Transcription Factor IIA Derepresses TATA-binding Protein (TBP)-associated Factor Inhibition of TBP-DNA Binding*

Josef OzerDagger §, Katherine Mitsourasparallel , Dennis ZerbyDagger , Michael Carey, and Paul M. LiebermanDagger **

From the Dagger  The Wistar Institute, Philadelphia, Pennsylvania 19104 and  Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The interaction of the general transcription factor (TF) IIA with TFIID is required for transcription activation in vitro. TFIID consists of the TATA-binding protein (TBP) and TBP associated factors (TAFIIs). TFIIA binds directly to TBP and stabilizes its interaction with TATA-containing DNA. In this work, we present evidence that TAFIIs inhibit TBP-DNA and TBP-TFIIA binding, and that TFIIA stimulates transcription, in part, by overcoming this TAFII-mediated inhibition of TBP-DNA binding. TFIIA mutants modestly compromised for interaction with TBP were found to be significantly more defective in forming complexes with TFIID. Subtle changes in the stability or conformation of the TFIIA-TBP complex resulted in a failure of TFIIA to overcome TAFII-mediated inhibition of TBP-DNA binding and transcription function. Inhibition of TBP-DNA binding by TAFIIs could be partially relieved by limited proteolysis of TFIID. Proteolysis significantly stimulated TFIIA-TFIID-TATA binding in both electrophoresis mobility shift assay and DNase I footprinting but had little effect on complexes formed with TBP. Recombinant TAFII250 inhibits TBP-DNA binding, whereas preincubation of TFIIA with TBP prevents this inhibition. Thus, TFIIA competes with TAFII250 for access to TBP and alters the TATA binding properties of the resulting complex. Transcriptional activation by Zta was enhanced by temperature shift inactivation of TAFII250 in the ts13 cell line, suggesting that TAFII250 has transcriptional inhibitory activity in vivo. Together, these results suggest that TAFIIs may regulate transcription initiation by inhibiting TBP-TFIIA and TBP-DNA complex formation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Transcription initiation by RNA polymerase II is regulated by the formation of a preinitiation complex consisting of general transcription factors (reviewed in Refs. 1 and 2). The interaction of the TATA-binding protein (TBP)1 with promoter DNA is an early and highly regulated step in preinitiation complex assembly at TATA containing promoters (Refs. 3-8 and reviewed in Refs. 9 and 10). Several factors bind directly to TBP and modulate its ability to form an active preinitiation complex. TAFIIs and TFIIA bind directly to TBP and are required for transcriptional activation from most promoters in vitro (reviewed in Refs. 11-14). Both TFIIA and TAFIIs can interact directly with promoter-specific activators and mediate their stable interaction with TBP and the preinitiation complex (15-20). TAFIIs can bind directly to core promoter sequences and recruit TBP to promoters lacking consensus TATA elements (21-23). TFIIA makes direct contact with DNA sequences upstream of the TATA box of some promoters and stabilizes TBP binding to TATA-containing DNA (24-27). Thus, both TFIIA and TAFIIs can modulate the interactions of TBP with activators, DNA, and other general transcription factors. However, the potential interactions between TFIIA and TAFIIs have been less extensively examined and are likely to be an important component of eukaryotic transcriptional regulation.

TFIID consists of TBP and at least eight TAFIIs (reviewed in Refs. 11 and 14). TAFII250 binds directly to TBP and multiple other TAFIIs, thus serving as a potential scaffold for the multiprotein complex (28, 29). Other TAFIIs can bind TBP directly, including a subset of TAFIIs with homology to histone proteins, which form a histone fold motif (30). Histones have generally been associated with transcriptional repression by limiting activator and general factor access to promoter sequences, and it is possible that some TAFIIs share histone-like features (31). Purified TAFII250 has been shown to inhibit the DNA binding activity of TBP (21, 28, 32). The functional significance of this inhibition has not been well characterized. TAFII250 is identical to CCG1, a gene identified by a mutation that causes arrest in the G1 phase of the cell cycle. TAFII250 has been shown to have both protein kinase and histone acetylase activities, but the functional significance of these activities is not yet clear (33, 34). Mutations in TBP, which abrogate transcription activation function in vivo, were found to disrupt the ability of TBP to bind to TAFII250 in vitro (35). More recent studies in yeast and hamster cell lines indicate that TAFII250 is required for coordinating the interaction between promoter-specific activators and core promoter elements (36-39).

