c-Jun Binds the N Terminus of Human TAFII250 to Derepress RNA Polymerase II Transcription in Vitro*

Tricia N. LivelyDagger, Heather A. Ferguson, Shelly K. Galasinski, Anita G. Seto, and James A. Goodrich§

From the Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215

Received for publication, January 11, 2001, and in revised form, April 18, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAFII250. The region of TAFII250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAFII250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAFII250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAFII250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAFII250 to derepress RNA polymerase II transcription in vitro.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Regulation of mRNA synthesis in eukaryotes is a complex process involving gene specific activators, coactivators, and general transcription factors. Understanding how this multitude of factors sets levels of transcription of individual genes is the goal of much research. Emerging evidence indicates that transcriptional activators have the potential to stimulate transcription via many different mechanisms. For example, activators can exert their effects by binding components of the transcription machinery, including subunits of the TFIID1 and mediator complexes (for review, see Refs. 1 and 2) as well as by recruiting chromatin modifying complexes (for review, see Ref. 3).

Whereas unique combinations of activators and coactivators function at different promoters, RNA polymerase II and the general transcription factors (TFIIA, -B, -D, -E, -F, and -H) are thought to function at most mRNA-encoding genes (for review, see Ref. 4). The TFIID complex plays a central role in core promoter recognition and the assembly of preinitiation complexes regardless of the manner by which the remaining basal transcriptional components are recruited, either via a stepwise pathway or as a holoenzyme (5). TFIID is composed of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs) whose sizes range from 18 to 250 kDa (for review, see Ref. 6). Subunits of TFIID contact core promoter elements including the TATA box, initiator, and downstream promoter element (7-10).

At some promoters, the recruitment of TFIID limits the overall level of transcription in vitro. Efficient recruitment of TFIID can be achieved by upstream activators that interact with TFIID components (11-13). In addition, activators can elicit a conformational change in the TFIID-TFIIA-promoter complex, as observed by DNase I footprinting (14, 15). Although direct interactions between TBP and activators have been noted, the primary functional targets of activators within TFIID are the TAF subunits. TAFs were coined coactivators in recognition of their requirement for activated transcription in some in vitro systems (16, 17). Recently, the ability of TAFs to adopt multifunctional roles as components of distinct complexes including TFIID, Spt-Ada-Gen5-acetyltransferase (SAGA), p300/CBP-associated factor (PCAF), and TBP-free TAFII complex (TFTC) has been demonstrated (for review, see Ref. 1). The validity of the coactivator function of TAFs has been firmly established from examples in yeast, Drosophila, and human systems.

Although TAFs can function as coactivators, regulated transcription of some promoters is not dependent on TAFs in vitro and in vivo (18-22). The mediator and other complexes are also targets of transcriptional activators and serve to coactivate transcription (for review, see Ref. 2). In the context of chromatin, nucleosome-remodeling complexes and histone acetyltransferases such as cAMP-response element-binding protein (CREB)-binding protein (CBP) function to coactivate transcription (3). It is likely that activators can stimulate transcription by many different mechanisms depending upon the conditions and the context of the promoter at which the activator binds.

One mechanism by which activators can function is by relieving the effects of factors that repress basal transcription. Although this is certainly true of activators in the context of chromatin, mechanisms of transcriptional repression and derepression have also been observed in the absence of chromatin. For example, the binding of TFIID to core promoters is internally repressed by the largest subunit of TFIID, TAFII250 (23). Most of the work on the mechanism of this repression has been carried out with dTAFII230 and yTAFII145, the Drosophila and yeast homologs of human TAFII250. Repression is mediated by a small domain (~150 amino acids) in the N termini of these proteins that binds directly to TBP and inhibits its ability to bind TATA boxes (23, 24). Furthermore, the N-terminal domain of dTAFII230 can repress TBP-driven transcription when added in excess of TBP in an in vitro transcription system (23). Mutational analysis identified the concave DNA binding surface of TBP as a binding site of the N terminus of dTAFII230 (25). In NMR studies, amino acids 11-77 of dTAFII230 appeared to mimic DNA by binding to the DNA binding cleft of TBP (26). A second region of dTAFII230 (amino acids 118-143) interacts with the convex surface of TBP and further stabilizes the dTAFII230-TBP interaction (27). The importance of TAFII250 repression to transcriptional regulation in cells and the potential for targeting the N terminus of TAFII250 as a means to derepress transcription are currently under study. TFIIA, which aids TFIID in binding core promoters, can prevent TAFII250 from inhibiting TBP-DNA binding in vitro (28). In addition, yTAFII145 and yTFIIA bind competitively to TBP (24). The acidic activation domain of VP16 also binds the underside of TBP and can block the interaction of dTAFII230 with TBP (25, 29). Activators have not, however, been shown to bind directly to the N terminus of TAFII250 to remove its repressive effects on TBP-DNA binding.

AP-1 transcription factors of the Jun and Fos families activate many genes including those involved in cell growth and proliferation (for review, see Ref. 30). AP-1 proteins each contain an extended alpha -helical dimerization/DNA binding domain of ~62 amino acids that consists of a leucine zipper found C-terminal of a basic region (31). Dimers bind DNA through sequence-specific contacts between amino acids in the basic regions and the major groove of the DNA helix (32). Members of the Jun family can form homo- and heterodimers. Members of the Fos family cannot form homo- or heterodimers among themselves but can heterodimerize with Jun proteins. Although there is high sequence homology in the basic leucine zipper domains of AP-1 proteins, there is much less homology in other regions, which contain activation domains. The activation domains of c-Jun and c-Fos, which are essential for transcriptional activation in vitro, have been characterized functionally (33-35) but not structurally and are known to bind other proteins such as the coactivator CBP (36). Despite a wealth of studies, the mechanisms by which c-Jun and c-Fos stimulate transcription are not well understood.

