MOLECULAR AND CELLULAR BIOLOGY, May 1996, p. 2110–2118 0270-7306/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 16, No. 5
Fos-Jun Dimerization Promotes Interaction of the Basic Region with TFIIE-34 and TFIIF MITCHELL L. MARTIN,1 PAUL M. LIEBERMAN,2
AND
TOM CURRAN3*
Roche Institute of Molecular Biology, Nutley, New Jersey 071101; Wistar Institute, Philadelphia, Pennsylvania 19104-42682; and St. Jude Children’s Research Hospital, Memphis, Tennessee 38101-03183 Received 28 July 1995/Returned for modification 31 January 1996/Accepted 9 February 1996
The regulation of RNA polymerase II-mediated transcription involves both direct and indirect interactions among regulatory proteins and the general transcription factors (GTFs) that assemble at TATA-containing promoters. Here we show that the oncogenic transcription factors Fos and Jun make direct physical contacts with three proteins of the basal transcription apparatus, TFIIE-34 (TFIIE-b), TFIIF-30 (RAP30), and TFIIF-74 (RAP74). The interactions among the activator proteins and these three GTFs were not detected with other transcription factors, including some bZIP protein family members. Both coimmunoprecipitation and protein blotting experiments demonstrated that the interactions were strongly favored by dimerization of Fos and Jun and that they involved the basic region and basic region-proximal domain of both proteins. Mutations within the DNA-binding domains of Fos and Jun abolished binding to GTFs, although the presence of DNA was not required for the association. Surprisingly, only a single basic region in the context of a protein dimer was sufficient for the interaction. Squelching of AP-1-dependent transcription in vitro by an excess of Fos-Jun dimers was relieved by the addition of TFIIE, indicating that it is a direct functional target of Fos and Jun. These results suggest that dimerization induces a conformational alteration in the basic region of Fos and Jun that promotes an association with TFIIE-34 and TFIIF, thus contributing to transcription initiation. (GTFs) and associated proteins referred to as coactivators, mediators, and adapters (20, 39, 73). Substantial evidence suggesting that activator proteins such as Fos and Jun stimulate promoter-specific transcription by recruiting one or more GTFs into a preinitiation complex has accumulated (11, 43). The recruitment of GTFs into a stable preinitiation complex is an ordered process that may be regulated at multiple steps during assembly (44, 50). In higher eukaryotes, TFIID binds directly to the TATA element of the promoter and nucleates the binding of TFIIA, TFIIB, TFIIF in association with Pol II, TFIIE, and TFIIH. Additional GTFs and activator-specific cofactors have also been isolated, but their precise roles in the formation of the preinitiation complex are not yet clear. Nevertheless, several studies have provided evidence for direct interactions between activator proteins and GTFs, supporting the notion that activators stimulate distinct steps in the assembly of preinitiation complexes. For example, the herpes simplex virus acidic activator protein VP16 binds directly to TFIID, TFIIB, and the p62 subunit of TFIIH (31, 45, 78). The Epstein-Barr virus transactivator Zta binds directly to TBP and TFIIA (43, 61); another Epstein-Barr virus gene product, EBNA 2, binds to TFIIH (74); and the Drosophila zinc-finger protein Kru ¨ppel interacts with both TFIIB and TFIIE (67). It is also clear that many components of the GTFs are multiprotein complexes themselves, and interactions with more than one polypeptide in the same GTF have been documented. For example, VP16 associates with two polypeptides in the multiprotein TFIID complex, the TBP and TBP-associated factor 40 (23). Similarly, the glutamine-rich cellular activator SP1 binds directly to TBP and to TBP-associated factor 110 (10, 29). The characterization of interactions among viral and cellular transcription activators with different subsets of GTFs is likely to give us a more complex but clearer understanding of the transcription activation process. This is a necessary first step that will ultimately lead to an elucidation of the molecular mechanisms that underlie the selective regulation of gene expression. Two previous studies suggested that Fos and Jun activate
The proto-oncogene products Fos and Jun are DNA-binding proteins of the bZIP family that function as transcription regulators in signal transduction pathways in many different cell types (15). The c-jun proto-oncogene is the cellular homolog of the transforming oncogene v-jun from avian sarcoma virus 17 (49). Gene knockout studies indicate that c-jun is required for normal mouse hepatogenesis (28). The c-fos gene is the cellular homolog of the transforming gene present in the FinkelBiskis-Jinkins murine sarcoma virus, which causes osteosarcomas in mice (14). Gene knockout experiments indicate that c-Fos is an essential regulator of osteoclast-macrophage lineage determination in mice (25). However, in addition to these genetically defined functions, Fos, Jun, and the related proteins Fra1, Fra2, FosB, JunB, and JunD participate as heterodimeric DNA-binding complexes in stimulus-evoked alterations in gene expression in a great many circumstances (13). Each protein contains a conserved core structure comprising a leucine zipper and an adjacent N-terminal basic region. Dimerization is mediated by a coiled-coil interaction and results in the juxtaposition of basic regions from each protein that form a bipartite DNA-binding domain (21, 