Several lines of evidence indicate that a dynamic interplay between TFIIA and TAFIIs regulates the binding of TFIID to promoter DNA. Cross-linking studies indicate that TFIIA significantly alters the interaction of TAFIIs with promoter DNA (40). TFIIA mediates an activator-dependent conformational change in TFIID that results in the interaction of TAFIIs with sequences near and downstream of the transcriptional initiation site (20, 41-43). The recruitment of TFIIA by an activator significantly enhanced the ability of TFIID to interact with TFIIB, further indicating that TFIIA can alter the properties of TFIID (43). TFIIA can derepress the inhibitory effects of several factors that bind directly to TBP (5, 44-47). The direct binding of TFIIA to TBP is likely to preclude interaction of these inhibitory factors with TBP. The derepression function of TFIIA is mediated by the subunits of TFIIA that contact TBP directly, but these subunits are not sufficient for TFIIA-mediated transcriptional activation function in vitro (48).

The crystal structure of the yeast TFIIA-TBP-DNA ternary complex reveals that TFIIA makes direct contact with TBP through several hydrophobic residues in the small subunit of TFIIA (Toa2) (26, 27). Mutagenesis of the homologous residues in the human TFIIA small subunit (gamma  ) interrupt TFIIA stimulation of TBP-DNA binding and TFIIA-mediated transcription stimulation in vitro (49). Interestingly, conservative phenylalanine substitution mutation of these residues (TFIIA gamma  Y65F and W72F) had little detectable effect on the formation of a TFIIA-TBP DNA complex but were found to be defective for transcription activation in vitro (49). The biochemical basis for these defects was not clear but suggest that subtle changes in the TFIIA-TBP interface have dramatic effects on transcription function. In this work, we further investigated the mechanism underlying the transcription defect caused by these TFIIA mutations. We showed that these mutants are substantially more defective in the presence of TAFIIs and that TAFIIs are generally inhibitory for TBP-TFIIA complex formation. We also show that the ability of TFIIA to overcome TAFII-mediated inhibition correlates with transcription activation function in vitro.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Plasmid Constructs-- The Escherichia coli expression constructs for human wild type and mutant TFIIA-gamma (pRSET A-IIA-gamma ) and TBP were previously described (49). The E. coli expression constructs for human TFIIA-alpha beta (pQE-IIA-alpha beta ) was described previously (20). Baculovirus expressed human TAFII250 was kindly provided by R. Tjian (29). Eukaryotic expression vector for TAFII250 (pCMV-TAFII250) was a gift from E. Wang, and Zta was expressed from the SV40 promoter in pBXGo.

Protein Preparations-- The pQE-IIA-alpha beta and pRSET A-IIA-gamma wild type or mutant constructs were expressed in M15 and BL21 E. coli strains, respectively. Expressed proteins were purified under denaturing conditions on nickel nitrilotriacetic acid-agarose columns (Qiagen) and dialyzed into D100 buffer as described (20, 49). Recombinant TFIIA polypeptides were used at a final concentration of ~0.2 µM for in vitro transcription reactions. Human recombinant TBP and recombinant Zta proteins were prepared as described previously (41). The GAL4-AH fusion protein was purified as described (50). Baculovirus-expressed human TAFII250 was purified from baculovirus-infected cell lysates using the 12CA5 monoclonal antibody for affinity purification and peptide elution essentially as described (51). Affinity-purified holo-TFIID was purified as described (51).

DNA Binding Reactions-- Polyacrylamide EMSA conditions for T-A complex formation were described (52). Conditions for Mg2+-agarose EMSA were described (41, 53). For Mg2+-agarose EMSA Zta-hIID-IIA complex formation, 0.2 unit of human IID, 80 nM TFIIA, and 33 nM Zta were used (41). DNase I footprinting reactions were performed as described previously (54). For protease experiments, protein and DNA were preincubated for 30 min at 30 °C before the addition of 40 ng of protease K for 20 min at 30 °C (unless noted otherwise). The relative mobility of the TBP-DNA (T) and TFIIA-TBP-DNA (T-A) complexes varied dramatically in EMSA depending upon the probe used (E1B or E4T) and whether magnesium was included in gels (46, 49). The positions of T and T-A are indicated in each figure.

In Vitro Transcription Reactions-- In vitro transcription reactions contained 100 ng of G5E4TCAT template, ~200 ng of purified recombinant-GAL4-AH activator protein, and 40 µg of TFIIA-depleted HeLa nuclear extract in a 50 µl final reaction volume incubated for 1 h at 30 °C. Primer extension reactions were described previously (51). The TFIIA-depleted nuclear extracts were described (20). Wild-type or mutant pRSET A-IIA-gamma was added back to transcription reactions as described (49).