Here we have investigated the mechanism by which human c-Jun activates transcription using a highly purified human RNA polymerase II transcription system. Activation of transcription by recombinant c-Jun was found to be dependent on TAFs. Interactions between c-Jun and TAFs were identified and characterized using protein-protein affinity chromatography, which revealed that c-Jun binds to the N terminus of human TAFII250. The function of c-Jun in derepressing transcription driven by TFIID and the ability of c-Jun to block TAFII250 repression of TBP transcription and DNA binding were characterized in vitro. Our results reveal a unique interaction between c-Jun and TAFII250 that results in derepression of TFIID-driven transcription in vitro. These studies lead to a novel model for activation in which c-Jun interacts with TAFII250 to relieve repression of TFIID-DNA binding, thereby contributing to transcriptional activation.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmids-- pET-Jun, a plasmid for expressing full-length human c-Jun, was a gift from T. Hoey (Tularik, Inc.). In creating this plasmid an NcoI site was generated that changed the fourth base pair in the c-Jun-coding region from AT to GC, resulting in a point mutation in the second amino acid of c-Jun (Thr to Ala). pGEX-Jun was created by digesting pET-Jun with NcoI and EcoRV and subcloning the c-Jun cDNA into the NcoI and StuI sites of pGEX-2TKN (a kind gift of S. Ruppert and R. Tjian).

Plasmids for expressing TAFs (pTbeta -hTAF250, pTbeta -dTAF40, pTbeta -dTAF30alpha , and pTbeta -dTAF30beta ) were gifts from R. Tjian. HA-hTAFII250 was expressed in SF9 cells as previously described (37). pTbeta -hTAF250-(1-560) and pTbeta -hTAF250-(1-163) were created by digestion of pTbeta -hTAF250 with EcoRI and SmaI, respectively, followed by gel purification of the linearized plasmid and ligation. pGEX-hTAF250-(1-163) was created by digestion of pTbeta -hTAF250-(1-163) with NdeI and EcoRI and insertion of the DNA fragment into pGEX-2TKN.

Plasmid p(AP-1)5-E1b-CAT was created by inserting five direct copies of a double-stranded oligonucleotide containing the AP-1 element from the human metallothionein IIA promoter (-105 to -96) into the XbaI site of plasmid E1b-CAT (38). Plasmid p(AP-1)5-E1b-G-less, used as template DNA in the in vitro transcription assays, was created by inserting a 377-base pair G-less cassette (excised from p(GAL4)5-E1b-G-less (a kind gift from M. G. Peterson and R. Tjian) with SacI) into the SacI site of p(AP-1)5-E1b-CAT. Plasmid p(GAL4)5-AdMLP-G-less (39), which contains five GAL4 sites upstream of the adenovirus major late core promoter (AdMLP; -53 to +10) and a 380-base pair G-less cassette, was generated by subcloning a HindIII-EcoRI fragment isolated from plasmid G5-MLP-G-less (a kind gift from Mike Carey) into the HindIII and EcoRI site of pBluescript-KS(+).

Expression and Purification of Recombinant c-Jun and GST Fusion Proteins-- Recombinant full-length human c-Jun was expressed in Escherichia coli BL21:DE3. Cultures (500 ml) were grown in LB containing 0.3 mM ampicillin at 37 °C until the A600 nm was 0.4. Expression was induced with 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside for 2 h at 37 °C. Cells were harvested and resuspended in 10 ml of a solution containing 20 mM Tris (pH 7.9), 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, and sonicated 4 times for 15 s. Samples were centrifuged for 30 min in a Sorval SS34 rotor at 18,000 rpm and 4 °C. Precipitated material (inclusion bodies) containing c-Jun was resuspended in 10 ml of 5 mM DTT and sonicated 2 times for 30 s. Samples were centrifuged for 10 min in a Sorval SS34 rotor at 15,000 rpm and 4 °C. Insoluble material was washed three more times by resuspending in 10 ml of 5 mM DTT, followed by centrifugation. Pellets from the final wash were resuspended in 5 ml of buffer A (20 mM Tris (pH 7.9), 1 mM EDTA, 5 mM DTT, and 7 M urea) containing 0.1 M NaCl. Soluble protein was loaded on an 8-ml column packed with sulfopropyl-Sepharose fast flow (Amersham Pharmacia Biotech) and washed with three column volumes of buffer A containing 0.1 M NaCl followed by three column volumes of buffer A containing 0.2 M NaCl. c-Jun was then eluted with buffer A containing 0.3 M NaCl and subjected to three sequential dialyses in buffer B (20 mM Tris (pH 7.9), 0.1 mM EDTA, 10% glycerol, 5 mM DTT) containing the following additions: 1) 1 M urea and M NaCl; 2) 1 M NaCl; 3) 0.1 M NaCl. After dialysis, the purified c-Jun was separated into aliquots and stored at -80 °C.

