The CREB Constitutive Activation Domain Interacts with TATA-binding Protein-associated Factor 110 (TAF110) through Specific Hydrophobic Residues in One of the Three Subdomains Required for Both Activation and TAF110 Binding*

Edward A. Felinski and Patrick G. QuinnDagger

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cAMP response element-binding protein (CREB) mediates both basal and PKA-inducible transcription through two separate and independently active domains, the constitutive activation domain (CAD) and the kinase-inducible domain, respectively. The CREB CAD interacts with the general transcription factor TFIID through one or more of the TATA-binding protein-associated factors (TAFs), one of which is TAF110. The CAD is composed of three subdomains, rich in either serine, hydrophobic amino acids, or glutamine. In the present study, analysis of deletion mutants of the CAD showed that all three CAD subdomains were required for effective interaction with TAF110 in a yeast two-hybrid assay. Therefore, a library of random point mutations within the CAD was analyzed in a reverse two-hybrid screen to identify amino acids that are essential for interaction with the TAF. Interaction defects resulted solely from mutations of hydrophobic amino acid residues within the hydrophobic cluster to charged amino acid residues. Together, the deletion and mutation analyses suggest that the entire CAD provides an environment for a specific hydrophobic interaction with TAF110 that is crucial for interaction. Our results provide further evidence for a model of basal activation by CREB involving interaction with TAF110 that promotes recruitment or stabilization of TFIID binding to the promoter, which facilitates pre-initiation complex assembly.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cAMP response element is an enhancer that mediates both basal and cAMP-induced transcriptional activation of a variety of genes in many cell types (1-6). cAMP response element-binding protein (CREB)1 binds constitutively as a dimer to CREs in the promoter of the target gene and activates basal transcription even in the absence of hormonal stimuli (3). Extracellular stimuli that activate protein kinases lead to phosphorylation of CREB on serine 133 by activated PKA, resulting in further enhancement of transcriptional activation (7, 8). Mutation of the serine 133 phosphorylation site in CREB abolishes kinase inducible activation (7, 9) but leaves basal activation unaltered (3, 10). In addition, previous work showed that CREB contains two separate and independently acting transcriptional activation domains: a constitutive activation domain (CAD) responsible for basal activation and a kinase-inducible domain that mediates activation in response to cAMP-activated PKA (3, 11). When the CREB CAD is fused to the DNA-binding domain (DBD) of the yeast Gal4 protein, it activates basal transcription from a Gal4 promoter nearly as well as the complete unphosphorylated CREB (3, 11). Activation by CREB kinase-inducible domain (KID) is proposed to be mediated by interaction with CREB-binding protein, which, in turn, interacts with polymerase complex components (12, 13). The CAD is composed of three subdomains with distinct amino acid compositions; a serine/threonine rich region (ST3), a hydrophobic cluster (HC), and a glutamine-rich region (Q3). Deletion of any of these regions results in a substantial loss of basal activation by the CAD (14). It is not clear whether these subdomains target a single factor or interact with different targets in the preinitiation complex (PIC). We also showed that the CREB CAD binds TFIID through interaction with one or more TAFs, rather than directly with the TATA-binding protein (TBP) (15). Subsequently, Ferreri et al. (17) showed that dTAF110, the interaction of which with the glutamine-rich/hydrophobic domain of Sp1 had been extensively characterized (16), also interacts with CREB.

Transcription of a protein-coding gene by RNA polymerase II requires the assembly of a large complex of factors around the transcriptional start site in the promoter of the gene. The characterization of these general transcription factors (GTFs) has been an ongoing, ambitious project in many laboratories and has lead to two basic models for basal transcriptional activation (18, 19). In the first model, factors assemble in a stepwise fashion (TFIID, TFIIB, TFIIA, TFIIF-polII, TFIIE, and TFIIH) into a functional PIC with the ultimate goal of positioning the polymerase complex at the proper start site and allowing synthesis of an mRNA transcript to begin (20). The specific order of addition in this model is based on in vitro transcription assays and template binding assays with purified GTFs (18, 21-23). A second model is based on recent in vivo work that suggests that the majority of the GTFs, except TFIID (and perhaps TFIIB), exist in the cell as large preformed complexes termed holoenzymes (19) and that holoenzymes may be recruited en bloc to the promoter (24, 25). Many activators have been shown to interact directly with one or more of the GTFs, and this interaction is currently thought to be a major mechanism for gene activation. Importantly, for either the stepwise addition or holoenzyme models, TFIID is proposed to be an obligatory first step in PIC formation.