41). In addition to the bZIP region, several distinct functional regions in Fos and Jun that influence transcription regulation and cellular transformation have been identified (1, 5, 72). The regulation of eukaryotic gene transcription from TATA-containing promoters by RNA polymerase II (Pol II) is a multistep process that is governed by both promoter- and regulator-specific mechanisms (80). The minimal complex that is capable of supporting basal transcription consists of the TATA-binding protein (TBP), transcription factor IIB (TFIIB), TFIIF-30 (RAP30), and Pol II (8, 76). Activated transcription requires additional general transcription factors * Corresponding author. Mailing address: St. Jude Children’s Research Hospital, 332 N. Lauderdale, P.O. Box 318, Memphis, TN 38101-0318. Phone: (901) 495-2255. Fax: (901) 495-2270. Electronic mail address:
[email protected]. 2110
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transcription, in part, through an association with TBP (53, 65). However, each study identified a distinct domain in Fos that mediated this association. In one case, a C-terminal domain that is not required for transcription activation or for cell transformation was implicated (53), and in the other the core bZIP region was demonstrated to be responsible for TBPbinding activity (65). Here we have focused on the interaction of dimers of Fos and Jun with several components of the preinitiation complex. To our surprise, this analysis revealed an unexpected dimerization-dependent interaction between the highly conserved basic and basic region-proximal domains of both Fos and Jun with TFIIE-34 (TFIIE-b), TFIIF-30, and TFIIF-74 (RAP74). The association was stimulated by dimer formation and required the presence of at least one intact basic region, but it was not dependent on DNA binding activity. This suggests that a conformational alteration occurs after Fos-Jun dimerization that promotes an association with the GTFs TFIIE-34 and TFIIF. Squelching analysis in in vitro transcription assays indicated that the interaction of Fos and Jun with TFIIE was required for AP-1-dependent transcription activity. Thus, the interaction of Fos-Jun dimers with TFIIE and TFIIF provides a functional link between these two oncogenic transcription factors and components of the cellular transcription machinery. MATERIALS AND METHODS Expression and purification of Fos, Jun, and GTFs from Escherichia coli. Full-length rat c-Fos residues (1 to 380), rat c-Jun (residues 1 to 334), and truncated proteins, containing residues 118 to 211 of Fos and 225 to 334 of Jun, were expressed in E. coli as histidine-tagged fusion proteins and purified to greater than 95% homogeneity by nickel chelate affinity chromatography as described previously (1). Histidine fusion proteins with the residues KRRIRR (139 to 144) from Fos deleted and the residues RKRMRNR (260 to 266) from Jun deleted were as described previously (32). The recombinant GTFs TBP (43), TFIIA (abg) (61), TFIIB (26), TFIIE (64), and TFIIF (9) were expressed in E. coli and purified as described previously. RNA Pol II, generously provided by M. Sheldon and D. Reinberg, was purified as described previously (46). Radiolabeling of Fos and Jun. Fos and Jun proteins were phosphorylated with radiolabeled phosphate by incubating 150 pmol of purified protein with 50 mM ATP (Pharmacia), 0.1 mCi of [g-32P]ATP (ICN), and 50 U of catalytic subunit protein kinase A (PKA) (Promega) in buffer containing 20 mM Tris (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 2 mM dithiothreitol, and 0.1% Nonidet P-40 at 308C for 30 min. Labeled proteins were separated from unincorporated ATP by centrifugation through G-25 columns (5 Prime-3 Prime) preequilibrated with buffer containing 20 mM HEPES (N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid) (pH 7.9), 50 mM NaCl, 0.1% bovine serum albumin, 0.02% NaN3, 0.05% Tween 20, and 5 mM dithiothreitol (B buffer). Typical specific activities obtained were 100 to 200 Ci/mmol for both Fos and Jun. The dimerization and DNA-binding properties of PKA-labeled Fos and Jun proteins were indistinguishable from those of unlabeled proteins (4, 6). Far-Western protein association assay. Purified recombinant GTFs were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (9% polyacrylamide) (SDS–9% PAGE) together with approximately 2.5 pmol of fulllength Fos or Jun as positive controls. Proteins were then transferred to 0.45mm-pore-size nitrocellulose membranes (Schleicher and Schuell) at 100 to 150 mA for 12 h in 192 mM glycine–25 mM Tris base–0.01% SDS–20% methanol at 48C. All subsequent steps were conducted at room temperature. Filters were rinsed briefly in B buffer and then were denatured in B buffer containing 6 M guanidine-HCl (ultrapure; U.S. Biochemical) for 30 min. Transferred proteins were renatured by incubation in 3 M guanidine-HCl for 30 min followed by incubations for 30 min each in 1.5 and 0.75 M guanidine-HCl. Blots were rinsed twice and then blocked by incubation in a 3% solution of nonfat dry milk in B buffer for 60 min. For heterodimer probes, 5 to 10 pmol of radiolabeled Fos (*Fos) was allowed to dimerize with an equimolar concentration of unlabeled Jun in the presence or absence of a 33-bp double-stranded oligonucleotide containing an AP-1 site by incubation in B buffer at 378C for 10 min. The probe complex was then added to a minimum volume of 1% milk–B buffer and incubated for 60 min at room temperature. Blots were briefly washed in B buffer, dried, and analyzed by autoradiography or with a Molecular Dynamics PhosphorImager. Coimmunoprecipitation assay. Plasmids containing the coding regions of cfos, c-jun, CREB341, ATF2, C/EBP, CTF1, serum response factor (SRF), Zta, TFIIE-34, TFIIE-56, TFIIF-30, and TFIIF-74 were transcribed by SP6 or T3 or T7 polymerase in vitro and translated in rabbit reticulocyte lysates (Promega) at 308C for 90 min. Reaction mixtures were supplemented either with 1 mM com-