GST-Jun was expressed in E. coli strain XA90. Cultures were grown in LB containing 0.3 mM ampicillin at 37 °C until the A600 nm was 0.4. Expression was induced with 0.5 mM isopropyl-1-thio-beta -D-galactopyranoside for 3 h at 37 °C. Cells were harvested and resuspended in a solution containing 20 mM Tris (pH 7.9), 20% glycerol, 1 mM EDTA, 5 mM MgCl2, 0.1 M NaCl, 5 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride and sonicated 4 times for 15 s. Insoluble material was washed with 5 mM DTT as described for c-Jun above. Pellets from the final wash were resuspended in a solution containing 20 mM Tris (pH 7.9), 1 mM EDTA, 5 mM DTT, and 6 M guanidine-HCl, and protein was extracted by overnight nutation at 4 °C. After centrifugation to remove the remaining insoluble material, extracts were diluted to give final concentrations of 200 ng/µl of GST-Jun. Samples were subjected to three sequential dialyses in buffer B containing 0.1 M NaCl and guanidine-HCl at 2, 1, and 0.5 M. The guanidine-HCl was then slowly lowered to 50 mM by pumping (over 10 h) buffer B containing 0.1 M NaCl into the third dialysis listed above. After dialysis, samples containing GST-Jun were separated into aliquots and stored at -80 °C. Just before use in protein-protein interaction assays, GST-Jun was further purified by incubation with glutathione-Sepharose for 1 h at 4 °C followed by two washes with 10 column volumes of buffer B containing 1 M NaCl and 0.1% Nonidet P-40 and three washes with the appropriate protein-protein interaction assay buffer (see below and figure legends). The affinity resin contained ~1 mg of protein/ml of beads.

GST-TAFII250-(1-163) and GST were expressed in E. coli strain XA90 as described for GST-Jun above. Harvested cells were resuspended in 1/50 culture volume of lysis buffer containing 20 mM Tris (pH 7.9), 20% glycerol, 1 mM EDTA, 0.1 M NaCl, 1 mM DTT, and 0.2 mM phenylmethylsulfonyl fluoride. Samples were sonicated 4 times for 15 s and centrifuged for 30 min in a Sorval SS34 rotor at 18,000 rpm and 4 °C. Supernatants from the centrifugation were incubated with glutathione-Sepharose at 4 °C for 1 h with constant nutation. Resin was washed two times with 10 column volumes of buffer B containing 1 M NaCl and 0.1% Nonidet P-40. For use in protein-protein interaction assays, the resin was washed three times with ten column volumes of the appropriate protein-protein interaction assay buffer (see below and figure legends). For elution, resin was washed five times with 10 column volumes of buffer C (100 mM Tris (pH 7.9), 120 mM NaCl). Protein was eluted by incubating resin at 4 °C for 10 min in 1 column volume of buffer C containing 20 mM glutathione (reduced) with constant nutation. Eluates were dialyzed overnight at 4 °C in a buffer containing 20 mM Tris (pH 7.9), 10% glycerol, 1 mM EDTA, 0.1 M NaCl, and 1 mM DTT. Samples were separated into aliquots and stored at -80 °C.

Protein-Protein Interaction Assays-- Each reaction contained 1 µg of GST-Jun or GST immobilized on 10 µl of glutathione-Sepharose beads (Amersham Pharmacia Biotech). Immobilized proteins and extracts containing target proteins were incubated with micrococcal nuclease at 30 °C for 10 min. Target proteins were then incubated with GST or GST-Jun beads at 4 °C for 2 h with constant nutation in TGEMD buffer (20 mM Tris (pH 7.9), 10% glycerol, 1 mM EDTA, 5 mM MgCl2, and 1 mM DTT) containing 0.1% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, and 0.1 M NaCl. After incubation, beads were washed at least 3 times with 10-15 bead volumes of buffer. Protein was eluted from the beads in SDS sample buffer and resolved by SDS-PAGE.

In Vitro Transcription-- General transcription factors (TBP, TFII-B, -E, -F, and -H) and RNA polymerase II were expressed and purified as previously described (40). Human TFIID was immunopurified (39). Recombinant human TFIIA was prepared as previously described (41, 42). The amounts of general factors and buffer conditions used for transcription were as previously described (39). The amounts of the TFIID (10 ng) and TBP (0.5 ng) used in transcription reactions contained equal moles of TBP, as determined by quantitative Western analysis with polyclonal antibody directed against TBP (39).

To study transcriptional activation (see Fig. 1B) c-Jun was incubated with p(AP-1)5-E1b-G-less for 5 min on ice. After the addition of general transcription factors, reactions (20 µl) were transferred to 30 °C for 25 min before adding nucleoside triphosphates. RNA synthesis was allowed to proceed for 20 min at 30 °C. To study derepression (see Fig. 4), c-Jun was incubated with TBP or TFIID in 13-µl reactions for 10 min on ice. p(GAL4)5-AdMLP-G-less template DNA and the remaining general transcription factors where then added to reactions in a final volume of 20 µl.

To study transcriptional repression (see Fig. 5A) GST-TAFII250-(1-163) and GST were added to transcription reactions containing p(GAL4)5-AdMLP-G-less template DNA before the addition of TBP, TFIIB, TFIIF, and RNA polymerase II. In Fig. 5B, c-Jun was incubated with GST-TAFII250-(1-163) (lanes 3 and 4) in 3 µl for 13 min on ice. p(GAL4)5-AdMLP-G-less template DNA was then added, followed immediately by the addition of the general transcription factors to a final volume of 20 µl.