TFIID consists of the TBP and a large group of TBP-associated factors or TAFs, ranging in size from 15 to 250 kDa, which are generally well conserved from yeast to mammals (26-28). In vitro transcription experiments have demonstrated that many activators will function only if the complete TFIID complex is added and cannot activate transcription if TBP alone is included (29, 30). Cellular TBP is required for transcription by all three RNA polymerases, and it is partitioned in vivo among multiprotein complexes for each polymerase, including TFIID (28, 31). Many diverse transcriptional activators have been shown to interact with the TAF subunits of TFIID, including Sp1 (32) and bicoid (33) with TAF110, estrogen receptor with TAF30 (34), CAAT transcription factor with TAF55 (35), NF-kappa B with TAF105 (36), vitamin D receptor with hTAF130 (37), thyroid receptor with TAF110 (38), and p53 with TAF40 and TAF60 (39).

In the case of CREB, the CAD alone bound to an enhancer upstream of the transcriptional start site activates transcription and the CAD interacts with TFIID (3, 15). Because binding of TFIID must precede the association of other general transcription factors with the promoter, activators such as the CAD in CREB are thought to enhance transcription initiation by facilitating the binding of the TFIID complex. This general mechanism for gene activation has been suggested for many activators (41).

The present study was designed to map the region(s) of the CAD involved in protein-protein interaction with TAF110 and ultimately determine the amino acid residues of the CAD that make crucial contacts with the TAF. A yeast two-hybrid assay was used to determine which subdomains of the CAD are required to support interaction with TAF110. In addition, the CAD was subjected to random point mutagenesis and the mutated CAD library was screened in a reverse two-hybrid system to identify amino acids of the CREB CAD that are crucial for interaction with this TAF.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Plasmids and Yeast Strains-- The same yeast expression vectors were used in the two-hybrid and reverse two-hybrid experiments. One, pGAD-110, contains the coding region for the N-terminal 308 amino acids of the TAF110 protein fused to the activation domain of the yeast Gal4 protein (amino acids 768-881) (42). This plasmid carries the LEU2 selectable marker gene and the ampr gene. A second yeast expression plasmid, pGN-1 (43), was modified to contain the coding regions for CREB, the CREB CAD, or the various deletion mutations and point mutations of the CAD fused to the Gal4 (DBD amino acids 4-94). This plasmid carries the TRP1 selectable marker, as well as the ampr gene. The yeast strain Y-153 (gal4-, leu-, trp-, Gal:LacZ, etc.) is that described by the S. Elledge laboratory (44). The MaV203 yeast strain is a derivative of Y-153 and was described by Vidal et al. (45). This strain is ura3- and contains the URA3 gene under control of a promoter that is kept silent by a sporulation repressor element unless induced by binding of activator to the Gal4 containing upstream activation sequence.

Yeast Transformation-- A LiAc protocol was used to introduce plasmids into yeast (46). The appropriate yeast strain was grown in 50 ml of YEPD (0.5% yeast extract, 1.0% peptone, 25 µg/ml adenine, 2% glucose) at 30 °C to an A600 of 0.2. Yeast were centrifuged at 5000 rpm for 5 min at 4 °C, and the pellet was washed with TE and re-centrifuged. The pellet was resuspended in 10 ml of LiAc solution (10 mM Tris, pH 7.5, 1 mM EDTA, 100 mM LiAc) and incubated for 1 h at 30 °C with gentle shaking. Cells were centrifuged as above and resuspended in 2 ml of LiAc solution. 200 µl of these competent yeast cells was combined with 10 µg of each appropriate plasmid DNA and incubated at room temperature for 10 min. One ml of 50% PEG 3350 was added, followed by mixing and incubation at 30 °C for 1 h. Cells were heat-shocked at 42 °C for 5 min, pelleted, and washed with dH2O. Yeast were resuspended in 200 µl of dH2O and plated on the appropriate selective medium. Plates were incubated at 30 °C for 2-3 days to allow colony formation.