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plete amino acid mixture or with mixture lacking methionine but supplemented with 40 mCi of [35S]methionine (Amersham). For in vitro binding assays, equal volumes of translation products were mixed gently in association buffer (20 mM HEPES [pH 7.9], 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 10% glycerol), incubated at 308C for 30 min, and then clarified by centrifugation at 1,200 3 g for 15 min at 48C. The cleared supernatants were diluted into 100 ml of NP-40 buffer (10 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.2% Nonidet P-40) containing 2 to 3 ml of antisera (Santa Cruz Biotechnology) and rotated for 2 h at 48C. Immune complexes were then incubated for 20 min at 48C with 20 to 30 ml of StaphA (Staphylococcus aureus) cells (Calbiochem), precipitated, and washed three times with buffer. Samples were resuspended in 40 ml of SDS sample buffer, boiled 5 min, and resolved by SDS–9% PAGE. Dried gels were analyzed by autoradiography or with a PhosphorImager (Molecular Dynamics). In vitro transcription assay. The transcription activity of recombinant proteins was assayed in vitro by primer extension of templates (69). Nuclear extracts were prepared from Namalwa cells (generous gift of J. Ozer) by the procedure of Dignam et al. (16). Namalwa extracts contain low endogenous levels of AP-1 activity. Fos and Jun proteins were dimerized in D100 buffer (20 mM HEPES [pH 7.9], 0.2 mM EDTA, 100 mM KCl, 20% glycerol) at 308C for 10 min. Purified recombinant GTFs were preincubated in nuclear extract at 308C for 10 min and then mixed with Fos-Jun-containing samples. Transcription reactions were initiated by the addition of 400 mM ribonucleoside triphosphates (Pharmacia) and 150 ng of supercoiled DNA (pFC53EG1). The template was constructed by inserting an oligonucleotide containing three AP-1 sites upstream of the human c-fos promoter in the plasmid pFC53 (18). Dimerized Fos-Jun complexes and GTFs were added to the reaction mixtures to final concentrations of 1 to 10 nM and approximately 0.2 to 2 mM, respectively. Transcription reactions were stopped after 40 min at 308C and were followed by primer extension with the oligonucleotide T35570 (59-ACACACTCGACACGGCA-39) and mouse mammary tumor virus reverse transcriptase (Bethesda Research Laboratories). Extension products were resolved on 10% acrylamide–8 M urea gels, and the dried gels were analyzed by autoradiography or with a PhosphorImager (Molecular Dynamics).
RESULTS Fos-Jun heterodimers bind to TFIIE-34, TFIIF-30, and TFIIF-74. A protein blotting (far-Western) assay was performed to investigate the association of the bZIP core region of Fos and Jun with a selection of GTFs. This assay permits the use of assembled protein dimers and protein-DNA complexes as probes for direct interactions with target proteins of interest (43, 48). Equivalent amounts of TBP, TFIIA, TFIIB, TFIIE34, TFIIE-56 (TFIIE-a), TFIIF-30, and TFIIF-74 were resolved by SDS–9% PAGE, and one gel was stained with Coomassie brilliant blue whereas the other was transferred to nitrocellulose, cycled through denaturation and renaturation steps, and incubated with labeled full-length Fos-Jun complexes. Probes were prepared by labeling purified recombinant Fos or Jun proteins to a high level of specific activity with [g-32P]ATP by incubation with PKA. For the experiments using heterodimeric complexes as probes, Fos was first labeled with PKA and then associated with unlabeled Jun. Phosphorylation by PKA has no effect on the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers (4, 6). As indicated in Fig. 1 (lanes 4, 6, and 7), Fos-Jun heterodimers formed a stable association with TFIIE-34, TFIIF30, and TFIIF-74. Consistently, the strongest interaction was observed with TFIIE-34. The additional bands in the TFIIF-74 sample represent proteolytic products that also interact with Fos-Jun dimers. In some experiments, a weak association was observed with TBP and TFIIB (lanes 1 and 3); however, this was not reproducible and, when it occurred, it was detected at a much lower level than was the association with TFIIE-34. This may reflect incomplete renaturation of these GTFs prior to incubation with probe, or it is possible that TBP and TFIIB interact with Fos-Jun with a much lower apparent affinity than does TFIIE-34 or TFIIF. It is difficult to assess the specificity of such low affinity interactions between TBP and highly charged proteins like Fos and Jun. The assays typically used for these studies, particularly affinity column chromatography, may detect nonspecific ionic interactions. Thus, while we par-
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FIG. 1. Direct physical interaction between Fos-Jun heterodimers with TFIIE-34, TFIIF-30, and TFIIF-74. Purified recombinant proteins TBP, TFIIA, TFIIB, TFIIE-34, TFIIE-56, TFIIF-30, and TFIIF-74 were fractionated by SDS–9% PAGE and stained with Coomassie brilliant blue (top panel) or transferred to a nitrocellulose membrane and probed with 32P-labeled full-length Fos dimerized to unlabeled full-length Jun (*FOS/JUN; bottom panel).
