©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
cAMP Response Element-binding Protein (CREB) Interacts with Transcription Factors IIB and IID (*)

(Received for publication, October 27, 1994; and in revised form, April 5, 1995)

Lianping Xing , Venkatesh K. Gopal , Patrick G. Quinn (1)(§)

From the Department of Cellular and Molecular Physiology and the Program in Cell and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

cAMP response element-binding protein (CREB) participates in both constitutive and cAMP-induced transcription of cAMP-responsive genes. CREB-mediated constitutive transcription requires only CREB-binding sites and a minimal promoter region (containing the TATA through start sequences), indicating that CREB interacts directly with components of the general transcription machinery. In this study, a coimmunoprecipitation assay was used to test for interaction of CREB with the general transcription factors (TF) TFIIB and TFIID and the core component of TFIID, TATA-binding protein (TBP). Human TFIIB and TBP, tagged with distinct epitopes (eTFIIB and eTBP), were expressed in and purified from Escherichia coli, and holo-eTFIID, containing eTBP, was obtained from the HeLa cell line LTR3. S-Labeled CREB, synthesized in vitro and incubated with eTFIIB, was coimmunoprecipitated with antibody recognizing eTFIIB, indicating that CREB specifically binds to TFIIB. S-CREB was coimmunoprecipitated with antibody against eTBP, but only when incubated with the holo-eTFIID complex, not with eTBP alone. TFIIB interacted with TBP, but CREB was not coprecipitated with the eTBP antibody when incubated with eTBP plus TFIIB, so CREB did not form a stable ternary complex with TFIIB and TBP. Conversely, depletion of TFIIB from the holo-TFIID preparation did not diminish the level of interaction between CREB and TFIID. Thus, CREB interacts independently with TFIIB and TFIID, but not directly with TBP. A protein kinase A phosphorylation site mutant of CREB and wild-type CREB exhibited equivalent interaction with TFIIB, indicating that this phosphorylation is not required. Consistent with the role of CREB in promoting constitutive or basal transcription, the constitutive activation domain of CREB was sufficient for interaction with both TFIIB and TFIID.


INTRODUCTION

cAMP response element-binding protein (CREB)()is a transcription activator that binds to the cAMP response element within a variety of cAMP-responsive promoters to regulate gene expression(1, 2, 3) . Hormone-induced accumulation of cAMP activates protein kinase A (PKA), which phosphorylates CREB at serine 133 and further enhances transcription activation. Mutation of the cAMP response element reduces cAMP-mediated transcription and diminishes constitutive transcription as well, indicating that the cAMP response element functions both as an inducible enhancer and a general promoter element(4) . A protein consisting of the CREB activation domain fused to the GAL4 DNA-binding domain (CRG) can restore both constitutive and PKA-inducible transcription to a promoter containing a GAL4 site in place of the cAMP response element, and the constitutive activity of CRG is unaffected by mutation of the PKA phosphorylation site or by inhibition of PKA activity by PKI(5, 6) .

CREB contains independent domains that mediate constitutive and kinase-inducible transcription(5) . The constitutive activation domain (CAD) of CREB maps to the C-terminal portion of the protein (amino acids 165-255). Deletion of the CREB CAD abolishes constitutive transcription without impairing inducibility by PKA, and fusion of the CAD to the GAL4 DNA-binding domain restores constitutive activity to the level observed for wild-type CRG. Expression of either the constitutive or PKA-inducible CREB activities required only GAL4 sites to bind CRG ligated to a minimal promoter encompassing the TATA footprint through the transcription start site of either the phosphoenolpyruvate carboxykinase or E1b gene(5) . This result indicates that CREB need not interact with other promoter-bound regulators outside of the TATA-based initiation complex. Together, these data suggest that the CREB CAD stimulates basal or constitutive transcription through interaction with one or more general transcription factors bound to the minimal promoter at the TATA box.

The general transcription factors are defined as those required for accurate initiation of in vitro transcription and are named TFIIA, TFIIB, etc. based on their chromatographic properties (7, 8, 9) . Regulatory factors, such as CREB, bound to upstream sites in the promoter are thought to stimulate transcription through interactions with the general transcription factors, assembled with RNA polymerase II into a preinitiation complex at the start site of transcription(9, 10, 11) . ATF/CREB family members qualitatively alter the binding of TFIID and associated proteins to a target promoter, facilitating subsequent interaction with TFIIB and other general transcription factors and increasing protection downstream of the TATA element(12, 13) . The activation domains of both VP16 (14) and the E1A protein (15, 16) interact directly with TBP. In addition, the VP16 activation domain (14) and others (14, 17, 18) interact directly with TFIIB. VP16 also has been shown to play a role in recruiting TFIIB to the initiation complex(19, 20) .