Yeast Protein Lysate Preparation-- A glass bead lysis method was used to make yeast protein lysates (47). The yeast clone of interest was grown in 50 ml of double-selective liquid medium to an A600 of approx. 0.8. Yeast were centrifuged at 5000 rpm at 4 °C for 10 min in a JA17 rotor, and the pellet was resuspended in breaking buffer (50 mM Na2HPO4, 50 mM NaH2PO4, 10 mM EDTA, pH 7.2) with protease inhibitors added (175 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 5 µg/ml pepstatin, 1 mM dithiothreitol, 1 mM e-amino-n-caproic acid). The pellet was transferred to a 15-ml Corex tube on ice. Glass beads (0.5 mm) were added up to the fluid meniscus, and the slurry was vortexed eight times for 30 s with 2 min on ice between each round. The resulting crude yeast lysate was transferred to a microcentrifuge tube and centrifuged at 13,000 rpm for 5 min at 4 °C to pellet insoluble cellular debris and residual glass beads. A standard Bradford assay was used to estimate total protein concentration to ensure that comparable amounts of lysates were used in quantitative beta -galactosidase assays and for SDS-polyacrylamide gel electrophoresis and Western blotting experiments.

beta -Galactosidase Assays-- To lift colonies for the qualitative filter assay, a Whatman filter paper circle was placed on the plate until wet. Yeast were lysed by placing the filter into liquid nitrogen for 1 min. The filter was placed in a Petri dish and incubated with 2 ml of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 40 mM beta -mercaptoethanol, 0.005% SDS) containing 20 mg/ml of 5-bromo-4-chloro-3-indolyl beta -D-galactopyranoside at 37 °C until a blue color was evident. For the quantitative liquid assay (48), 2-5 mg of protein lysate was added to 1 ml of Z buffer and incubated with 200 µl of 4 mg/ml O-nitrophenyl-beta -D-galactopyranoside at 30 °C until a yellow color was evident. After color development, 0.5 ml of 1 M Na2CO3 was added to stop the reaction, and the absorbance at 420 nm was determined spectrophotometrically. Values were adjusted for incubation time and protein concentration to give the specific activity of the sample.

PCR Mutagenesis-- A relaxed stringency PCR protocol that promotes polymerase errors was adjusted to introduce <= 1 error per CAD sequence of 300 base pairs (49). The plasmid pGEM3Z-CAD94, containing the CREB CAD coding region, was used as a template for PCR with two primers that annealed to opposite strands on either side of NcoI and ClaI restriction sites that remove the CAD from the plasmid. The PCR mixture contained 10 ng of template DNA, 5pmole of each primer, 10 mM of each dNTP, 4 mM MgSO4, 50 mM KCl, 10 mM Tris, pH 8.4, and 5 units of Taq polymerase. Amplification of the CAD was carried out for 33 cycles under the following conditions: denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 2 min, followed by a final extension at 72 °C for 10 min. The resulting amplicon pool was digested with NcoI/ClaI, and the DNA fragments were subcloned into the pGN-CAD-G4 yeast expression vector in place of the wild type CAD NcoI/ClaI fragment. The resulting library was transformed into Escherichia coli and grown, and plasmid DNA for transformation of yeast was prepared by a standard CsCl method.