tially confirm a previous study (65) reporting an association between the bZIP core of Fos or Jun with TBP, we feel that the binding we observe with TFIIE-34 and TFIIF is much more robust and is more likely to be physiologically important. No interactions with TFIIA or TFIIE-56 were detected (lanes 2 and 5), even when they were used in excess relative to the other factors. Neither Saccharomyces cerevisiae TFIIA nor human TFIIA interacted with Fos-Jun dimers. Similar results were obtained whether probing was performed with Fos-Jun heterodimers or with Jun homodimers, in the presence or absence
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of an excess of oligonucleotides containing AP-1 DNA-binding sites (data not shown). Coimmunoprecipitation of TFIIE-34, TFIIF-30, and TFIIF-74 with dimerized Fos-Jun complexes. To determine whether the interactions observed with immobilized proteins also occurred in solution, Fos-Jun heterodimers and Jun homodimers were incubated with each of the subunits of TFIIE and TFIIF and treated with specific antibodies. Fos, Jun, and another member of the bZIP family, CREB, were expressed in vitro by using a coupled transcription-translation procedure and were labeled with [35S]methionine. Similarly, the GTFs TFIIE-34, TFIIE56, TFIIF-30, and TFIIF-74 were expressed in vitro in the presence of either [35S]methionine or unlabeled methionine. Antisera specific for each of the GTFs were added to protein mixtures, and the coimmunoprecipitation products were resolved by SDS–9% PAGE as described in Materials and Methods. Approximately equal amounts of protein were used in each case. As a positive control, a sample containing only the labeled GTF was included for each antiserum used (Fig. 2A, lanes 1, 6, 11, and 16). No nonspecific immunoprecipitation was detected in these experiments, as labeled Fos, Jun, and CREB were only precipitated in the presence of unlabeled GTFs (data not shown). Figure 2A shows that Jun homodimers interact with TFIIF30, TFIIF-74, and TFIIE-34 (lanes 3, 8, and 13) but not with TFIIE-56 (lane 18). In contrast, little or no CREB was coimmunoprecipitated in any situation (lanes 2, 7, 12, and 17). Although some Fos coimmunoprecipitated with the same three GTFs (lanes 5, 10, and 15), dimerization with Jun strengthened the interactions at least three- to fourfold (lanes 4, 9, and 14). Thus, some aspect of heterodimerization significantly favors the association with Fos-Jun dimers. In contrast to the situation with TFIIE-34 and TFIIF, no interaction was observed between any of the factors tested and TFIIE-56 (lanes 17 to 20). Similar results were obtained in the presence
FIG. 2. Coimmunoprecipitation of TFIIE-34, TFIIF-30, and TFIIF-74 with dimerized Fos-Jun complexes. (A) [35S]methionine-labeled in vitro-translated CREB (*CREB), Jun (*JUN), Fos alone (*FOS), or Fos dimerized with unlabeled in vitro-translated Jun (*FOS/JUN) was incubated with unlabeled in vitro-translated TFIIF-30 (lanes 2 to 5), TFIIF-74 (lanes 7 to 10), TFIIE-34 (lanes 12 to 15), or TFIIE-56 (lanes 17 to 20). Antibodies (Ab) specific for each of the GTFs were added to the reaction mixtures as indicated, and samples were precipitated with protein A-Sepharose. As a positive control, each antibody was incubated with the cognate [35S]methionine-labeled in vitro-translated GTFs (lanes 1, 6, 11, and 16). (B) [35S]methionine-labeled in vitro-translated Jun, ATF2, C/EBP, SRF, CTF, CREB, and Zta were incubated with unlabeled in vitro-translated TFIIF-30, TFIIF-74, or TFIIE-34 and subjected to immunoprecipitation with antibodies specific for each of the GTFs. Lane INPUT contains 50% of the protein used in each of the reactions prior to immunoprecipitation.
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or absence of oligonucleotides containing an AP-1 binding site, indicating that DNA binding was not essential for the interaction of Fos and Jun with GTFs. All of these interactions have been shown to be resistant to ethidium bromide (50 mg/ml), eliminating the possibility that there was a nonspecific association mediated by contaminating DNA (40). These findings indicate that TFIIE-34, TFIIF-30, and TFIIF-74 are capable of interacting either with DNA-bound or with free Fos-Jun and Jun-Jun complexes. These results confirm the observations made when the far-Western approach was used, i.e., that FosJun heterodimers and Jun homodimers can interact stably with TFIIE-34, TFIIF-30, and TFIIF-74. Furthermore, it appears that the interaction of Fos with each of these general factors is enhanced by dimerization with Jun. TFIIE-34 and TFIIF interact preferentially with Fos-Jun family members. To determine whether other bZIP proteins and transcription factors interact with TFIIE-34 and TFIIF, the coimmunoprecipitation experiments were extended to include the bZIP family members ATF2 (CREBP-1) (27), C/EBP (33), Zta (42), and CREB (55) as well as the unrelated SRF (56) and the proline-rich activator CTF (NF1) (66). As shown in Fig. 2B, Jun homodimers were precipitated to a similar extent by TFIIE-34, TFIIF-30, and TFIIF-74. JunB gave results identical to those with Jun (data not shown); however, ATF2, a protein very closely related to Jun, was found to interact preferentially with TFIIF-30, albeit with a lower apparent affinity than that of Jun. Although Fos and Jun bound to TFIIE-34 and each of the TFIIF subunits with approximately equal affinities, ATF2 consistently discriminated among these three GTFs. Of the bZIP proteins tested that were less structurally similar to Fos and Jun, C/EBP and CREB exhibited much weaker interactions and the more distantly