The minimal factor requirement to support transcription from a promoter is (i) TBP, (ii) TFIIB, and (iii) the RNA polymerase II-TFIIF complex (9, 21) . In at least one case, the initiator recognizing element YY1 can substitute for TBP and requires only TFIIB and polymerase II for basal transcription(22) . Moreover, the TFIIF requirement has been further refined to only the RAP30 component of TFIIF(23) . However, many transcription activators require TFIID rather than TBP to mediate regulated, as opposed to basal, transcription(10, 24, 25) . Given that CREB interacts with general transcription factors to promote constitutive activation and that TBP/TFIID and TFIIB are indispensable for RNA polymerase II-mediated transcription, we tested the ability of CREB to interact with TBP, TFIID, and TFIIB in this study. To determine which targets or combinations of targets would interact with CREB in a coimmunoprecipitation assay, we used TBP and TFIIB tagged with distinct epitopes and the eTFIID fraction from a HeLa cell line (LTR3) expressing eTBP. We show here that CREB interacts directly with TFIIB and TFIID, but not with TBP.


MATERIALS AND METHODS

Plasmids

pRT-CR and its mutants have been described elsewhere(5) . For in vitro transcription-translation, the wild-type CREB cDNA or CREB S133A, in which the PKA phosphorylation site is mutated, was cloned into pET3d (Novagen) between the NcoI and BamHI sites, creating pET-CR and pET-CR-S133A, respectively. The pET-CAD vector contains amino acids 1-8 of CREB (to provide peptide initiation) fused to amino acids 165-243, encoding the CAD, followed by the 6-amino acid linker region of CRG and an in-frame stop codon.

Human eTFIIB

The human TFIIB expression plasmid was obtained from Dr. Kent W. Wilcox (Medical College of Wisconsin, Milwaukee, WI). This plasmid vector contains a T7 phage gene 10 epitope tag and a 10-histidine tag at the N terminus of the human TFIIB cDNA. Recombinant eTFIIB was purified from Escherichia coli (BL21(DE3)) as follows. eTFIIB-transformed cells were grown at 37 °C in super broth containing 200 µg/ml ampicillin until A = 0.6 was reached. Isopropyl-1-thio--D-galactopyranoside was added to a final concentration of 0.4 mM, and the cells were grown at 30 °C for an additional 3 h. Cell lysates from 50 ml of culture were incubated for 1 h at 4 °C with 1 ml of Ni-agarose resin (QIAGEN, Inc.) in 2 ml of buffer A (20 mM Tris-HCl (pH 7.9), 0.1 M KCl, 20% glycerol, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride) containing 1 M ammonium sulfate. The matrix was washed with 10 column volumes of buffer A containing 25 mM imidazole and with 10 column volumes of buffer A containing 37.3 mM imidazole. Bound eTFIIB was eluted in 2 column volumes of the same buffer containing 125 mM imidazole(26) .

Human eTBP

A human eTBP expression plasmid, containing an epitope of 9 amino acids from the hemagglutinin antigen of influenza virus fused in frame to the amino terminus of human TBP, was a generous gift of Dr. A. Berk (UCLA)(15) . pET-eTBP-transformed cells (BL21(DE3)) were grown at 37 °C in ZY medium (1% Bactotryptone, 0.5% yeast extract, 0.086 M NaCl) containing 0.4% glucose in the presence of 100 µg of ampicillin/ml until A = 0.8 was reached. Isopropyl-1-thio--D-galactopyranoside was added to a final concentration of 0.5 mM, and the cells were harvested 3 h later. Fractionation of the cell lysate was performed essentially as described previously(27, 28) .