Yeast Plasmid DNA Isolation-- The method described by Nasmyth and Reed (50) was used for plasmid isolation from yeast. The yeast clone of interest was grown in 5 ml of single selective medium (without Trp, to select for only the CAD-G4 containing plasmid) to an A600 of approximately 0.6. An aliquot of 1.5 ml of the culture was centrifuged at 13,000 rpm at 4 °C for 5 min, and the pellet was resuspended in 0.5 ml of S buffer (10 mM KPO4, 10 mM EDTA, 50 mM beta -mercaptoethanol, pH 7.2). Yeast cell walls were digested by adding 5 µl of 20 mg/ml zymolase and incubating at 37 °C for 60 min. Lysis buffer (0.1 ml) (0.25 M Tris, pH 7.5, 25 mM EDTA, 2.5% SDS) was added, and the sample was vortexed and incubated at 65 °C for 30 min. After precipitation with 166 µl of 3 M KOAc (pH 5.8) on ice for 10 min, samples were centrifuged, and nucleic acid was precipitated from the supernatant with 0.8 ml of EtOH. The pellet was washed, dried, and resuspended in 40 µl of dH2O. An aliquot of 2 µl of this preparation was used to transform E. coli by electroporation. Clones were screened for the proper plasmid construct and sequenced.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The CREB CAD Interacts with the N Terminus of TAF110 in a Yeast Two-hybrid System-- We first established a functional yeast two-hybrid assay to test mutants for interaction (51). CREB contains two independent activation domains, kinase-inducible domain (KID) and CAD, that were defined by functional analysis of CREB-Gal4 (CRG) fusion proteins (Fig. 1A) (3). The CAD can be further subdivided into three subdomains, based on sequences required for activation and prevalent amino acid composition: ST3, HC, and Q3 (14). Based on previous reports of interaction between TAF110 and SP-1 or CREB (16, 17, 32), we used two yeast expression plasmids, one in which the N-terminal 308 amino acids of TAF110 are fused to the Gal4 activation domain and that carries a selectable marker (Trp), and a second expression plasmid carrying a different selectable marker (Leu) and expressing the CREB activation domain-Gal4 DNA-binding domain fusion proteins (CRG, CAD-G4, etc.). These plasmids were co-transformed into the yeast strain Y-153 (leu-, trp-, gal4-), which contains the lacZ gene under control of a Gal4 responsive promoter (44). Activation of this hybrid gene, as a result of interaction between the two fusion proteins, leads to the production of beta -galactosidase, which can be measured by qualitative and quantitative assays. The extent of this interaction can be qualitatively determined by using the synthetic substrate 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside in a filter lift assay to give a blue color to those colonies expressing beta -galactosidase (data not shown). In addition, a quantitative beta -galactosidase assay of yeast extracts was used to determine the relative strength of the interactions, which is directly proportional to the amount of beta -galactosidase activity (beta -galactosidase activity/mg of protein) (Fig. 1B). Neither of the test plasmids, CRG or TAF-AD, had any activity alone, but significant beta -galactosidase activity was observed when full-length CAD-G4 was cotransformed with a TAF110-AD expression construct (Fig. 1B). This response indicates that CAD-G4 recruited TAF110-AD to the upstream activation sequence-lacZ gene promoter, resulting in activation of the lacZ gene. The specificity of this interaction, together with the lack of activity of CREB in yeast, provides a useful model for determining interactions between various CAD mutants and TAF110.


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Fig. 1.   Domains of CRG and interaction with TAF110 in a yeast two-hybrid system. A, schematic representation of the CRG fusion protein indicating the various domains within the amino acid sequence. The kinase inducible domain (KID) is indicated, including its PKA phosphorylation site at serine 133, and the three subdomains of the CAD are shown: ST3, HC, and Q3. CRG has the CREB DNA-binding domain replaced with the DBD of the yeast GAL4 protein, amino acids 4-94. B, CAD and the N-terminal 308 amino acids of TAF110 were tested for interaction in a yeast two-hybrid system. Yeast strain Y153 (see under "Experimental Procedures") was transformed with the indicated plasmid DNA and plated on double-selective medium. Individual clones were picked from each plate, and whole-cell protein lysates were assayed for beta -galactosidase activity, which is indicative of the strength of the interaction. Values are adjusted for concentration of lysates and incubation time in the assay. Data represent three independent experiments with three clones assayed for each. Error bars depict S.E.

The Three Subdomains of CAD Are Required for Full Interaction with TAF110-- The results in Fig. 1B show that the interaction of TAF110 with CREB maps to the CREB CAD. Previous work in our laboratory showed that constitutive activity in vivo required all three subdomains of the CAD, which may interact with one or more different targets in the polymerase complex (14). In addition, several different TAFs in TFIID have been shown to interact with transcriptional activators. Thus, we were interested in determining whether a specific subdomain in the CAD would mediate the interaction with TAF110 or whether all three subdomains were required. We first tested CADs deleted of each of the three subdomains, ST3, HC, and Q3, for interaction with TAF110 in an attempt to identify a smaller region of interaction, with the ultimate goal of mapping the amino acid residues involved. Site-directed mutagenesis was used to introduce restriction sites flanking each region in question so that it could be deleted. Each mutant was tested for interaction with TAF110 and compared with the activity obtained with the full-length CAD (Fig. 2). Deletion of any of the three CAD subdomains reduced the interaction with TAF110, as compared with full-length CAD. In fact, Delta HC and Delta Q3 showed little, if any, activity. However, Delta ST3 did retain approximately 35% of full-length CAD activity, so we further truncated the remaining region to see whether a discreet interaction target for TAF110 might reside within the HC/Q3 region. We deleted the N-terminal half of HC (Delta NHC), the C-terminal half of Q3 (Delta CQ3), and both (Delta NHC/Delta CQ3). When these mutants were tested for interaction with TAF110, a gradual loss of activity was observed for successive truncation of the HC/Q3 region of the CAD (Fig. 2). The results of these yeast two-hybrid experiments lead to the conclusion that elements throughout the entire CAD are necessary for interaction with TAF110, either for a conducive conformation or through direct amino acid contacts with the TAF.