Transfection and CAT Assay-- The hamster ts13 cell line was purchased from ATCC. Approximately 5 × 105 cells were transfected in a 100-mM dish using calcium phosphate precipitation with 250 ng reporter, 5 µg of SV-Zta and 30 µg of CMV-TAFII250 (where indicated). Vector DNA was used to compensate for differences in DNA concentration of effector plasmid. Transfected cells were incubated for 8-12 h at 34 °C, washed with phosphate-buffered saline, and then incubated at 34 or 39.5 °C as indicated. Cells were harvested for CAT assay 36 to 48 h post-transfection. CAT reactions analyzed by thin layer chromatography and quantitated with a PhosphorImager 445SI screen. All transfections were repeated at least three times.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Mutations in TFIIA Disrupt Complex Formation with TFIID-- In a previous study, we examined a large panel of TFIIA-gamma mutants for the ability to stimulate TBP binding to the adenovirus E1B core promoter and to mediate activator stimulated transcription in vitro. We found that alanine substitution of Tyr-65 completely eliminated the TBP-TFIIA DNA complex (T-A), whereas W72A caused an aberrant migrating complex in acrylamide EMSA. Alanine substitution of these residues and neighboring aromatic residues reduced transcription activation by several activators in vitro (49). To explore the possible effect of TFIIA mutations on interactions with TAFIIs in the TFIID complex, we compared the panel of TFIIA mutants for their ability to support an interaction between the Zta transcriptional activator and TFIID (Fig. 1). Zta stimulates a highly stable interaction between TFIIA, TFIID, and promoter DNA (referred to as the Z-D-A complex) that correlates with transcriptional activation function for a subset of promoters (55). TFIIA mutants were first tested for their ability to form a stable complex with TBP (referred to as T-A) on the E4T promoter. Under limiting conditions, TBP did not form a stable Mg2+-agarose EMSA complex by itself but forms a weak smear (Fig. 1A, lane 2). The addition of wt TFIIA results in a stable well resolved T-A complex (Fig. 1A, lane 3). Similar stable complexes were resolved for most TFIIA-gamma substitution mutants, with the exception of gamma Y65A, which failed to form T-A (lane 10). Several substitution mutations were reduced for T-A complex formation, and gamma W72A produced a T-A complex with a slower mobility in these Mg2+-agarose EMSA (Fig. 1A, lane 13 and data not shown). We previously reported an altered mobility complex with the gamma W72A mutant in polyacrylamide-EMSA (49). These Mg2+-EMSA results are consistent with previous studies examining the ability of these TFIIA mutants to form a T-A complex with the E1B 30-base pair oligonucleotide in polyacrylamide gel EMSA (49).


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Fig. 1.   Comparison of the ability of the TFIIA-gamma mutant to form T-A and Z-D-A. A, detection of the T-A ternary complex on the Z7E4T promoter fragment by Mg2+-agarose EMSA. TFIIA wt or mutant (~80 nM) was used to stimulate TBP (~70 nM) in reactions incubated for 30 min at 30 °C. TFIIA alanine mutant substitutions are indicated above each lane. The T-A shift is indicated by an arrow. B, detection of the Z-D-A complexes on the Z7E4T promoter fragment by Mg2+-agarose EMSA. 50 ng of TFIIA mutant (80 nM), 1 µl of hIID (0.2 units), and 12.5 ng Zta (33 nM) were used for reactions incubated at 30 °C for 30 min.

The same panel of TFIIA-gamma mutants were examined for the ability to form Z-D-A by Mg2+-agarose EMSA. Under conditions where affinity-purified TFIID (hIID) was limiting for DNA binding, wt TFIIA significantly stimulated Z-D-A complex formation (Fig. 1B, lane 2). Most TFIIA-gamma substitution mutations that support T-A were capable of forming Z-D-A. TFIIA mutant gamma Y65A, which could not form T-A, was similarly incapable of forming Z-D-A (lane 9). Interestingly, gamma Y6A, gamma F67A, gamma C68A, gamma W72A, and gamma F74A were significantly reduced in Z-D-A complex formation. The gamma Y6A, gamma F67A, gamma C68A, gamma W72A, and gamma F74A mutants have defects in mediating regulated transcriptional activity in vitro, indicating that the failure to form Z-D-A correlates with the transcription defects of these TFIIA mutants (49). Quantitation of complex formation revealed that TFIIA gamma Y6A, gamma F67A, gamma C68A, gamma W72A, and gamma F74A were significantly more disruptive to Z-D-A complex formation compared with T-A complex formation (compare Fig. 1, A and B). None of these TFIIA-gamma mutants disrupted the interaction with Zta in a glutathione S-transferase binding assay, suggesting that failure to form Z-D-A is a result of a failure of TFIIA to interact properly with TFIID (data not shown). The fact that these mutations were more defective with TFIID than with TBP raised the possibility that some of these TFIIA mutations were incapable of interacting with TBP in the presence of TAFIIs.

