Vol. 10, No. 10

MOLECULAR AND CELLULAR BIOLOGY, Oct. 1990, p. 5532-5535 0270-7306/90/105532-04$02.00/0 Copyright © 1990, American Society for Microbiology

Fos and Jun Cooperate in Transcriptional Regulation via Heterologous Activation Domains CORY ABATE,' DANIEL LUK,1 ELIZABETH GAGNE,2 ROBERT G. ROEDER,2 AND TOM CURRAN'* Department of Molecular Oncology and Virology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110-1199,1 and Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 100812 Received 22 May 1990/Accepted 9 July 1990 The products of c-fos and c-jun (Fos and Jun) function in gene regulation by interacting with the AP-1 binding site. Here we have examined the contribution of Fos and Jun toward transcriptional activity by using Fos and Jun polypeptides purified from Escherichia coli. Fos contained a transcriptional activation domain as well as a region which exerted a negative influence on transcriptional activity in vitro. Moreover, distinct activation domains in both Fos and Jun functioned cooperatively in transcriptional stimulation. Thus, regulation of gene expression by Fos and Jun results from an integration of several functional domains in a bimolecular complex.

The proto-oncogenesfos and jun are rapidly induced by a great variety of extracellular stimuli (9). Their protein products, Fos and Jun, function cooperatively as a heterodimeric protein complex to regulate the expression of genes containing AP-1 binding sites (5, 14, 17, 23, 26-29). Fos and Jun dimerize via the leucine zipper structure, which serves to juxtapose regions of each protein rich in basic amino acids that constitute the DNA-binding domain (2, 15, 17, 24, 32). While it is clear that Fos and Jun function synergistically in DNA binding (23, 27) and in transient transfection assays (5, 28, 29), their respective contributions to transcriptional activation are not known. Here we used purified proteins to demonstrate that Fos and Jun have distinct transcriptional activation domains. Furthermore, the interaction of these heterologous domains determines the overall transcriptional activity. Full-length and truncated Fos and Jun polypeptides were expressed and purified from Escherichia coli to study their contribution to transcriptional regulation in vitro (2; C. Abate, D. Luk, and T. Curran, Cell Growth Different., in press). The regions of Fos and Jun expressed as histidine fusion proteins are illustrated in Fig. 1A. All of the constructs contained the leucine zipper and basic regions that are required for dimerization and DNA binding (15, 17, 32). Highly purified protein preparations were obtained from E. coli cell lysates by affinity chromatography with a nickel chelate matrix (Fig. 1B) (2). DNase footprinting analysis revealed that full-length Fos and Jun and truncated proteins containing Fos amino acids 116 to 121 and Jun amino acids 224 to 234 (Fosll6-211 and Jun224-334, respectively) interacted with the same region of the human metallothioneinllA (HMTIIA) enhancer (Fig. 2). Thus, the truncated Fos and Jun polypeptides were sufficient for an appropriate interaction with DNA. When assayed alone, neither Fos nor Fosll6-211 exhibited DNA-binding activity, whereas Jun and Jun224-334 protected the same region of DNA as heterodimeric Fos-Jun complexes (Fig. 2). The apparent affinities of the full-length and truncated Jun proteins for DNA were lower than those of the heterodimeric complexes (2; Abate et al., in press), as has been observed for proteins translated in vitro in reticulocyte lysates (27). The contribution of the Fos and Jun polypeptides toward

transcriptional activation was determined in a cell-free system. HeLa nuclear extracts, prepared as described by Dignam et al. (12), were depleted of endogenous AP-1 binding activity by adsorption with an AP-1 oligonucleotide resin. Western immunoblot analysis with anti-Fos and anti-Jun antisera confirmed that the extracts were depleted of endogenous Fos and Jun, and gel retardation assays confirmed that AP-1 binding activity was depleted (not shown). The depleted HeLa extracts exhibited the same low basal level of transcriptional activity measured by using either a DNA template containing six AP-1 sites (Fig. 3A, Table 1) or one with six mutated AP-1 sites (Fig. 3B). Although the DNA-binding activities of the full-length and truncated Fos and Jun proteins were equivalent, they exhibited a differential ability to stimulate transcription in vitro (Fig. 3A). Jun224-334, when assayed alone, did not stimulate transcription, although this protein did bind to DNA (Fig. 2). This was also the case when up to 10-fold-higher concentrations of Jun224-334 were assayed for transcriptional activity. Thus, the inability of Jun224-334 to stimulate transcription was not due to the lower affinity of this protein for DNA. Jun224-334 lacks sequences rich in proline and glutamine that contribute to transcriptional activation (4). In contrast, full-length Jun exhibited a low but reproducible level of stimulation (Fig. 3A). Fosll6-211 did not stimulate transcription by itself; however, it produced a threefold increase in transcriptional activity in the presence of Jun224-334 (Fig. TABLE 1. Transcriptional activities of full-length and truncated Fos and Jun proteins Protein(s)

Fosll6-211 ........................................... Jun224-334 ........................................... Fosll6-211 + Jun224-334 .......................................... Fos ........................................... Jun ...........................................