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Fig. 2.   Interaction of CAD deletion mutations with TAF110. Deletion mutants of CAD were generated by inserting restriction sites at the indicated boundaries using site-directed mutagenesis. The indicated regions of CAD were then deleted, and the resulting constructs cloned into the yeast expression vector. Each mutation was then tested for interaction with TAF110 in the yeast two-hybrid system, and beta -galactosidase activity was assayed as indicated previously. Data are representative of three experiments with three clones assayed in each. Error bars depict S.E.

Synthesis and Screening of a CAD Random Point Mutation Library-- To determine the amino acid residues of CAD involved in the interaction with TAF110, the CAD was subjected to random point mutagenesis, and the resulting library was screened in a reverse two-hybrid assay for interaction-deficient mutants. The lack of a specific region within CAD responsible for TAF110 interaction led to the decision to use the entire 87-amino acid CAD for point mutagenesis. We employed a PCR-based mutagenesis protocol to introduce random point mutations into the CAD coding region. This approach exploits the high error rate of the native Taq polymerase and can be fine-tuned by altering the Mg2+ concentration and cycle number to introduce mutations into a given template at a desired rate (49). PCR primers that flank the CAD coding sequence were chosen (Fig. 3A), and the region was amplified under conditions designed to introduce, on average, one base change error for each 300 base pairs synthesized (see under "Experimental Procedures" for exact conditions used). DNA sequencing of clones from the mutagenized pool confirmed the expected error rate. The resulting mutagenized amplicon pool was then digested with restriction enzymes with recognition sites flanking the CAD coding sequence and the DNA fragments were subcloned into the yeast expression vector as a fusion with a wild type Gal4 DBD (Fig. 3A).


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Fig. 3.   Generation and screening of a CAD point mutation library. A, the CREB CAD coding region was amplified using polymerase chain reaction with two primers that bind flanking the sequence as indicated. Conditions and amplification cycle number were chosen to favor the introduction of <= 1 point mutation per CAD sequence of 300 base pairs amplified (see under "Experimental Procedures" for conditions). The resulting amplicon pool was then subcloned into the yeast expression vector in fusion with a wild type Gal4 DBD. The library of CAD point mutations was transformed along with the TAF110 1-308-AD expression plasmid into the reverse two-hybrid yeast strain MaV203 (see under "Experimental Procedures") and plated on double selective medium containing 5-FOA at concentrations of 0.0, 0.005, 0.010, 0.015, 0.020, 0.025, and 0.050%. B, yeast clones containing the indicated expression constructs were streaked on double-selective medium with or without 0.025% 5-FOA. C, colony number was determined for yeast containing the CAD point mutation library clones and TAF110-AD at the indicated concentrations of 5-FOA. The results of a typical experiment are depicted.

To screen the library for interaction-deficient CAD mutants, we employed a reverse two-hybrid method (45) (Fig. 3A). This method uses a yeast strain (MaV203, a derivative of the Y-153 used above) that contains a URA3 gene under control of a Gal4 promoter in addition to the GAL4-lacZ reporter gene. Addition of the drug 5-fluoroorotic acid (5-FOA) is toxic to yeast when the URA3 gene product is expressed. Therefore, in a two-hybrid assay in the presence of 5-FOA, cells containing CAD-G4 that interacts with TAF-AD will activate Gal4-URA3, resulting in cell death. Those clones with an interaction-defective CAD-G4 will survive 5-FOA treatment. Double selective medium ensures the presence of both expression plasmids.

Preliminary experiments showed that the cytotoxicity of 5-FOA was specific, because yeast containing the wild type CAD-DBD, and TAF110-AD expression vectors or an expression plasmid for full-length Gal4 showed normal growth without 5-FOA but no growth with 0.025% 5-FOA added (Fig. 3B). In contrast, clones containing DBD plus TAF110-AD, or CAD-DBD plus empty-AD, or both empty vectors are able to grow normally in the absence or presence of 5-FOA. These preliminary results established the utility of the reverse two-hybrid system for screening our mutated CAD library to identify interaction-defective CADs.