DNase I Footprinting-- DNase I footprinting reactions were performed with a 183-base pair DNA fragment containing the AdMLP (-53 to +33) 32P-labeled on the 5'-end of the nontemplate strand. The buffer conditions were identical to those used for transcription (before the addition of NTPs). When present, GST-TAFII250-(1-163) and c-Jun were incubated together in a reaction volume of 2 µl for 10 min on ice. These mixtures were then added to reactions containing promoter DNA followed immediately by the addition of TBP at a final concentration of 6 nM in a final volume of 20 µl. After 10 min at 30 °C, 1 µl of a solution containing 0.15 units/µl of DNase I (Promega) and 10 mM CaCl2 was added to each reaction. Reactions were stopped, and DNA products were analyzed as described (39).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of Transcription by Human c-Jun in a Highly Purified in Vitro System Requires TAFs-- To study the mechanism of c-Jun transcriptional activation, we developed a human RNA polymerase II transcription system reconstituted from highly purified recombinant and native general transcription factors. This system is useful for studying the roles of individual subunits of the general RNA polymerase II transcription machinery, including the TFIID TAFs, in basal and activated transcription. Recombinant human c-Jun was expressed in E. coli and purified to near homogeneity (Fig. 1A). To test the activity of c-Jun for activating transcription in vitro we created a DNA template, p(AP-1)5-E1b-G-less, containing five AP-1 elements upstream of the adenovirus E1b TATA box and a 377-base pair G-less cassette. We detected basal transcription from this template in vitro (Fig. 1B) in reactions reconstituted with equal molar concentrations of either immunopurified native TFIID (lane 1) or recombinant TBP (lane 3). Interestingly, reactions reconstituted with TBP reproducibly showed higher levels of basal transcription than those containing TFIID even though equivalent molar concentrations of TBP and TFIID were used (39). This is likely to be due to activities present in the TFIID complex that inhibit its binding to DNA, such as the N terminus of TAFII250 (23). c-Jun (30 ng) activated transcription ~10-fold in reactions reconstituted with TFIID (Fig. 1B, compare lanes 1 and 2) but did not stimulate transcription in reactions reconstituted with TBP (Fig. 1B, compare lanes 3 and 4). Thus, in a highly purified in vitro transcription system, TAFs are required for transcriptional activation by human c-Jun.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1.   The TAF subunits of TFIID are required for activation of transcription by c-Jun in a RNA polymerase II transcription system reconstituted from highly purified transcription factors. A, purified recombinant human c-Jun (2 µg) was subjected to SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. B, c-Jun activates transcription in a highly purified in vitro transcription system containing TFIID but not TBP. Reactions in lanes 1 and 2 contained 10 ng of immunopurified TFIID. Reactions in lanes 3 and 4 contained 0.5 ng of recombinant TBP. 30 ng of recombinant c-Jun was added to the reactions in lanes 2 and 4. The DNA template was p(AP-1)5-E1b-G-less, which contained five AP-1 sites upstream of the adenovirus E1b TATA box and a 377-base pair G-less cassette.

c-Jun Binds TAFII250 with High Specificity-- The finding that TAFs were required to mediate transcriptional activation by c-Jun in our in vitro transcription system led us to hypothesize that c-Jun interacted with one or more of the TAFs. To identify interactions between c-Jun and TAFs, we performed protein-protein affinity chromatography utilizing immobilized GST-Jun and in vitro translated 35S-labeled TAFs. TAFs were incubated with GST-Jun or control GST resin, and after extensive washing, proteins bound to the resins were analyzed by SDS-PAGE and autoradiography. These studies revealed that TAFII250 interacted with immobilized GST-Jun but not control GST (Fig. 2A, lanes 1-3). Other TAFs, for example dTAFII40, dTAFII30alpha , and dTAFII30beta , did not interact with GST-Jun (Fig. 2A, lanes 4-12).


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 2.   c-Jun binds TAFII250 with high specificity. A, protein-protein affinity chromatography was used to identify TAFs that interact with GST-Jun. 35S-Labeled human TAFII250 (lanes 1-3), Drosophila TAFII40 (lanes 4-6), Drosophila TAFII30alpha (lanes 7-9), and Drosophila TAFII30beta (lanes 10-12) were expressed by in vitro translation and tested for interaction with immobilized GST-Jun or GST. 10% of the input 35S-labeled proteins were included in lanes 1, 4, 7, and 10. B, recombinant human TAFII250 was specifically purified from cell lysates using GST-Jun affinity resin. HA-TAFII250 was expressed in insect cells using a recombinant baculovirus. Portions of cell lysate containing TAFII250 were incubated with GST-Jun and control GST resins. After extensive washing, protein bound to the affinity resins was resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. An aliquot of insect cell lysate equal to 100% of the input was included in lane 1.