Holo-eTFIID

The eTFIID fraction of the human eTBP-expressing LTR3 cell line was isolated by chromatography on phosphocellulose as described previously(24) . The LTR3 cell line, in which 80% of the TBP is present as eTBP, was a generous gift of Dr. A. Berk. Nuclear extracts were prepared by lysis of the cells with 0.5% Nonidet P-40, collection of the nuclei, and extraction of the nuclei with 420 mM NaCl as described by Hurst et al.(29) . TFIIB-depleted LTR3 nuclear extracts were prepared by incubation of nuclear extract with a polyclonal anti-TFIIB antibody for 1 h at 4 °C, followed by addition of protein A/G-agarose beads and further incubation for 1 h at 4 °C. The beads were collected by centrifugation to remove antibody-bound TFIIB from the extracts, and the supernatant nuclear extract was used for interaction assays. Complete and TFIIB-depleted nuclear extracts were analyzed for TFIIB by Western blotting with primary antibody against TFIIB and secondary antibody coupled to horseradish peroxidase. Blots were developed with chemiluminescence reagents (DuPont NEN).

Coimmunoprecipitation

S-Labeled proteins were synthesized in a coupled in vitro transcription-translation system using rabbit reticulocyte lysate (Promega) in the presence of [S]methionine. Equimolar amounts of S-labeled in vitro translated CREB or mutated CREB protein were mixed with 10 pmol of eTFIIB in 125 µl of buffer D (20 mM Hepes (pH 7.9), 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM, phenylmethylsulfonyl fluoride) containing 0.1 M KCl and incubated at 4 °C for 1 h. 2 µg of monoclonal antibody recognizing the T7 Tag epitope on eTFIIB was added, and the mixture was incubated at 4 °C for 1.5 h. 20 µl of a 50% slurry of protein A/G-agarose beads was added and mixed for 1 h at 4 °C. Protein complexes immobilized on protein A/G beads were washed three times with buffer D and suspended in 1 Laemmli sample buffer. One-half of the bound fraction of each sample was loaded onto 15% SDS-polyacrylamide gels. One-sixtieth of the original sample (input) or the supernatant (free) was included as indicated. S-Labeled proteins were visualized by autoradiography.


RESULTS

Lack of Interaction of CREB with TBP

The observation of Zhou et al.(24) that E1A interacts directly with TBP prompted us to investigate the possibility that CREB may interact directly with TBP. CREB, E1A, and luciferase proteins were synthesized in vitro and examined, together with a control lysate, for binding to eTBP. As in the studies of Zhou et al., E1A interacted strongly with eTBP, and luciferase did not (Fig. 1). Control lysate, to which no template was added, did not synthesize any significant amount of protein, and none bound to eTBP. CREB protein was synthesized, but did not bind to eTBP, indicating that there is no direct interaction between CREB and TBP.


Figure 1: Assay for interaction of TBP with E1A, luciferase, and CREB. Plasmids containing cDNAs for E1A, luciferase (LUC), nothing, or CREB were used to program a coupled in vitro transcription-translation system. Equimolar amounts of S-labeled proteins were incubated with eTBP in buffer D for 1 h at 4 °C and then incubated with antibody against the HA epitope of eTBP (12CA5) for 1.5 h at 4 °C. Protein A/G-agarose was used to collect the immunoprecipitates, which were washed three times with binding buffer. Fractions of the input (I; one-sixtieth), free (F; one-sixtieth), and bound (B; one-half) proteins were resolved by SDS-PAGE and visualized by autoradiography.



Interaction of CREB with TFIIB

We next examined the possibility that CREB interacted directly with the general transcription factor TFIIB. S-Labeled in vitro translated CREB was incubated without or with eTFIIB protein and then with a monoclonal antibody that recognizes the T7 Tag epitope on eTFIIB (Fig. 2). No binding was observed in the absence of eTFIIB. In the presence of eTFIIB, CREB was coimmunoprecipitated with the T7 Tag antibody recognizing eTFIIB. Thus, CREB formed a stable complex with the TFIIB protein that allowed it to be precipitated with antibody recognizing the epitope on TFIIB.


Figure 2: Interaction assay for CREB with eTFIIB. [S]Methionine-labeled CREB was incubated in the absence (firstfourlanes) or presence (last four lanes) of 10 pmol of eTFIIB in buffer D for 1 h at 4 °C and immunoprecipitated with a monoclonal antibody (Ab) directed against the T7 Tag on the eTFIIB protein. The protein-antibody complex was isolated with protein A/G-agarose beads and washed three times with binding buffer. Fractions of the input (I), free (F), wash (W), and bound (B) proteins were resolved by SDS-PAGE and visualized by autoradiography. First and fifthlanes, one-twentieth of the input; second and sixthlanes, one-tenth of the free CREB in the supernatant; third and seventhlanes, one-thousandth of the wash; fourth and eighthlanes, one-half of the bound protein.