The MaV203 yeast strain was cotransformed with the mutagenized CAD library and TAF110-AD and plated on selective medium containing increasing concentrations of 5-FOA ranging from 0.005 to 0.05%. As shown in Fig. 3C, the number of colonies formed by the library in a representative experiment decreased as 5-FOA concentrations increased. The CAD-G4 plasmids were recovered from colonies isolated from plates with different drug concentrations, and the CAD sequence was determined. The sharp drop in colony number between 0 and 0.015% 5-FOA reflects the elimination of wild type CAD clones or phenotypically wild type mutations. The majority of colonies found on the plate with the lowest concentration of 5-FOA (0.010%) were wild type. Importantly, many were found to be phenotypically wild type despite having acquired amino acid mutations (Fig. 4A). Colonies from plates selected at intermediate drug concentrations (0.015-0.025% 5-FOA) contained CADs that were impaired for interaction with TAF110, as discussed in detail below. Intermediate concentrations of 5-FOA (0.015-0.025%) resulted in a significant number of clones that produced full-length CAD-DBD fusion proteins with varying degrees of reduction in their ability to interact with TAF110 (Fig. 4). The small number of colonies surviving the highest dose of 0.050% 5-FOA was found to contain CAD-G4s with stop codon insertions or, in a few cases, frameshifts. These clones would not produce a protein capable of binding DNA because the Gal4 DBD is fused downstream of the CAD.


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Fig. 4.   Analysis of the CAD point mutation library in a yeast reverse two-hybrid system. A, yeast clones containing the CAD point mutation library and TAF110-AD were chosen from plates with various concentrations of 5-FOA. Plasmid DNA was recovered from the yeast and sequenced to determine the presence and identity of the mutation. B, the mutations depicted in the graph without an asterisk were isolated at 0.015-0.025% 5-FOA. Those mutations with an asterisk were isolated at 0.010% 5-FOA. Yeast clones bearing point mutations were grown in liquid selective medium and assayed for beta -galactosidase activity using an O-nitrophenyl-beta -D-galactopyranoside liquid assay as described under "Experimental Procedures." C, yeast clones with interaction-defective CAD mutants were grown in selective liquid medium, and whole-cell protein lysates were made. Proteins were separated by SDS-polyacrylamide gel electrophoresis, and a Western blot was performed using a primary antibody directed against the Gal4 DBD and visualized with chemiluminescence.

To further characterize these mutations, the yeast colonies from which plasmid DNA had been isolated and sequenced were grown and assayed for beta -galactosidase activity. Interestingly, all mutations that reduced interaction with TAF110 lie within the HC domain and represent changes in hydrophobic amino acid residues to charged amino acid residues. These mutations decreased interaction with TAF110 to varying degrees, ranging from near complete elimination of interaction with mutations G189R, L193Q, and L207Q to less severe disruption of the interaction in I191R, I191T, L204Q, and L207S. Mutations in the ST3 and Q3 domains of CAD were isolated only under less stringent conditions (low 5-FOA concentrations). These mutations, along with a few mutations of polar amino acids within the HC domain (indicated in Fig. 4 by asterisks), produced little or no effect on TAF110 binding. This result was not surprising because it is likely that many clones that are mutated, but phenotypically normal for binding to TAF110, would survive a very low dose of 5-FOA. No mutations in ST3 or Q3 or of hydrophilic amino acids in HC were isolated from plates with higher doses of 5-FOA (>= 0.020%). Thus, it would appear that the central hydrophobic residues of the CAD are most crucial to its interaction with TAF110.