related Zta did not interact at all with any subunit of TFIIE or TFIIF. The non-bZIP transcription factor CTF did not bind to the GTFs tested in this study, but SRF did coprecipitate weakly with TFIIF-74. This is consistent with the observation reported previously by Joliot et al., using a yeast two-hybrid assay (34). None of the transcription regulators assayed here interacted with TFIIE-56 (data not shown). These data suggest that the interactions observed are not general properties of all bZIP family members or transcription activators, but rather they appear to be specific for proteins within the immediate Fos and Jun families. Interaction with TFIIE-34 and TFIIF requires the bZIP core domain of Fos and Jun. To identify the region of Fos and Jun required for the interaction with GTFs, TFIIE-34, TFIIE-56, TFIIF-30, and TFIIF-74 were transferred onto nitrocellulose filters and incubated in the presence of several radiolabeled Fos-Jun complexes. Probes consisted either of full-length Fos (residues 1 to 380) dimerized with full-length Jun (residues 1 to 334), as shown in Fig. 1, or of truncated Fos (residues 118 to 211) dimerized with truncated Jun (residues 225 to 334). The truncated Fos and Jun core proteins contain only the leucine zipper and DNA-binding domains, along with 36 to 38 residues immediately N terminal of the basic region. Previous studies demonstrated that dimers of these truncated proteins behave very similarly to the full-length forms with regard to their dimerization and DNA-binding properties and retain the ability, though reduced, to activate transcription in vitro (2, 3). Figure 3 demonstrates that TFIIE-34, TFIIF-30, and TFIIF-74 interacted in a similar fashion with the full-length forms and with the bZIP core of the heterodimeric protein complexes. The associations observed with the truncated proteins were qualitatively and quantitatively similar to those detected with the full-length proteins. As indicated previously, no interaction with TFIIE-56 was observed. The interactions between GTFs and the core dimers were not affected by the presence of a
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FIG. 3. Interaction with TFIIE-34 and TFIIF maps to core domain of Fos and Jun, containing only leucine zipper and basic region. Purified recombinant proteins TFIIF-30, TFIIF-74, TFIIE-34, and TFIIE-56 were fractionated by SDS–9% PAGE and stained with Coomassie brilliant blue (top panel) or transferred to a nitrocellulose membrane and probed with 32P-labeled full-length Fos dimerized to unlabeled full-length Jun (*FLFOS/FLJUN) or 32P-labeled truncated Fos dimerized to unlabeled truncated Jun (*FOS118-211/JUN225-334).
10-fold molar excess of an oligonucleotide containing an AP-1 binding site under conditions in which the concentration of free Fos or Jun was very low. Identical results were obtained with Jun homodimers, either the full-length form or the bZIP core (data not shown). These results indicate that TFIIE-34 and TFIIF bind to the bZIP regions of Fos and Jun regardless of whether they are bound to DNA or in free solution. Dimerization enhances interactions with GTFs. To assess the relative contribution of Fos and Jun to the association with GTFs, far-Western analysis was carried out with labeled monomeric Fos and Jun. In the absence of a partner, Fos exists primarily in a monomeric state in solution (60, 62). Figure 4B (upper panel) demonstrates that undimerized Fos bound to recombinant full-length Jun, but the interaction with TFIIF-30, TFIIF-74, and TFIIE-34 was dramatically diminished. The modest binding activity detected may represent a lower-affinity interaction of Fos monomers with the GTFs, or it could be contributed by the low level of Fos homodimers that exist in solution. Identical results were observed with labeled fulllength Fos monomers (data not shown). A mutated Jun protein, lacking the third leucine of the zipper [Jun225-334 (D297)], that was deficient in dimerization activity did not interact with any of the GTFs examined (Fig. 4B, bottom panel). Thus, as determined by far-Western analysis, TFIIF-30, TFIIF-74, and TFIIE-34 interacted preferentially with dimerized Fos and Jun. However, these results do not rule out the possibility that there is a direct interaction between the GTFs and an intact Jun leucine zipper. Association with GTFs requires at least one basic domain within a dimer. The data presented above present a conundrum: the interaction of Fos and Jun with GTFs prefers protein dimers, but it is independent of DNA-binding activity. To determine whether the DNA-binding region is required for association with GTFs, a core Jun homodimer that contained an intact leucine zipper but had seven residues deleted from the DNA-binding domain [Jun225-334(D260-266)] was used as a probe in far-Western assays. Although it was capable of
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MOL. CELL. BIOL. FIG. 4. Basic domain within Fos-Jun dimer is required for interaction with GTFs. (A) Diagram of full-length and truncated Fos and Jun proteins. (B) Purified recombinant proteins TFIIF-30, TFIIF-74, TFIIE-34, and TFIIE-56 were fractionated by SDS–9% PAGE, transferred to a nitrocellulose membrane, and probed with 32P-labeled truncated Fos alone (*FOS118-211) or with 32Plabeled truncated Jun with leucine deleted from the zipper [*JUN225334(DL297)]. (C) Far-Western blot, as shown in panel B, probed with 32P-labeled truncated Jun containing a deletion within the basic domain [*JUN225334(D260-266)] or with 32P-labeled truncated Fos dimerized with unlabeled Jun basic domain mutant [*Fos118-211/Jun225-334(D260-266)].