Interaction of CREB with TFIID

The majority of TBP is found in complexes with TATA-associated factors (TAFs) in eukaryotic cells (10, 24) . To determine whether CREB might interact with TBP indirectly through TAFs, rather than directly with TBP, we tested the interaction of CREB with a holo-eTFIID fraction, the eTBP upon which it is based, and eTFIIB. The eTFIID fraction was purified from LTR3 cells, in which 80% of the TBP present is eTBP(24) . As shown above, CREB was coimmunoprecipitated with antibody recognizing eTFIIB (Fig. 3A). Antibody recognizing eTBP precipitated CREB incubated with eTFIID, but not with eTBP alone. The composition of the eTFIID fraction precipitated from LTR3 cells with the 12CA5 antibody is shown in Fig. 3B. This silver-stained gel shows that, in addition to eTBP (39 kDa), bands corresponding to TAFs of 250, 125, 78, and 50 kDa and faint bands at 95 and 30/28 kDa are coimmunoprecipitated from LTR3 cells. The band expected at 70 kDa is obscured by residual carrier bovine serum albumin. These coimmunoprecipitated proteins correspond to the TAFs identified in LTR3 cells by Zhou et al.(24) . Thus, our data suggest that CREB associates with TBP indirectly through interactions with one or more TAFs, rather than directly interacting with TBP.


Figure 3: Interaction of CREB with holo-eTFIID. A, [S]methionine-labeled CREB was incubated with eTFIIB, eTBP, or holo-eTFIID (fractionated from the nuclear extracts of LTR3 cells stably expressing eTBP) in buffer D for 1 h at 4 °C and immunoprecipitated with a monoclonal antibody (Ab) directed against the T7 Tag on eTFIIB (first and secondlanes) or the HA epitope tag on eTBP/eTFIID (third through sixthlanes). Antibody-bound protein complexes were isolated on protein A/G-agarose beads and washed. The input (I; one-sixtieth of the starting reaction) and bound (B; one-half of the bound protein) CREB fractions were resolved by SDS-PAGE and visualized by autoradiography. B, nuclear extracts were prepared from LTR3 cells expressing eTBP. The TFIID-containing fraction was obtained by phosphocellulose chromatography and subjected to immunoprecipitation with monoclonal antibody against the HA epitope on eTBP under the same conditions used for interaction assays. The immunoprecipitates were resolved by SDS-PAGE, and proteins were visualized by silver staining. The asterisks indicate gel artifacts observed in all lanes. BSA, bovine serum albumin.



TFIIB Does Not Permit Interaction of CREB with TBP

TFIIB has been shown to interact with TBP(9, 30) . Therefore, the TBPTFIIB complex is a potential target for interaction with CREB. To determine whether TFIIB can interact with both CREB and TBP and thus recruit TBP to a complex with CREB, S-labeled CREB was incubated with TFIIB and eTBP, alone or in combination. CREB was immunoprecipitated when incubated with eTFIIB, but not with TBP (Fig. 4A), as observed above (Fig. 3). Preincubation of eTBP with an excess of TFIIB still did not permit immunoprecipitation of CREB with antibody against eTBP (Fig. 4A), indicating that the TBPTFIIB complex is not a strong target for CREB interaction. To verify that eTFIIB interacted stably with eTBP under our experimental conditions, S-labeled eTBP was incubated with eTFIIB and precipitated with antibody against eTFIIB. Interaction between eTBP and eTFIIB was observed (Fig. 4B) and was more pronounced than that seen between CREB and TFIIB. A similar degree of interaction was observed when eTBP precipitates were electrophoresed and probed with the TFIIB antibody (data not shown). Thus, TFIIB and TBP interact under these conditions, but the TBPTFIIB complex does not bind CREB.