To rule out the possibility that mutations that show up as interaction-deficient in this screen are actually mutations that destabilize the protein, the mutant CAD-DBD fusion proteins were assayed for their stability in yeast. This was especially important for those clones that appeared to be completely impaired for binding to TAF110. Western blotting with antibody directed against the Gal4 DBD was used to determine the stability of CAD-G4 proteins. Yeast containing the putative interaction-defective clones were grown in liquid selective medium, and whole-cell yeast protein lysates were made and analyzed. The results in Fig. 4C show that the seven CAD mutants identified in Fig. 4A that are most severely impaired for interaction with TAF110 produce proteins of the proper size in yeast. In addition, the amounts of the mutant fusion proteins were approximately equal to that of wild type CAD-DBD, as indicated by the CAD control lanes on Western blots (Fig. 4C). However, this was not the case with all mutants isolated. One mutant that decreased binding to TAF110 did not produce a detectable, appropriately sized fusion protein (data not shown) and was therefore excluded from the mutant set. This mutation (G188R) presumably destabilized the CAD-DBD fusion protein in yeast and led to a false positive in the screen for interaction defective mutants.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The TAFs of TFIID have been shown to interact with several different transcription factors and are required for function of the activator both in vitro and in vivo (32-36, 39). In vitro experiments using the Drosophila activators bicoid and hunchback, performed by Tjian et al. (33), showed that the function of these activators is dependent on the presence of TAF110 and TAF60 in the reaction, respectively. This same study also showed that truncation of either TAF could inhibit activation by the factor binding to it, both in vitro and in vivo. More recent studies have demonstrated that TAFs interact with and serve as coactivators for a wide range of transcription factors in mammalian cells (32-36, 39), where they are proposed to facilitate recruitment of the TFIID complex to the promoter.

In addition, there is evidence that TAFs play diverse roles at different promoters during transcription initiation. Recent studies in yeast indicate that TAFs are dispensable for activated transcription of many genes in vivo (53, 54). TAFs appear to play a more general role in promoter selectivity in that organism. In mammalian cells, also, some TAFs bind to DNA and serve as promoter selectivity factors at TATA-less promoters (28, 57). Other TAFs possess enzymatic activities, such as protein kinase or histone acetyltransferase activity (55). It is therefore likely that some TAFs are more important than others for serving as coactivators for specific transcription factors. In particular, TAF110 seems to be playing a major coactivator role. Many reports have shown that TAF110, or its human homolog hTAF130, interacts with various transcriptional activators, including Sp1 (32), vitamin D receptor (37), thyroid hormone receptor (38), adenovirus E1A (58), Drosophila bicoid (33), and the CCAAT-binding factor (59). Sp1 was the first transcription factor shown to interact with TAF110 (32). The strength of the interaction between Sp1 and TAF110 correlates with the strength of the activator in vivo (16). In our model (Fig. 5), we propose that TAF110 serves as a coactivator by providing a target for the CAD to recruit TFIID to the promoter and/or stabilize its association with the TATA box. In either case, the interaction would increase the association of TFIID with the promoter, a proposed rate-limiting step in PIC assembly, thereby increasing the overall rate of PIC formation.


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Fig. 5.   Model for basal activation by the CREB CAD. The proposed model shows CREB interacting with the TAF110 subunit of TFIID through the CAD. This interaction would recruit TFIID to the promoter DNA and/or stabilize its binding to the TATA-box. Once this step in PIC formation is achieved, the remainder of the complex can form, ultimately positioning RNA polymerase II properly at the transcriptional start site.

Given that the CAD interacts with holo-TFIID in vitro (15) and that all three CAD subdomains are required for function in vivo, we were interested to know whether the CAD-TFIID interaction was mediated by multiple TAFs. We observed no interaction with TAF55 (data not shown), a TAF that has been shown to interact with multiple transcription factors, including Sp1 (60). Also, others observed that CREB did not interact with TAF250.2 In addition, we found that our previous report of an interaction between CREB and TFIIB (15) cannot be replicated in vitro or in a two-hybrid assay (data not shown). At this point, the finding that all three CAD subdomains are required for interaction with TAF110, together with the lack of evidence for association with other GTFs, suggests that the CAD interaction with a single GTF component, TAF110, may be its primary mode of action.

TAF110 (or dTAF110) is the Drosophila homolog of the human hTAF130, which was recently cloned (37). Our study with TAF110 was undertaken before the human clone had been obtained and was based on preliminary observations made by others (17). dTAF110 and hTAF130 share much sequence identity, especially at the N terminus, where CREB interacts with the TAF, and at the C terminus, where the TAF binds to TAF250, the scaffolding TAF that integrates it in the TFIID complex (37). It was recently shown by Saluja et al. (61) that hTAF130 and dTAF110 interact with transcriptional activators, such as Sp1 and CREB, in a similar manner, suggesting that our studies of CREB interactions with dTAF110 will be directly applicable to a mechanism that involves hTAF130 in vivo.