To further characterize the affinity and specificity of the c-Jun-TAFII250 interaction, we utilized extracts of insect cells containing HA-tagged TAFII250 that was expressed from a recombinant baculovirus. Insect cell extracts were incubated with immobilized GST-Jun and control GST. After extensive washing, bound protein was resolved by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue. As shown in Fig. 2B, the GST-Jun resin specifically purified recombinant HA-TAFII250 from the crude insect cell extract. The numerous and abundant insect cell proteins in the extract were not retained on the GST-Jun resin.

c-Jun Binds the N-terminal Region of TAFII250 That Is Involved in Transcriptional Repression-- To gain insight into the role of the c-Jun-TAFII250 interaction in transcriptional activation, we delimited the region of TAFII250 that is bound by c-Jun. Two C-terminal deletions of TAFII250 were expressed by in vitro translation and tested for interaction with immobilized GST-Jun: TAFII250-(1-560) and TAFII250-(1-163). As shown in Fig. 3A, TAFII250-(1-560) and TAFII250-(1-163) each bound GST-Jun at a level comparable with that observed for full-length TAFII250 (amino acids 1-1893).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3.   c-Jun binds the N-terminal 163 amino acids of TAFII250. A, 35S-Labeled full-length TAFII250 and two C-terminal deletions were expressed by in vitro translation and tested for interaction with immobilized GST-Jun and GST. Bound protein was analyzed by SDS-PAGE and autoradiography. B, immobilized GST-TAFII250-(1-163) binds purified c-Jun. Immobilized GST-TAFII250-(1-163) and control GST were incubated with c-Jun. Bound protein was analyzed by SDS-PAGE and silver staining (lanes 4 and 5). Purified GST and GST-TAFII250-(1-163) are shown in lanes 2 and 3, respectively.

As an additional test of the ability of the N-terminal 163 amino acids of TAFII250 to bind c-Jun, we expressed and purified a GST-TAFII250-(1-163) fusion protein and incubated the immobilized GST-TAFII250-(1-163) with purified c-Jun. The silver-stained SDS gel in Fig. 3B shows GST-TAFII250-(1-163) bound c-Jun (lane 5). c-Jun was not retained on control GST resin (lane 4). Thus, purified c-Jun can bind directly to GST-TAFII250-(1-163).

These results revealed that the region of TAFII250 bound by c-Jun is the N-terminal domain, which is known to inhibit TBP-DNA binding and transcription. Based on this finding, we hypothesized that c-Jun binds the N terminus of TAFII250 and inhibits its repression of TBP-DNA binding, thereby contributing to transcriptional activation.

c-Jun Derepresses Basal Transcription Directed by TFIID-- It is possible that the lower level of basal transcription observed with TFIID compared with recombinant TBP (see Fig. 1) was due to the N-terminal region of TAFII250 interacting with the DNA binding surface of TBP to repress the binding of TBP to TATA DNA. In this case, c-Jun may bind directly to the N terminus of TAFII250 in TFIID to derepress transcription. To test this we used a DNA plasmid that lacks known AP-1 sites, p(GAL4)5-AdMLP-G-less, which contains the adenovirus major late core promoter (-53 to +10) upstream of a 380-base pair G-less cassette. TFIID and TBP were incubated separately with increasing concentrations of c-Jun and then added to transcription reactions with p(GAL4)5-AdMLP-G-less. As shown in Fig. 4, lanes 7-10, c-Jun stimulated transcription when preincubated with TFIID. The level of transcription with TFIID approached that observed with TBP at the highest concentrations of c-Jun. c-Jun did not increase the level of transcription when preincubated with TBP (Fig. 4, lanes 2-5).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   c-Jun derepresses TFIID-directed transcription from a template that lacks AP-1 sites. Recombinant c-Jun was incubated with TBP (lanes 2-5) or TFIID (lanes 7-10) before adding p(GAL4)5-AdMLP-G-less (consisting of the adenovirus major late core promoter upstream of a 380-base pair G-less cassette) and the remaining general transcription factors. The amounts of c-Jun added to the transcription reactions were 30 ng (lanes 2 and 7), 200 ng (lanes 3 and 8), 400 ng (lanes 4 and 9), and 700 ng (lanes 5 and 10).

c-Jun Overcomes Repression of TBP-DNA Binding and Transcription Caused by the N Terminus of TAFII250-- To demonstrate that the N terminus of TAFII250 can repress in vitro transcription and to directly test whether c-Jun can inhibit TAFII250-mediated transcriptional repression, we added purified GST-TAFII250-(1-163) to transcription reactions. The ability of TAFII250-(1-163) to repress basal transcription was determined by comparing the effects of GST-TAFII250-(1-163) and GST on basal transcription in a minimal in vitro transcription system consisting of TBP, TFIIB, TFIIF, RNA polymerase II, and negatively supercoiled p(GAL4)5-AdMLP-G-less template DNA. As shown in Fig. 5A, the addition of GST-TAFII250-(1-163) repressed basal transcription by more than 95%, whereas GST did not affect transcription when titrated over the same concentration range. Thus, the N terminus of TAFII250 can repress basal transcription in a highly purified minimal transcription system.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5.   c-Jun blocks the repression of basal transcription and TBP-DNA binding caused by the N terminus of TAFII250. A, GST-TAFII250-(1-163) specifically represses basal transcription in vitro. GST-TAFII250-(1-163) and control GST were added to transcription reactions before the addition of the transcription factors TBP, TFIIB, TFIIF, and RNA polymerase II. The final concentrations of GST (lanes 2-4) and GST-TAFII250-(1-163) (lanes 6-8) in reactions before the addition of nucleoside triphosphates were 10 nM (lanes 2 and 6), 50 nM (lanes 3 and 7), and 100 nM (lanes 4 and 8). B, c-Jun blocks transcriptional repression by GST-TAFII250-(1-163). GST-TAFII250-(1-163) was incubated alone (lane 2) or with c-Jun (lanes 3 and 4) before being added to reactions containing p(GAL4)5-AdMLP-G-less, followed immediately by the addition of the general transcription factors. The amounts of c-Jun added to transcription reactions were 100 ng (lane 3) and 200 ng (lanes 4 and 5). C, c-Jun blocks the ability of GST-TAFII250-(1-163) to repress DNA binding by TBP. GST-TAFII250-(1-163) (18 ng) and c-Jun (530 ng) were incubated together before adding TBP and AdMLP promoter DNA. After treatment with DNase I, samples were subjected to denaturing PAGE, and the results were analyzed and quantitated by phosphor-imaging. Plots are shown of relative intensities of bands (y axis) versus position on gel (x axis) for four different reactions: no proteins, TBP alone, TBP with GST-TAFII250-(1-163), and c-Jun with TBP and GST-TAFII250-(1-163). Left to right in the plots represents top to bottom of the gel. The TATA box region is indicated with a bracket.