Figure 4: Effect of addition of TFIIB on CREB binding to eTBP. A, S-labeled CREB was incubated with the indicated target proteins, eTFIIB and eTBP, alone or together, and then immunoprecipitated with antibody (Ab) against the appropriate epitopes, indicated at the top. Protein A/G-agarose was used to collect the immunoprecipitates, which were washed three times with binding buffer. Fractions of the input (I; one-sixtieth) and bound (B; one-half) proteins were resolved by SDS-PAGE and visualized by autoradiography. B, a cDNA encoding eTBP was used to program in vitro transcription-translation, and S-labeled eTBP was incubated with eTFIIB in buffer D for 1 h at 4 °C and then immunoprecipitated with antibody recognizing the T7 Tag epitope on eTFIIB. Protein A/G-agarose was used to collect the immunoprecipitates, which were washed three times with binding buffer. Fractions of the input (one-sixtieth) and bound (one-half) proteins were resolved by SDS-PAGE and visualized by autoradiography.



CREB Interacts with TFIID Immunodepleted of TFIIB

It is possible that the interaction observed between the TFIID fraction and CREB was due to association of TFIIB with eTFIID. The TFIID fraction used in the above studies contained readily detectable TFIIB, as judged by Western blotting with antibody recognizing native TFIIB (data not shown). To determine whether TFIIB influenced the interaction of CREB with TFIID, we prepared nuclear lysates containing eTFIID from LTR3 cells, in which 80% of the TBP is eTBP. Half of the extract was depleted of TFIIB by incubation with antibody recognizing native TFIIB and protein A/G-agarose beads and by removal of the beads by centrifugation. Both the complete nuclear extract and the TFIIB-depleted nuclear extract were examined for interaction with CREB (Fig. 5A, NE and NE-TFIIB, respectively). Depletion of TFIIB did not alter CREB interaction with eTFIID in the nuclear extract, indicating that TFIIB is not required for interaction between CREB and TFIID. Complete and immunodepleted nuclear extracts (Fig. 5B, NE and NE-TFIIB, respectively) were subjected to Western blotting with a polyclonal TFIIB antibody, and the bands were detected by enhanced chemiluminescence. Immunodepletion appeared to have been effective (<0.3 fmol). We estimate that 0.3-0.6 fmol of CREB is bound to TFIIB or TFIID in these assays, based on 3% incorporation of 10-20 fmol of input protein. Therefore, it is unlikely that residual TFIIB is responsible for the interaction of CREB with eTFIID.


Figure 5: Effect of immunodepletion of TFIIB on CREB interaction with TFIID. S-Labeled CREB was incubated with nuclear extract from LTR3 cells (NE) or with LTR3 nuclear extract immunodepleted of TFIIB (NE-TFIIB) and then with antibody (Ab) against the HA epitope in eTFIID. Protein A/G-agarose was used to collect the immunoprecipitates, which were washed three times with binding buffer. Fractions of the input (I; one-sixtieth) and bound (B; one-half) proteins were resolved by SDS-PAGE and visualized by autoradiography. B, nuclear extracts from LTR3 cells were treated with protein A/G-agarose alone (left lane) or with a polyclonal anti-TFIIB antibody plus protein A/G-agarose (right lane). Following removal of the agarose beads, proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with a polyclonal TFIIB antibody.



Effect of Phosphorylation of CREB by PKA on Its Interaction with TFIIB or TFIID

Transcription activation by CREB is enhanced by cAMP-dependent PKA-mediated phosphorylation of Ser-133, and reticulocyte lysates are known to have endogenous PKA(31) . To determine whether phosphorylation of CREB by PKA in the lysate contributed to the interaction of CREB with TFIIB or TFIID, we used a PKA site mutant of CREB, CREB S133A, in which Ser-133 is changed to alanine. This mutation completely abolishes phosphorylation by PKA (2, 32) and PKA inducibility in transfection assays, but has no effect on constitutive transcription activity in these assays(5, 6) . Mutation of the CREB phosphorylation site in CREB S133A had no effect on its ability to interact with TFIIB (Fig. 6) or TFIID (data not shown) in vitro in the coimmunoprecipitation assay. The lack of effect of mutation of the PKA phosphorylation site demonstrates that phosphorylation by PKA is not crucial for the interaction of CREB with either eTFIIB or holo-eTFIID.