dimerizing with a full-length Fos positive control, this mutant protein failed to interact with any of the GTFs tested (Fig. 4C, top panel). Because the leucine zipper is intact in this probe, these data together with the results shown in Fig. 4B (bottom panel) suggest that the failure of this protein to bind to GTFs is a consequence of a requirement for dimerization per se rather than a requirement for direct contact between the zipper and the GTFs. Similarly, a bZIP core Fos protein with six residues deleted from the basic domain [Fos118-211(D139144)] did not interact with the GTFs even when dimerized to Jun225-334(D260-266) (data not shown). It is possible that the deleted residues are required for a direct interaction with the GTFs or that they may be required to maintain a particular structure that is necessary for the association. Remarkably, Fos-Jun complexes in which one dimerization partner had an intact basic domain and the other had a mutated basic region exhibited reduced but reproducible levels of GTF-binding activity (Fig. 4C, bottom panel). Thus, although neither the monomeric Fos nor the mutated Jun core protein could interact with the GTFs by itself, a dimer formed from the same two
proteins clearly scored as positive in the assay (compare Fig. 4B, top panel, and 4C, top panel, with 4C, bottom panel). This suggests that one intact basic region from either Fos or Jun is necessary and sufficient for association with the GTFs, but only in the context of a protein dimer. One explanation for these results is that the GTFs interact with at least one other region of the protein dimer in addition to the basic region. Alternatively, dimerization may induce a structural alteration in the basic domain that is required for the association with the GTFs. Indeed, it has been demonstrated that in the dimeric state Fos adopts a much greater overall a-helical character (62). Thus, it is possible that the GTFs bind to the basic or basic region-proximal domains of Fos and Jun only when they adopt a stable a-helical conformation upon dimerization. Fos-Jun bZIP core heterodimers activate transcription at low concentrations and repress transcription at high concentrations. To assess the functional role of the interaction observed between TFIIE-34, TFIIF-30, and TFIIF-74 and Fos and Jun in the context of cell nuclear extracts, we employed an in vitro transcription assay. Namalwa cell nuclear extracts were
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FIG. 5. Fos-Jun core heterodimers at low concentrations activate and at high concentrations repress transcription in vitro. (A) AP-1-dependent in vitro transcription of pFC53EG1 from Namalwa nuclear extracts in the absence of exogenous proteins (lane 1) or from increasing concentrations of heterodimers of truncated Fos-Jun (Fos118-211/Jun225-334; lanes 2 to 4) or truncated Fos-Jun basic domain deletion mutants [Fos118-211(D139-144)/Jun225-334(D260-266); lanes 5 to 7]. Transcription was assayed by primer extension. Transcripts were resolved on 10% polyacrylamide–8 M urea gels; the slow-mobility band (arrow) represents correctly initiated transcripts. (B) Purified GTFs were added to transcription reaction mixtures in the absence of added Fos-Jun as indicated (lanes 1 to 8). Fos118-211/Jun225-334 heterodimers (DFOS/DJUN) were added at concentrations of 1, 2, 4, and 10 nM in the absence of added GTFs (lanes 9 to 12). Equivalent amounts of purified GTFs were added to reaction mixtures which contained either 4 nM (lanes 13 to 19) or 10 nM (lanes 20 and 21) truncated Fos-Jun heterodimers. Transcription was assayed as described for panel A and quantitated with a PhosphorImager. The solid bars represent fold activation of transcription in the presence of different exogenous proteins, expressed relative to a reaction in the absence of recombinant proteins (lane 1). The averages and standard deviations (error bars) of three to eight separate experiments are each plotted directly above the corresponding lane from a representative experiment (lane 19 was assayed twice).
used because of their relatively low basal level of transcription from an AP-1-dependent template in the absence of exogenous Fos or Jun (Fig. 5A, lane 1). The addition of bZIP core FosJun heterodimers to a final concentration of 1 nM in nuclear extracts reproducibly activated transcription approximately 3.5-fold above the basal level (lane 2). At higher levels of heterodimer (.3 to 4 nM) specific transcription was decreased in a concentration-dependent manner to a level below that of the basal value (lanes 3 and 4). The squelching of AP-1 sitedependent transcription is most likely due to the titration of limiting amounts of factors present in the extracts that are required for transcription activity. As expected, heterodimers comprising bZIP core Fos-Jun proteins [Fos118-211(D139144)–Jun225-334(D260-266)] that did not bind to AP-1 sites did not elevate the level of transcription at any concentration tested (lanes 5 to 7). The apparent difference in signals in lanes 1 and 5 was not reproducible in other experiments. The reduced signal observed in the presence of increasing concentrations of the mutant dimers defective in DNA-binding activity is a consequence of a low level of exchange between the mutant proteins and endogenous AP-1 proteins. Previously, using fluorescence energy transfer techniques, we demonstrated that Fos and Jun can readily exchange subunits (63). TFIIE-34 restores specific transcription from extracts re-
