-length Fos was lower than that of several of the truncated polypeptides (Fig. 1A and 5A). Thus, Fos contains inhibitory domains as well as activating regions. This inhibitory effect was associated predominantly with a proline-rich region (amino acids 58 to 116) adjacent to the acidic activator. A C-terminal serineand threonine-rich region (amino acids 270 to 380) also reduced transcription, but to a lesser degree. For example, a truncated Fos polypeptide containing both of these inhibitory regions (e.g., Fos58-380) exhibited very low levels of transcriptional stimulation in association with Jun (twofold), although this protein also contained both activator domains (Fig. 1A and 5A). Similarly, polypeptides that contained either inhibitory region (e.g., Fosl-270 and Fosl-321) were less active in transcriptional stimulation than other Fos polypeptides (Fig. 1A and 5A), although in each case these proteins interacted equivalently with DNA (Fig. 2A and 3A). Transcriptional stimulation by Fos and Jun occurs by integration of positive and negative domains. Heterodimeric complexes in which both the Fos and Jun polypeptides contained activator domains produced levels of transcriptional stimulation higher than the sum of the individual activator regions. For example, the transcriptional activity of Jun or Jun9O-334 in the presence of a Fos polypeptide that contained an activator domain (Fosll6-211) was greater (40-fold and 42-fold, respectively) than in the presence of Fosl39-211 (18-fold and 30-fold, respectively) (Fig. 1B and 6). Conversely, the transcriptional activity of heterodimers containing the various Fos polypeptides was greater in the presence of Jun or Jun9O-334 (Fig. 1A and 5B) than in the presence of Jun224-334 or Junl86-334 (Fig. 1A and 5A). Thus, activation domains in Fos and Jun may function synergistically to stimulate transcription. The cooperativity among activation domains was distinct from the cooperative interaction of the heterodimeric complex with DNA. Interestingly, both Jun and Jun9O-334 were less active (13-fold and 19-fold, respectively) in transcriptional stimulation in combination with Fos polypeptides that contained inhibitory domains (e.g., Fos) than in combination with a Fos polypeptide that did not contain activator domains (e.g., Fosl39-211) (Fig. 1A, 1B, and 6). This finding demonstrates that the inhibitory regions of Fos function in trans to influence the overall transcriptional activity of the heterodimer. DISCUSSION Leucine zipper interactions among members of thefos and jun gene families generate heterodimeric complexes that interact cooperatively with the AP-1 binding site (15, 42a, 48, 66). Fos-Jun heterodimers also function cooperatively in vivo to stimulate transcription when measured indirectly in transient-transfection assays (13, 25, 35, 50, 54, 56). Here, using a sensitive and quantitative in vitro assay, we have defined multiple domains in Fos and Jun that function additively and perhaps cooperatively in transcriptional regulation (Fig. 7). Distinct domains in both partners of the heterodimeric complex promote cooperative interaction with DNA and stimulation of transcription. While the activation domains in Fos and Jun functioned independently, they appeared to interact cooperatively in the heterodimer to stimulate transcription. The activity of these regions was modulated by other domains that exerted a negative influence on transcription in vitro. These data demonstrate that

3630

ABATE ET AL. Fos

MOL. CELL. BIOL. LIL l--P-I

_-

I

...+ LLLLL

Jun

[

Activation Domains

*

Regulatory DomainS

E

Ancillary Binding Domain

FIG. 7. Summary of the multiple functional domains in the Fos-Jun heterodimer that contribute to DNA binding and transcriptional stimulation. Dimerization via the leucine zipper structure (LLL) in Fos and Jun promotes DNA binding of adjacent basic regions (+ + +). Jun also contains an ancillary DNA-binding domain that is rich in prolines and glutamines (P-Q). Fos contains two activation domains; one of these is acidic ( - -), and one is rich in proline and acidic residues (- -P- -). Jun contains a single activation domain that is rich in prolines and glycines (P-G). In addition to these, both Fos and Jun contain regions that inhibit transcription in vitro. One of these putative regulatory regions in Fos is rich in prolines (P) and the other is rich in serines and threonines (S).