Figure 6: Effect of mutation of the PKA phosphorylation site in CREB on interaction with eTFIIB or holo-eTFIID. CREB S133A contains alanine in place of serine at the PKA phosphorylation site. S-Labeled wild-type (WT) CREB or mutant CREB S133A was incubated with eTFIIB, immunoprecipitated with T7 Tag antibody and protein A/G-agarose beads, and washed three times in binding buffer. Fractions of the input (I; one-sixtieth) and bound (B; one-half) proteins were resolved by SDS-PAGE and visualized by autoradiography.



Binding of the CAD of CREB to TFIIB and TFIID

Constitutive activation and kinase-inducible activation by CREB are mediated by independent domains in the protein. The CAD located between amino acids 165 and 243 of CREB provides the majority of constitutive activity(5, 33, 34) . The CAD was fused to the 8 amino-terminal amino acids of CREB to permit initiation of protein synthesis. The S-labeled CREB CAD protein was incubated with TFIIB and TFIID to determine whether the CREB CAD can mediate binding to these proteins. The CREB CAD bound to both TFIIB and TFIID (Fig. 7), indicating that the CAD is sufficient for interaction with these general transcription factors, as is consistent with the role of the CAD in promoting transcription activation in the absence of phosphorylation of CREB by PKA.


Figure 7: Interaction of the CREB constitutive activation domain with TFIIB and TFIID. The CREB CAD vector encodes the constitutive activation domain of CREB (amino acids 165-252) fused to the 8 amino-terminal residues in CREB that provide peptide initiation. The S-labeled CREB CAD was incubated with eTFIIB or eTFID and then immunoprecipitated with antibody directed against either the T7 Tag (for eTFIIB) or the HA epitope (for eTFIID) and protein A/G-agarose beads and washed three times in binding buffer. Fractions of the input (I; one-sixtieth) and bound (B; one-half) proteins were resolved by SDS-PAGE and visualized by autoradiography.




DISCUSSION

We previously demonstrated that CREB mediates both constitutive and kinase-inducible activities through distinct, independently acting domains(5) . Given that these activities of CREB require only binding sites for CREB and minimal promoters containing the TATA region and start site, we suggested that CREB interacts directly with one or more components of the general transcription machinery. Others have shown that the general transcription factors TBP/TFIID (or YY1), TFIIB, and RNA polymerase II/RAP30 are sufficient to initiate in vitro transcription(9, 21, 22, 23) . We now show that CREB interacts directly with both TFIIB and TFIID, but not with TBP. The CREB-TFIID interaction is presumably mediated through one or more TAFs in the TFIID complex. CREB interacts independently with TFIIB and TFIID. These interactions are not affected by mutation of the PKA phosphorylation site in CREB. Finally, the constitutive activation domain of CREB is sufficient for interaction with either TFIIB or TFIID.

The first step in the assembly of the preinitiation complex is the binding of TFIID to the TATA element(35, 36) . TFIID is composed of TBP and TAFs(37, 38) . TBP alone is capable of binding to the TATA sequence and mediating basal transcription in the absence of regulators(9) . However, TBP alone cannot mediate activated transcription regulated by SP1 and other factors(11, 25, 39, 40, 41) . Regulated transcription requires the TAFs in the TFIID fraction in addition to TBP(25, 40, 41) . Different activators interact with different TAFs of the multisubunit TFIID complex, contributing to the specificity of gene regulation(41, 42) . We show here that CREB does not interact with TBP, but that CREB does interact with holo-TFIID. The interaction between CREB and TFIID does not require TFIIB and can be mediated by the constitutive activation domain alone. Although we cannot rigorously exclude a requirement for other factors associated with TFIID, our data are consistent with a requirement for the TAFs to mediate CREB interaction with TFIID. While this study was in its final stages, Ferreri et al.(43) also showed that CREB does not interact directly with TBP and provided evidence for an interaction between CREB and TAF110 mediated by the CREB CAD. Our data are consistent with theirs, but neither set of data addresses the possibility that CREB interacts with more than one TAF to form a stable complex with TFIID. In the study of Ferreri et al., only TAF110 was examined, whereas in this study, the holo-TFIID fraction used consisted of many different TAFs associated with TBP. Thus, the possibility that more than one TAF or other coactivators are involved in CREB binding to TFIID cannot be ignored.