pressed by Fos-Jun dimers. To determine whether any of the GTFs were responsible for the squelching effect of high concentrations of exogenous Fos-Jun dimers, we first investigated which GTF activity was limiting for AP-1-dependent transcription in these nuclear extracts. Recombinant TBP, TFIIA, TFIIB, TFIIE (equimolar TFIIE-34 and TFIIE-56), TFIIF (equimolar TFIIF-30 and TFIIF-74), and Pol II purified from HeLa cells were added, individually or together as indicated in Fig. 5B, to Namalwa cell nuclear extracts, and AP-1-dependent in vitro transcription was measured. Basal transcription from this template increased only with the addition of TFIIB (lane 4) and, to a lesser extent, with the addition of TFIIE (lane 5), indicating that the activity of these factors was limiting in the extracts. Addition of TBP (lane 2), TFIIA (lane 3), TFIIF (lane 6), or Pol II (lane 7) individually or together (lane 8) had little or no effect on basal transcription activity. Thus, only the activities of TFIIB and TFIIE were limiting for AP-1-dependent transcription in the extracts under the conditions examined. As previously demonstrated in Fig. 5A, AP-1-dependent transcription was stimulated by the addition of bZIP core FosJun dimers to a final concentration of 1 to 2 nM (Fig. 5B, lanes 9, 10), repressed to below basal level at 4 nM (lane 11), and further squelched at a heterodimer concentration of 10 nM
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(lane 12). To test the ability of GTFs to overcome the FosJun-dependent repression, we maintained the heterodimer concentration at 4 nM and then added the specific GTFs. The addition of TBP (lane 13), TFIIA (lane 14), or TFIIB (lane 15) had no detectable effect on AP-1-dependent transcription under these conditions, despite the observation that TFIIB activity was limiting in the extracts. This suggests that the excess Fos-Jun titrated out a limiting factor(s) other than TFIIB required for transcription. However, the addition of TFIIE clearly overcame the transcription repression associated with 4 nM Fos-Jun (lane 16). In fact, in the presence of 4 nM Fos-Jun the effect of added TFIIE on AP-1 dependent transcription was cooperative compared to that of TFIIE alone (lane 5) or that obtained using Fos-Jun at 4 nM (lane 11). These results suggest that the squelching effect observed in vitro is most likely a consequence of the direct interaction of Fos-Jun heterodimers with TFIIE. The restoration of transcription was dependent on the concentration of Fos-Jun added and not on some other aspect of TFIIE activity, as the same amount of TFIIE was unable to restore transcription to extracts in which 10 nM Fos-Jun was added (lane 20). The addition of exogenous TFIIF (lane 17) or Pol II (lane 18) to samples containing 4 nM Fos-Jun had little effect on AP-1-dependent transcription. However, TFIIF is known to associate tightly with Pol II, and when added as a complex TFIIF-Pol II did restore transcription to a level roughly twice that of the basal value (lane 19). TFIIF was unable to restore transcription activity to reaction mixtures containing 10 nM exogenous Fos-Jun (lane 21). TFIIF had a more modest effect in derepressing AP-1-dependent transcription than did TFIIE, most likely because TFIIF was not limiting in these extracts (compare lane 6 to lane 1). In summary, these results confirm the association of Fos-Jun dimers with TFIIE in cell extracts and they suggest that the interaction is required for AP-1dependent transcription activity. DISCUSSION Many prokaryotic and eukaryotic transcription regulatory proteins function as dimers. Heterodimerization generates combinatorial structural complexity, with consequences in terms of DNA-binding specificities, DNA-binding affinities, transcription activity, and, ultimately, cell physiology (71). Among proteins of the bZIP family, dimerization is a requirement for functional activity, and heterodimerization between family members has been well documented (27, 37). We have described novel high-affinity interactions of the basic region of Fos-Jun dimers with three GTFs: TFIIE-34, TFIIF-30, and TFIIF-74. At least one of these GTFs, TFIIE, appears to cooperate with Fos-Jun complexes to stimulate transcription from an AP-1-dependent promoter in vitro. This represents the first report of a functional association between Fos and Jun and these key GTFs. Dimerization of Fos and Jun significantly enhanced interactions with three GTFs in two different assays. Immunoprecipitation and protein blotting analysis revealed that TFIIE-34 and TFIIF formed a stable complex with FosJun dimers, whereas a reduced interaction was detected with Fos and Jun proteins deficient in dimerization activity. This property was not shared by the other transcription activators tested in this study, namely, C/EBP, Zta, CREB, CTF, or SRF. However, the bZIP regulator ATF2 was found to interact preferentially with TFIIF-30. Given the high degree of sequence similarity in the bZIP core region among Fos, Jun, and the Maf and Nrl protein families (35–37), it is likely that these proteins interact with GTFs in a similar manner. Mutation analysis of Fos and Jun demonstrated that the
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interactions with TFIIE-34 and with both subunits of TFIIF required an intact basic domain in at least one member of the dimer. A seven-residue deletion within the DNA-binding domain of Jun [Jun225-334(D260-266)] prevented interactions between TFIIE-34 and TFIIF, in the context of both Jun homodimers and heterodimers with a basic-region mutation of Fos [Fos118-211(D139-144)]. Thus the basic and basic regionproximal domains must be intact and be part of a dimeric complex to bind to TFIIE-34 and TFIIF. This may reflect a requirement for the increase in a-helicity of the basic region that accompanies dimerization and/or a preference for two precisely positioned basic region-proximal domains. The crystal structure of the bZIP Fos-Jun core bound to an AP-1 site indicates that the position occupied by the deleted residues in Jun225-334(D260-266) and Fos118-211(D139-144) begins within their respective DNA-contacting surfaces and extends just past the region that lies in the major groove (22). The sequences in the DNA-contacting regions of Fos (within residues 144 to 158) and Jun (within residues 262 to 276) are highly conserved across species (.95%). This amino acid conservation extends throughout the basic region-proximal domain to the N termini of the core proteins used in this study (residues 118 to 144 of Fos and 225 to 262 of Jun), although these basic region-proximal domains are dispensable for DNAbinding activity. In comparison, throughout the remainder of