The nature of the mutants isolated in the reverse two-hybrid screen (hydrophobic right-arrow charged) suggests that a hydrophobic surface on the CAD may contact a similar surface on TAF110. Gill et al. (16) showed that hydrophobic residues in the activation domain of Sp1, which are arranged similarly to those in the CREB HC (Fig. 4A), mediate interaction with TAF110. This pattern of hydrophobic residues is present in many activation domains and was first recognized in the viral activator VP16 (62). In VP16, mutation of key hydrophobic residues leads to a dramatic loss of activation.

Upon analysis of codon usage and the probabilities of obtaining hydrophobic versus charged residues for single base changes in the codons of the hydrophobic residues in HC, it was found that some, Gly-189, Ala-190, and Ile-191, had a 50% chance of being changed to a charged amino acid residue or of being changed to another hydrophobic residue. Other hydrophobic residues, however, had a much smaller probability of being changed to a charged residue with a single base change (Leu-193 = 33%, Leu-204 = 33%, and Leu-207 = 17%). These results show that the hydrophobic residues for which interaction-defective mutants were isolated were either random or were biased in favor of a conservative mutation to another hydrophobic residue. The fact that mutants were isolated in which each of these hydrophobic residues was changed to a charged residue (in the case of Ile-191 and Leu-207, to two different charged residues), even though the mutagenesis was either random or biased in favor of conservative mutations, makes it more likely that the interaction-defective phenotype between CAD and TAF110 is caused by the presence of charged residues on an interacting hydrophobic surface in CAD, which would have a repulsive effect.

Both the reverse two-hybrid screen and our deletion mutation analysis suggest that the remaining two regions of the CAD, ST3 and Q3, contribute to CAD conformation, rather than being directly involved in contacting TAF110. All point mutations isolated in the ST3 or Q3 subdomains were found only under a low concentration of 5-FOA (0.010%), and all were phenotypically wild type. However, deletion of either, or even part, of these two subdomains significantly reduced interaction with TAF110 (Fig. 2). In addition, all three subdomains of the CAD are required for full activation of transcription in mammalian cells (15). These results suggest that interaction with TAF110 is at least part of the mechanism of CAD activation.

The fact that our CAD-DBD fusion protein is incapable of activating transcription from a Gal4-lacZ reporter in yeast (Fig. 1B), whereas the same fusion construct activates transcription from a Gal4-CAT reporter in mammalian cells (3), is also very interesting. This same observation has been made for other activators that interact with TAF110, including Sp1 (32, 52, 56). Yeast contain homologs of most of the TAF subunits of human TFIID but does not contain a TAF110 homolog (40). Thus, if TAF110 is the target for Sp1 and CREB in the general transcription machinery, the lack of a TAF110 homolog in yeast could explain why these activators do not stimulate transcription in yeast.

We have presented here evidence that supports a mechanism for CREB activation of basal transcription that utilizes TAF110 as a coactivator to recruit TFIID to the promoter and facilitate PIC assembly (Fig. 5). Although additional experimentation is required to further test our current model for activation, there is mounting evidence in the literature that the TAF subunits of TFIID mediate selective activator function in higher eukaryotes. In particular, TAF110 (hTAF130) seems to be a widely used coactivator for transcriptional activators, including the CAD of CREB, that regulate transcription in metazoans but not in yeast.

    ACKNOWLEDGEMENTS

We thank Dr. J. Hopper and members of his laboratory for providing plasmids, yeast strains, technical assistance, and critical discussions of this work. We also thank Dr. A. Hopper and members of her laboratory for technical advice on yeast experiments. We thank the Tjian laboratory for providing TAF110 clones and David Spector for critical reading of the manuscript.

    FOOTNOTES

* 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 To whom correspondence should be addressed: Dept. of Cellular and Molecular Physiology, H166, The Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033. Tel.: 717-531-6182; Fax: 717-531-7667; E-mail: pquinn{at}psu.edu.

2 J. Goodrich, personal communication.

    ABBREVIATIONS

The abbreviations used are: CREB, cAMP response element-binding protein; DBD, DNA-binding domain; CRG, CREB-Gal4; CAD, constitutive activation domain; ST3, serine/threonine-rich region; HC, hydrophobic cluster; Q3, glutamine-rich region; GTF, general transcription factor; TBP, TATA-binding protein; TAF, TBP-associated factor; PIC, preinitiation complex; URA, uracil; 5-FOA, 5-fluoroorotic acid; PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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