Following the binding of TBP or TFIID to the TATA box, other general transcription factors associate with TFIID to form a preinitiation complex(9, 44) . The most crucial of these appears to be TFIIB because TBP, TFIIB, and RNA polymerase II are the minimal factors required to initiate transcription in vitro(9, 21) . TFIIB interacts with several transcription regulatory proteins (14, 17, 18) and with the RNA polymerase II-TFIIF complex(44) . The most thoroughly studied activator, herpes simplex virus transcription factor VP16, has been shown to directly bind to TFIIB and to increase its stable assembly into a preinitiation complex(19, 20) . We show here that CREB can interact directly with recombinant TFIIB, purified essentially to homogeneity from bacterial extracts. This interaction is independent of phosphorylation of the PKA phosphorylation site (Ser-133) in CREB and can be mediated by the constitutive activation domain alone. It is not clear at present whether CREB can affect recruitment of TFIIB to the promoter. It was demonstrated recently that an adaptor, CBP, specifically binds to CREB phosphorylated at Ser-133 (45) and is required for cAMP-induced gene transcription, at least in some cells (46) . CBP augments the effects of CREB phosphorylation and also binds to TFIIB(47) . Thus, CREB may interact with this crucial general transcription factor (TFIIB) directly to mediate constitutive transcription and indirectly through CBP to mediate kinase-induced transcription.

Some activators interact with both TFIIB and TBP or TFIID(18, 30) , and both the TFIIB and TBP proteins interact with VP16 through the same interaction domain(19, 48) . In addition, direct association of TBP with TFIIB has been demonstrated(9, 30) . Factors bound to the ATF-binding site have been shown to extend the footprint formed over the TATA box to include the initiation site and downstream sequences (12) . In addition, ATF-bound factors enhance the recruitment of TFIIB, TFIIE, and RNA polymerase II to the preinitiation complex(13) . The nature of the ATF-bound factors has not been characterized, but they presumably include CREB. Given that TBP and TFIID interact with each other and that CREB interacts with TFIIB but not TBP, we considered the possibility that a stable ternary complex (CREBTFIIBTBP) might be formed, allowing the immunoprecipitation of CREB by antibody recognizing epitope-tagged TBP in the presence of TFIIB. We found no evidence for such a complex. Although both CREB and TBP interact with TFIIB, they do not appear to form a stable ternary complex, at least under our experimental conditions. One possibility is that CREB and TBP have similar or overlapping recognition sites on TFIIB such that binding is mutually exclusive. In that case, CREB might interact with TFIIB and recruit it to the initiation complex, after which TFIIB would form a stable complex with TBP to facilitate transcription initiation. Consistent with this notion is the demonstration by Hai et al.(13) that ATF factors facilitate formation of the initiation complex, but are not required for maintenance of this complex. Thus, CREB may play a role in recruiting TFIIB to the initiation complex. Studies to determine whether CREB can recruit TFIIB are underway in our laboratory.

In summary, CREB specifically binds to both TFIID and TFIIB. These interactions are independent of kinase-induced phosphorylation of Ser-133 in CREB, and the pairwise interactions between CREB and TFIIB and TFIID occur independently of the third protein. We have not examined directly whether CREB can affect recruitment of TFIIB to the promoter, but this is likely in light of data in other experimental systems. Thus, CREB may promote basal transcription initiation both through direct interaction with the TFIID complex and by recruiting TFIIB to the promoter. The exact nature of the contacts between CREB and these targets remains to be elucidated, both in terms of which TAFs and other cofactors are involved and which domains in CREB are responsible for interaction with different components of the basal transcription complex.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant DK 43871 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 717-531-6182; Fax: 717-531-7667; pquinn{at}cmp.hmc.psu.edu

The abbreviations used are: CREB, cAMP response element-binding protein; PKA, protein kinase A; CAD, constitutive activation domain; TF, transcription factor; eTFIID, epitope-tagged TFIID; TBP, TATA-binding protein; eTBP, epitope-tagged TBP; TAFs, TATA-associated factors; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin; CRG, CREB activation domain-GAL4 DNA-binding domain; ATF, activating transcription factor.


ACKNOWLEDGEMENTS

We thank Lorraine Altland for excellent technical assistance, Drs. J. Hopper and D. Spector for critical discussions of the work, A. Berk for providing expression vectors for eTBP and E1A and the HeLa and LTR3 cell lines, K. W. Wilcox for the eTFIIB expression vector, R. Maurer for providing the PKAc expression plasmid, and M. Ptashne for the antibody to the GAL4 DNA-binding domain.


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