We next tested the ability of c-Jun to block transcriptional repression caused by GST-TAFII250-(1-163). c-Jun was preincubated with GST-TAFII250-(1-163) before adding the p(GAL4)5-AdMLP-G-less template (lacking known AP-1 sites) and the remaining transcription factors. c-Jun overcame the repression of basal transcription observed with GST-TAFII250-(1-163) (Fig. 5B, compare lanes 3 and 4 with lane 2). These data demonstrate that c-Jun can bind the N terminus of TAFII250 to block its ability to repress basal transcription.

It was possible that the transcriptional effects presented above resulted from c-Jun binding the N terminus of TAFII250 to inhibit its repression of TATA box binding by TBP. To test this, we performed DNase I footprinting with TBP using a DNA fragment containing the adenovirus major late core promoter but lacking AP-1 sites. GST-TAFII250-(1-163) and c-Jun were added to determine their effects on TBP-DNA binding. Fig. 5C shows plots of relative intensities (y axis) versus position on the gel (x axis) for the region spanning the TATA box (left to right represents top to bottom on the gel). TBP alone protected bands in the TATA box region from DNase I digestion by greater than 90%. GST-TAFII250-(1-163) almost completely repressed TBP-DNA binding. In the presence of GST-TAFII250-(1-163), the intensities of bands in the TATA box region were decreased by less than 10% from the control reaction performed in the absence of transcription factors. Incubation of c-Jun with GST-TAFII250-(1-163) blocked the repression of TBP binding, resulting in ~70% of the maximal protection in the TATA box region that was observed with TBP alone. Since the footprinting experiments were performed with recombinant TBP, GST-TAFII250-(1-163), and c-Jun, we can rule out a requirement for other general transcription factors in repression by GST-TAFII250-(1-163) and inhibition of this repression by c-Jun. Taken together, the transcription and DNase I footprinting experiments strongly support a model in which c-Jun binding to the N terminus of TAFII250 inhibits repression of TBP-DNA binding and transcription initiation.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we have elucidated one mechanism by which c-Jun activates transcription. c-Jun binds the N terminus of TAFII250 to derepress RNA polymerase II transcription. Not only did c-Jun derepress TFIID-directed transcription in vitro, but c-Jun was also capable of blocking repression of TBP-DNA binding by the N terminus of TAFII250. Our results are the first to characterize a mechanism by which c-Jun activates RNA polymerase II transcription in a cell-free system. In addition, we provide the first evidence that activators can directly bind the N terminus of TAFII250 to derepress transcription in vitro.

Derepression is emerging as a general mechanism for controlling levels of transcription in eukaryotic cells. This is not surprising since derepression is a prevalent mechanism for controlling gene expression in prokaryotes (43). The most widespread example of derepression in eukaryotes is thought to be the alteration of chromatin structure by histone acetyltransferases and ATP-dependent chromatin remodeling factors (3). In small nuclear RNA transcription the snRNA activating protein complex (SNAPc) has a built-in DNA binding damper that is deactivated by the transcriptional activator Oct-1 (44). Another general mechanism of transcriptional repression in eukaryotic cells results from the interaction of the N terminus of the largest TFIID subunit to the concave surface of TBP to prevent binding of TFIID to TATA boxes (23, 25). The finding that c-Jun binds the N terminus of TAFII250 to derepress TFIID-driven transcription in vitro further supports the generality of derepression as a means of regulating eukaryotic transcription. We are left wondering whether other transcriptional activators also bind the N terminus of TAFII250 to derepress transcription. We note that significant stimulation of transcription from the p(GAL4)5-AdMLP-G-less template (which lacks known AP-1 sites) required 400 ng or more of c-Jun. These amounts are far greater than the amounts of transcriptional activators typically used in in vitro transcription experiments. For example, we observe significant activation from p(AP-1)5-E1b-G-less with 15 ng of c-Jun. With this low amount of c-Jun we do not observe activation from any template that does not contain known AP-1 sites. Moreover, in early c-Jun activation experiments, we did not titrate c-Jun to high enough levels to observe AP-1 site-independent derepression. Perhaps other transcriptional activators that bind the N terminus of TAFII250 to derepress transcription will be revealed through a combination of protein-protein interaction assays (with activators and the N terminus of TAFII250) and in vitro transcription experiments aimed at testing for site-independent transcriptional stimulation in a TFIID-dependent transcription system.