Fos + Jun ........................................... Fos + Jun224-334 ........................................... Fosll6-211 + Jun ...........................................

1.2 0.9 3.0 1.2 2.1 2.5 1.7 6.5

In vitro transcription reactions, as discussed in the legend to Fig. 3, were performed six times with three different batches of extract. The fold stimulation represents an average of these experiments, relative to basal transcriptional activity, as determined by densitometric scanning. a

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3A, Table 1). As Jun224-334 was not transcriptionally active, this result demonstrates the presence of an activation domain in Fosll6-211. The stimulation of transcription obtained with Fosll6-211 in the presence of full-length Jun was greater (6.5-fold) than that observed in the presence of truncated Jun224-334 (Fig. 3A, Table 1). These results suggest that the transcriptional activation property of the Fos-Jun complex results from an integration of the activation domains present in each protein. Interestingly, in some cases full-length Fos alone produced a modest stimulation of transcription (Fig. 3A), although it did not bind DNA (Fig. 2). This activation may be due to the interaction of Fos with other proteins present in the extract that are not related immunologically to Jun. Indeed, the converse has been shown for Jun, which interacts with a cyclic AMP response element-binding protein (19). Although a heterodimeric complex consisting of fulllength Fos and full-length Jun stimulated transcription, surprisingly, the degree of stimulation (2.5-fold) was consistently lower than that obtained with Fosll6-211 in association with full-length Jun (Fig. 3A, Table 1). The reduced transcriptional activity of Fos relative to that of the truncated Fosll6-211 was even more apparent when fulllength Fos was assayed in combination with Jun224-334 (Fig. 3, Table 1). Thus, in addition to containing an activation domain, Fos contains a region(s) that exerts a negative influence on transcription in vitro. A similar finding has been reported recently for Jun (4).

These inhibitory domains in Fos and Jun are potential targets for regulatory factors that may modulate the transcriptional efficiency of the heterodimer. Both Fos and Jun have been shown to undergo extensive posttranslational

modification, including serine and threonine phosphorylation (4, 10, 11, 27), which may contribute to regulation of transcription. Interestingly, full-length Fos but not Fosll6211 can be phosphorylated by nuclear kinases in vitro (C. Abate and T. Curran, unpublished data). Although Fos is often referred to as a transcription factor, it has never been shown to function as such in a direct assay. In the present study we have determined that Fos contributes directly to the transcriptional activity of the Fos-Jun heterodimeric complex. Moreover, the transcriptional efficiency of the complex reflects a combinatorial interaction of the transcriptional activation domains of Fos and Jun as well as the regions that act negatively in vitro. This is in marked contrast to the suggestion that Fos and Jun act synergistically in transcriptional regulation solely as a consequence of their cooperative interaction with DNA (3). A transcriptional activation function for Fos was detected by analysis of LexA-Fos fusion proteins in Saccharomyces cerevisiae (18). Here we show that an activation domain is contained within Fosll6-211. This truncated Fos polypeptide contained a stretch of four glutamic acid residues that were contiguous with the DNA-binding domain (Fig. 1A). These acidic residues are highly conserved across species (1, 21) and in several Fos-related genes (6, 33). Similar structures, termed

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FIG. 3. In vitro transcription with purified Fos and Jun. HeLa nuclear extracts, depleted of AP-1 binding activity, were incubated with the purified Fos and Jun proteins (1 ,uM) in the presence of 0.5 mM each ATP, GTP, and TTP and 10 ,uM UTP supplemented with 5 ,uCi of [32P]UTP (Amersham), 40 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-NaOH (pH 7.6), 5 mM MgCl2, 70 mM KCI, 1 mM dithiothreitol, 12% glycerol, 200 U of RNasin (Promega), and 1.4% polyvinyl alcohol. The DNA template (10 ,ug/ml) contained (A) six AP-1 sites (TGACTAA) or (B) six mutated AP-1 sites (GTACTAG) linked to a truncated Fos promoter (-53 to +43) that was fused to a region of the chloramphenicol acetyltransferase (cat) gene as described before (13). The template DNA was linearized at the EcoRI site in the cat sequence (+288). Reactions proceeded for 30 min at 30°C. Radioactive RNA products were extracted, analyzed on an 8% acrylamide-7 M urea gel, and visualized by autoradiography. The autoradiograms were subjected to densitometric scanning to quantitate the results. NA, No addition. Arrows indicate specific RNA products.