Fos there is only 70% identity between the chicken and human sequences. This argues for an important functional role for the basic region-proximal domains. Our observation that the addition of oligonucleotides containing AP-1 binding sites did not disrupt the Fos-Jun interaction with GTFs suggests that any conformational changes of DNA and protein that accompany DNA binding do not preclude interactions with TFIIE and TFIIF. Furthermore, it suggests that the precise residues that contact DNA are not directly involved in the interactions with GTFs. Thus, it seems probable that, in the context of the intact cell, DNA-bound Fos-Jun complexes are able to interact with these GTFs. An important finding of this study is that the DNA-binding domains of Fos and Jun may overlap with transcription regulatory domains. There are functional data in support of this observation for the bZIP family Epstein-Barr virus transcription activator Zta (EB1) (54), the basic region helix-loop-helix protein MyoD (7), the zinc finger glucocorticoid receptor (68), and the yeast activators HAP1 (75) and ADR1 (12). Moreover, this observation has been suggested previously with respect to the binding of Fos to TBP (65). Interestingly, the basic regionproximal domain has also been proposed to make crucial intermolecular interactions with unknown cellular proteins to mediate the transformation and transactivation properties of Fos (77). To our knowledge this is the first report of a eukaryotic transcription regulator interacting with TFIIF-30. Mammalian TFIIF-30 exhibits sequence similarity with E. coli sigma factors, and it binds RNA Pol II avidly in a manner analogous to the interaction of s70 with bacterial RNA Pol (19, 52). Binding of TFIIF-30 to Pol II suppresses nonspecific DNA binding by RNA Pol II, and it is required for the stable interaction with the promoter-TFIID-TFIIA-TFIIB complex (38). TFIIF-30 is absolutely required for basal transcription from the Drosophila Adh promoter (76), whereas TFIIF-74 is dispensable for initiation although it is required for Pol II promoter clearance and for early transcript elongation (9). In the only other report of a transcription regulator binding to TFIIF-74, it was shown that the interaction with SRF correlates with transcription activation of an SRF-dependent promoter through an unknown mechanism (34). The interactions of Fos-Jun with
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TFIIF-30 and TFIIF-74 could facilitate loading of the Pol II complex onto the TFIID-TFIIA-TFIIB-promoter assembly. It is also possible that the interaction of Fos-Jun with TFIIF-74 promotes elongation of the nascent transcript. Our results clearly demonstrate both a physical and a functional interaction of the Fos-Jun core heterodimer with TFIIE34. Recently, the Drosophila zinc finger protein Kru ¨ppel was also shown to interact with TFIIE in a dimerization-dependent manner that resulted in repression of transcription (67). TFIIE exists as a heterotetramer composed of TFIIE-34 and TFIIE-56 (59). After TFIIF and Pol II have assembled at the promoter, the addition of TFIIE, TFIIH, and ATP is required for the formation of transcription complexes that are competent to proceed through the promoter clearance stage to transcript elongation (from nonsupercoiled templates) (24, 30). The large subunit of TFIIE is known to associate tightly with TFIIH and to recruit it to the preinitiation complex (51). Perhaps more importantly, TFIIE modulates the RNA Pol II carboxy-terminal domain kinase activity of TFIIH (47, 57, 70), stimulates the DNAdependent ATPase activity of TFIIH (58), and inhibits the DNA helicase activity of TFIIH (17). Thus, TFIIE exerts multiple effects on TFIIH and it represents an important control point for the action of transcription regulators (80). The interaction of Fos and Jun with TFIIE-34 may facilitate the assembly of the TFIIE-56-TFIIH complex with the promoter and/or the efficiency of transcript elongation. Indeed, other activators have been shown to enhance transcription processivity (79). It is also possible that Fos-Jun complexes interact directly with TFIIH, as has been reported for other activators (74, 78). The precise mechanisms responsible for RNA Pol II-mediated transcription are likely to be promoter and regulator dependent. This study has focused on the novel finding that Fos-Jun dimers can interact directly with TFIIE and TFIIF. This interaction requires intact bZIP core DNA-binding domains of Fos and Jun. Although maximal transcription activation requires additional protein domains outside of the DNAbinding domain which most likely interact with other proteins of the transcription machinery, it has become increasingly clear that the DNA-binding domains of transcription factors contribute significantly to interactions with regulatory proteins. On the basis of results of this study, it appears that Fos-Jun dimerization is a necessary prelude to the interaction with GTFs. This suggests that the surface of Fos and Jun that is required for the interaction with TFIIE and TFIIF adopts an appropriate conformation only after zipper dimerization. In this way highly ordered multisubunit protein complexes can be assembled through a consecutive series of relatively low-affinity interactions. A model in which a multiplicity of interactions among domains of activating proteins with one or more GTFs cooperate in the regulation of distinct stages of transcription initiation is emerging. ACKNOWLEDGMENTS We are grateful to M. Sheldon and D. Reinberg for generous sharing of reagents, to A. Bolden for technical support, to N. Kirov for critical reading of the manuscript, and to J. Ozer for reagents and helpful discussions. This work was supported in part by NIH Cancer Center Support CORE grant P30 CA21765 and the American Lebanese Syrian Associated Charities (ALSAC). REFERENCES 1. Abate, C., D. Luk, and T. Curran. 1991. Transcriptional regulation by Fos and Jun in vitro: interaction among multiple activator and regulatory domains. Mol. Cell. Biol. 11:3624–3632.
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