Other models for derepression of TFIID binding have been studied. For example, it has been shown that VP16 and dTAFII230 competitively bind the concave surface of TBP (25). A point mutation in TBP (L114K) that disrupted the TBP-VP16 interaction prevented VP16 activation in vivo. The same point mutation in TBP also disrupted interaction with dTAFII230. In addition, when the N terminus of yTAFII145 was replaced with an acidic activation domain, the fusion protein autoinhibited TFIID in yeast (29). A two-step "hand-off" model has been proposed to explain the function of acidic activation domains in derepressing DNA binding by TFIID (25, 29). In the first step, the acidic activation domain replaces the N terminus of dTAFII230/yTAFII145 by binding the underside of TBP. In the second step, TBP binds to the TATA box when the acidic activation domain releases its hold on TBP. This proposed mechanism of relieving the internal repression of TFIID binding is quite different from the mechanism of derepression by c-Jun that we have characterized here; however, both modes of derepression serve to increase TFIID-driven transcription.

Since TFIIA has been shown to stimulate TFIID binding to the TATA box and to compete with the largest subunit of TFIID for binding TBP (24, 28), it is possible that transcriptional activators could indirectly derepress TAFII250 inhibition of TATA box binding by helping TFIIA bind TBP. Although TFIIA may play a role in c-Jun derepression, it is not required under all conditions. In our highly purified in vitro transcription system we found that c-Jun blocked the repression of transcription by GST-TAFII250-(1-163) in the absence of TFIIA. Furthermore, using DNase I footprinting we found that c-Jun blocked the ability of GST-TAFII250-(1-163) to repress TATA box binding by TBP. Thus, TFIIA and other general transcription factors are not required for the function of c-Jun in derepressing TBP binding in vitro.

TBP has also been shown to dimerize in vitro and in vivo either alone or when part of TFIID (45, 46). Dimers form through interactions between the concave surfaces of two molecules of TBP, resulting in a complex that is not capable of binding DNA. The coordination of TBP dimerization and the N terminus of TAFII250 interacting with TBP is not understood. Perhaps these two distinct mechanisms of repressing TFIID-DNA binding function together to ensure that the repressed state is maintained in the absence of interaction with TFIIA and transcriptional activators.

    ACKNOWLEDGEMENTS

We thank Natalie Ahn, Lin Chen, Jennifer Kugel, and Irene Ota for helpful discussions and comments on the manuscript. J. A. G. is grateful to Robert Tjian for generous support, especially during the early stages of this work.

    FOOTNOTES

* This research was supported by National Institutes of Health Public Health Service Grant GM-55235, a Leukemia Society of America Special Fellowship, and a Scholarship from the Pew Charitable Trusts (to J. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported in part by a National Institutes of Health Predoctoral Training Grant T32 GM07135 and a Graduate Student Fellowship from the Colorado Institute for Research in Biotechnology, which is state-funded by the Colorado Advanced Technology Institute.

§ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, University of Colorado, Campus Box 215, Boulder, CO 80309-0215. Tel.: 303-492-3273; Fax: 303-492-5894; E-mail: james.goodrich@colorado.edu.