NOTES

VOL. 10, 1990

Transcriptional regulatory proteins may have multiple distinct activator regions (7, 20, 31), and the combination of multiple functional domains may govern the specificity of transcriptional activity (30, 31). Here we show that distinct activation domains in two partners of a heterodimeric complex cooperate in transcriptional activation. Indeed, analysis of the function of truncated proteins in in vitro transcription assays showed that the combination of a Fos acidic activator domain and a Jun proline- and glutamine-rich domain resulted in a stimulation that was greater than additive (Fig. 3). This indicates that these activation domains function cooperatively. At present it is not known how the activator domains of Fos and Jun interact, either directly or indirectly, with the basic transcriptional machinery. One possibility is that the distinct activator regions of Fos and Jun permit multiple contacts with the basic transcriptional apparatus, resulting in a cooperative stimulation of transcription. These interactions may be modulated by the regulatory domains that act as negative regions in vitro (4). c-fos and c-jun are members of families of related inducible genes (8, 9). The high degree of conservation among these families is primarily restricted to their respective dimerization and DNA-binding domains (1). These proteins form multiple homodimeric and heterodimeric complexes that have similar but not identical specificities for AP-1 binding sites and cyclic AMP-responsive elements (8, 17, 22, 27, 29). The interaction of several distinct regulatory and activation domains of the Fos and Jun families may contribute to the specificity of target gene selection in the many and varied situations in which these proteins are expressed. Reconstitution of the synergistic activities of these protein complexes in vitro is a first step towards defining the transcriptional function of the fos and jun proto-oncogenes. We thank J. Kadonaga for helpful discussion and advice on in vitro transcription. This work was supported, in part, by Public Health Service grants CA42567 and A127307 from the National Cancer Institute to R.G.R., and by general support from the Pew Charitable Trusts to the Rockefeller University. E.G. was supported by National Institutes of Health Fellowship F32 CA08648, with additional support from the Leslie Arps Cancer Research Fund. LITERATURE CITED 1. Abate, C., and T. Curran. 1990. Encounters with Fos and Jun on the road to AP-1. Sem. Cancer Biol. 1:19-26. 2. Abate, C., D. Luk, R. Gentz, F. J. Rauscher III, and T. Curran. 1990. Expression and purification of the leucine zipper and the DNA-binding domains of Fos and Jun: both Fos and Jun directly contact DNA. Proc. Natl. Acad. Sci. USA 87:1032-1036. 3. Angel, P., T. Smeal, J. Meek, and M. Karin. 1989. Transactivation domains of jun. New Biol. 1:35-43. 4. Bohmann, D., and R. Tjian. 1989. Biochemical analysis of transcriptional activation by Jun: differential activity of c- and v-jun. Cell 59:709-717. 5. Chiu, E., W. J. Boyle, J. Meek, T. Smeal, T. Hunter, and M. Karin. 1988. The c-fos protein interacts with c-jun/AP-1 to stimulate transcription of AP-1-responsive genes. Cell 54:541-552. 6. Cohen, D. R., and T. Curran. 1988. fra-J: a serum-inducible cellular immediate-early gene that encodes afos-related antigen. Mol. Cell. Biol. 8:2063-2069. 7. Courey, A. J., and R. Tjian. 1988. Analysis of SP-1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell 55:887-898. 8. Curran, T., and B. R. Franza, Jr. 1988. Fos and Jun: the AP-1 connection. Cell 55:395-397. 9. Curran, T., and J. I. Morgan. 1987. Memories offos. BioEssays 7:225-258. 10. Curran, T., A. D. Miller, L. Zokas, and I. M. Verma. 1984. Viral

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Fos and jun cooperate in transcriptional regulation via heterologous activation domains.

The products of c-fos and c-jun (Fos and Jun) function in gene regulation by interacting with the AP-1 binding site. Here we have examined the contrib...
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