the transcriptional function of the Fos-Jun heterodimer in vitro results from a complex integration of multiple functional domains (Fig. 7). Stimulation of transcription by Fos was associated with two regions, one of which has characteristics of an acidic activator domain (46), whereas the other has a high content of proline and acidic residues. The proline-rich and acidic region has also been shown to be important for transcriptional activation in cotransfection assays (24, 42). A single activation domain was identified in the N-terminal region of Jun that is rich in proline, acidic, and glycine residues. This region functions as a potent activator in vivo as well as in vitro (6, 7, 26, 28). In fact, the single Jun activator domain was more effective than both activation domains of Fos. Thus, Jun provides the major contribution to transcriptional activation of the heterodimer in vitro. This is similar to the heterotrimeric CAAT-binding complex, in which transcriptional activation was predominantly associated with one component of the complex (44). However, the combination of activation domains in Fos and Jun was slightly greater than additive, suggesting that they function cooperatively in the stimulation of transcription. Transcriptional regulatory proteins often contain several heterologous activation domains that function synergistically (see, e.g., references 16 and 60). Recent studies have indicated that heterologous activation domains interact with basal transcription factors via diverse mechanisms. For example, these may utilize "bridging" factors or "adaptors" to interact with the core transcription complex (9, 28, 47, 57, 59). Thus, the heterologous activation domains in Fos and Jun might promote interactions with several distinct basal transcription factors. The regions of Fos and Jun associated with transcriptional stimulation are only partially conserved among the other members of these gene families. For example, although the acidic region in Fos is conserved across species and among all members of the fos gene family, the proline-rich and acidic domain is unique to Fos (14, 38, 40, 43, 66). Similarly, the region of Jun containing the activation function is only partially conserved among the Jun family members (26, 49). Thus, other members of these gene families may have distinct transcriptional properties. These may confer a degree of specificity on the heterodimers formed among leucine zipper-containing proteins. The many possible combinations of heterologous activation functions generated by interac-

tions among these proteins expand the potential for transcriptional diversity and target gene selectivity. A surprising finding from this study was the identification of an ancillary DNA-binding domain in Jun. This region contains a high concentration of proline and glutamine residues, features that are typically associated with activation domains (39). Previously, this region was reported to function directly in transcriptional stimulation in vitro (10). However, in a subsequent report from the same laboratory, the proline-glutamine-rich region did not activate transcription on its own in cotransfection assays (7). In contrast, the N-terminal domain containing the proline-glycine region defined in this report as an in vitro activator domain also functions in vivo (6, 7, 26, 28). The contribution of the proline-glutamine-rich domain to binding activity was determined by comparing the DNA-binding activities of several truncated Jun polypeptides by several independent criteria. It is important to distinguish domains that promote DNAbinding activity from activation domains, as these are partially defined by exclusion of the DNA-binding regions. The reasons for the discrepancy between our results and those of Bohmann and Tjian (10) are not clear, although it is possible that their HeLa extracts contained low levels of Fos-like or CREB-like proteins that contributed to AP-1 activity. Here we have obtained very high levels of transcriptional stimulation in vitro (up to 40-fold) by using a procedure (oligonucleotide competition) that does not reduce overall levels of transcription. This allowed a relatively clear delineation of the activation regions in Fos and Jun without interference by endogenous AP-1 activity. Fos has been implicated in transcriptional repression as well as activation (23, 34, 63). In particular, Fos functions as an autorepressor by an indirect effect of its C terminus on the serum-responsive element (23, 63). Fos may also repress transcription of heterologous promoter elements containing serum-responsive element sequences (23). Additionally, a negative effect of Fos expression has also been observed on regulatory elements containing AP-1 binding sites (45, 53). Here we define domains in Fos that function in transcriptional repression in vitro. These regions are active in trans, as they reduce transcriptional stimulation mediated by the Jun activator region. Both inhibitory regions (the prolinerich and the C-terminal serine-rich region) can be extensively modified by several protein kinases, including a nuclear kinase that is present in HeLa extracts (1). Thus, it is possible that these inhibitory regions serve regulatory functions in vivo. Jun also contains an N-terminal domain that has a negative influence on transcription in vitro. It has been suggested that this domain interacts with a cell typespecific inhibitory factor (7). In the context of the Fos-Jun heterodimer, the inhibitory regions in Fos have a more potent effect on transcriptional repression than the N-terminal inhibitory region of Jun. This point underscores potential differences in the function of Jun homodimers and Fos-Jun heterodimers. Indeed, it has become increasingly apparent that transcriptional regulation involves multiple proteins that interact with complex DNA-regulatory elements. Thus, it is likely that the interactions among leucine zipper dimers and many other proteins underlie transcriptional specificity and

activity. ACKNOWLEDGMENTS We thank Philip Familletti and Dale Mueller of Hoffmann-La Roche Inc. for providing HeLa cells.

VOL. 11, 1991

TRANSCRIPTIONAL REGULATION BY Fos AND Jun IN VITRO

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61. 62.

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66.

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