Published, JBC Papers in Press, April 20, 2001, DOI 10.1074/jbc.M100278200

    ABBREVIATIONS

The abbreviations used are: TFII-, transcription factor for RNA polymerase II; TBP, TATA-binding protein; TAF, TBP-associated factor; AP-1, activator protein 1; VP16, herpesvirus protein 16; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; AdMLP, adenovirus major late promoter; y-, yeast; d-, Drosophila; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Albright, S. R., and Tjian, R. (2000) Gene 242, 1-13[CrossRef][Medline] [Order article via Infotrieve]
2. Malik, S., and Roeder, R. G. (2000) Trends Biochem. Sci. 25, 277-283[CrossRef][Medline] [Order article via Infotrieve]
3. Workman, J. L., and Kingston, R. E. (1998) Annu. Rev. Biochem. 67, 545-579[CrossRef][Medline] [Order article via Infotrieve]
4. Orphanides, G., Lagrange, T., and Reinberg, D. (1996) Genes Dev. 10, 2657-2683[CrossRef][Medline] [Order article via Infotrieve]
5. Carey, M. F. (1995) Curr. Biol. 5, 1003-1005[Medline] [Order article via Infotrieve]
6. Verrijzer, C. P., and Tjian, R. (1996) Trends Biochem. Sci. 21, 338-342[CrossRef][Medline] [Order article via Infotrieve]
7. Kaufmann, J., and Smale, S. T. (1994) Genes Dev. 8, 821-829[Abstract]
8. Weis, L., and Reinberg, D. (1992) FASEB J. 6, 3300-3309[Abstract/Free Full Text]
9. Verrijzer, C. P., Chen, J.-L., Yokomori, K., and Tjian, R. (1995) Cell 81, 1115-1125[Medline] [Order article via Infotrieve]
10. Burke, T. W., and Kadonaga, J. T. (1997) Genes Dev. 11, 3020-3031[Abstract/Free Full Text]
11. Wang, W., Gralla, J. D., and Carey, M. (1992) Genes Dev. 6, 1716-1727[Abstract]
12. Sauer, F., Hansen, S. K., and Tjian, R. (1995) Science 270, 1783-1788[Abstract]
13. Lieberman, P. M., and Berk, A. J. (1994) Genes Dev. 8, 995-1006[Abstract]
14. Horikoshi, M., Carey, M. F., Kakidani, H., and Roeder, R. G. (1988) Cell 54, 665-669[Medline] [Order article via Infotrieve]
15. Chi, T., and Carey, M. (1996) Genes Dev. 10, 2540-2550[Abstract]
16. Pugh, B. F., and Tjian, R. (1990) Cell 61, 1187-1197[Medline] [Order article via Infotrieve]
17. Dynlacht, B. D., Hoey, T., and Tjian, R. (1991) Cell 55, 563-576
18. Lee, T. I., Causton, H. C., Holstege, F. C., Shen, W. C., Hannett, N., Jennings, E. G., Winston, F., Green, M. R., and Young, R. A. (2000) Nature 405, 701-704[CrossRef][Medline] [Order article via Infotrieve]
19. Moqtaderi, Z., Bai, Y., Poon, D., Weil, P. A., and Struhl, K. (1996) Nature 383, 188-191[CrossRef][Medline] [Order article via Infotrieve]
20. Oelgeschlager, T., Tao, Y., Kang, Y. K., and Roeder, R. G. (1998) Mol. Cell 1, 925-931[Medline] [Order article via Infotrieve]
21. Walker, S. S., Reese, J. C., Apone, L. M., and Green, M. R. (1996) Nature 383, 185-188[CrossRef][Medline] [Order article via Infotrieve]
22. Wu, S. Y., Kershnar, E., and Chiang, C. M. (1998) EMBO J. 17, 4478-4490[Abstract/Free Full Text]
23. Kokubo, T., Gong, D.-W., Yamashita, S., Horikoshi, M., Roeder, R. G., and Nakatani, Y. (1993) Genes Dev. 7, 1033-1046[Abstract]
24. Kokubo, T., Swanson, M. J., Nishikawa, J. I., Hinnebusch, A. G., and Nakatani, Y. (1998) Mol. Cell. Biol. 18, 1003-1012[Abstract/Free Full Text]
25. Nishikawa, J., Kokubo, T., Horikoshi, M., Roeder, R. G., and Nakatani, Y. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 85-90[Abstract/Free Full Text]
26. Liu, D., Ishima, R., Tong, K. I., Bagby, S., Kokubo, T., Muhandiram, D. R., Kay, L. E., Nakatani, Y., and Ikura, M. (1998) Cell 94, 573-583[Medline] [Order article via Infotrieve]
27. Kotani, T., Miyake, T., Tsukihashi, Y., Hinnebusch, A. G., Nakatani, Y., Kawaichi, M., and Kokubo, T. (1998) J. Biol. Chem. 273, 32254-32264[Abstract/Free Full Text]
28. Ozer, J., Mitsouras, K., Zerby, D., Carey, M., and Lieberman, P. M. (1998) J. Biol. Chem. 273, 14293-14300[Abstract/Free Full Text]
29. Kotani, T., Banno, K., Ikura, M., Hinnebusch, A. G., Nakatani, Y., Kawaichi, M., and Kokubo, T. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 7178-7183[Abstract/Free Full Text]
30. Karin, M., Liu, Z., and Zandi, E. (1997) Curr. Opin. Cell Biol. 9, 240-246[CrossRef][Medline] [Order article via Infotrieve]
31. Turner, R., and Tjian, R. (1989) Science 243, 1689-1694[Medline] [Order article via Infotrieve]
32. Glover, J. N., and Harrison, S. C. (1995) Nature 373, 257-261[CrossRef][Medline] [Order article via Infotrieve]
33. Abate, C., Luk, D., Gagne, E., Roeder, R. G., and Curran, T. (1990) Mol. Cell. Biol. 10, 5532-5535[Medline] [Order article via Infotrieve]
34. Bohmann, D., and Tjian, R. (1989) Cell 59, 709-717[Medline] [Order article via Infotrieve]
35. Sutherland, J. A., Cook, A., Bannister, A. J., and Kouzarides, T. (1992) Genes Dev. 6, 1810-1819[Abstract]
36. Lee, J. S., See, R. H., Deng, T., and Shi, Y. (1996) Mol. Cell. Biol. 16, 4312-4326[Abstract]
37. Chen, J.-L., Attardi, L. D., Verrijzer, C. P., Yokomori, K., and Tjian, R. (1994) Cell 79, 93-105[Medline] [Order article via Infotrieve]
38. Lillie, J. W., and Green, M. R. (1989) Nature 338, 39-44[CrossRef][Medline] [Order article via Infotrieve]
39. Galasinski, S. K., Lively, T. N., Grebe de Barron, A., and Goodrich, J. A. (2000) Mol. Cell. Biol. 20, 1923-1930[Abstract/Free Full Text]
40. Kugel, J. F., and Goodrich, J. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9232-9237[Abstract/Free Full Text]
41. Ozer, J., Moore, P. A., Bolden, A. H., Lee, A., Rosen, C. A., and Lieberman, P. M. (1994) Genes Dev. 8, 2324-2335[Abstract]
42. Sun, X., Ma, D., Sheldon, M., Yeung, K., and Reinberg, D. (1994) Genes Dev. 8, 2336-2348[Abstract]
43. Struhl, K. (1999) Cell 98, 1-4[Medline] [Order article via Infotrieve]
44. Mittal, V., Ma, B., and Hernandez, N. (1999) Genes Dev. 13, 1807-1821[Abstract/Free Full Text]
45. Coleman, R. A., and Pugh, B. F. (1995) J. Biol. Chem. 270, 13850-13859[Abstract/Free Full Text]
46. Coleman, R. A., Taggart, A. K. P., Benjamin, L. R., and Pugh, B. F. (1995) J. Biol. Chem. 270, 13842-13849[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.