To further investigate the possible effect of TAFIIs on TFIIA association with TBP, we examined the properties of TFIIA mutants that specifically alter the TFIIA-TBP interface. Based on the crystal structure of the yeast TFIIA-TBP-DNA ternary structure, conserved human residues TFIIA-gamma Tyr-65 and gamma  Trp-72 are likely to make direct contact with TBP. Our previous analysis indicated that semiconservative substitution mutants TFIIA gamma Y65F and gamma W72F were able to stimulate T-A complex formation at nearly wt levels on a 30-base pair adenovirus E1B oligonucleotide in EMSA (Fig. 2B) (49). However, these mutants were defective in mediating regulated transcriptional activation for the GAL4-CTF, -AH, and -VP16 activators on the G5E1BTCAT template and AP1 on the collagenase promoter in vitro (49). In this study, the TFIIA gamma -W72A and Phe mutants were also shown to be incapable of coactivating transcription from the G5E4TCAT reporter with the GAL4-AH activator in vitro (Fig. 2C). Thus, these TFIIA mutants are defective for transcriptional activation on two different core promoters. Because these mutants were capable of forming T-A but were defective in mediating transcriptional activation, we suspected they may be incapable of interacting with TFIID. The ability of these mutants to interact with TFIID was tested by comparing their ability to form complexes with TBP, TFIID, or TFIID and Zta in Mg2+-agarose EMSA (Fig. 2A). The gamma Y65F and gamma W72F mutants stimulate T-A formation in Mg2+-agarose EMSA (Fig. 2A, lanes 4 and 6), just as they do in the polyacrylamide EMSA (Fig. 2B, lanes 3 and 5) (49). Both the Trp-72 mutants formed a slower, altered mobility complex in polyacrylamide EMSA as in the Mg2+-agarose gel EMSA (Fig. 2A, lanes 5 and 6). However, these TFIIA mutants were completely defective in forming stable complexes with TFIID in the absence (lanes 9-11) or presence (lanes 15-17) of the Zta transcriptional activator. Thus, the TAFIIs in TFIID inhibit these TFIIA mutants from forming a stable complex with TBP. Moreover, the failure of these mutants to form stable complexes with TFIID correlates with their defects in transcriptional activation function (Fig. 2C) (49).


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Fig. 2.   TFIIA mutants stimulate TBP but not TFIID DNA binding. A, comparison of TFIIA mutants stimulation of TBP, TFIID, or Zta-TFIID binding to the Z7E4T promoter by Mg2+-agarose EMSA. B, ability of TFIIA mutants to stimulate T-A on a 30-base pair oligonucleotide derived from the Ad E1B TATA box in polyacrylamide gel EMSA. C, in vitro transcription reactions reconstituted with HeLa nuclear extracts depleted for endogenous TFIIA, the GAL4-AH activator, and the G5E4TCAT template (lane 1). The addition of wt (lane 2), gamma  W72A (lane 3), or gamma  W72F (lane 4) is indicated above. Transcription was assayed by primer extension, and the correctly initiated product is indicated by the arrow.

Phenylalanine Substitution of Tyr-65 Decreases Stability of T-A-- The semiconservative substitutions at residues Tyr-65 and Trp-72 were particularly interesting because these form T-A but were defective for D-A formation and transcriptional coactivation (Fig. 2) (49). Based on crystal structure, we would predict that mutations at these residues should have a primary defect in the interaction with TBP. To determine whether these TFIIA mutants alter the stability of the T-A complex, we compared the dissociation rate of T-A formed with the wild-type and mutant TFIIAs. Dissociation rates were measured in polyacrylamide EMSA by the addition of excess unlabeled TATA oligonucleotide competitor to preformed T-A-DNA complexes (Fig. 3A). T-A complex stability was assayed at increasing times after the addition of competitor TATA oligonucleotide. We found that gamma  Y65F had a significantly increased dissociation rate relative to wild-type TFIIA because there was an ~8-fold difference in the reduction of T-A complex after 4 h of oligonucleotide challenge relative to the reduction of complex formed with wild-type IIA (Fig. 3A, lanes 18 and 19). In contrast, the gamma  W72A mutant showed no significant change in the dissociation rate relative to wild-type TFIIA after 4 h of oligonucleotide challenge (Fig. 3A). Thus, the transcriptional defect of gamma  Y65F may be attributed, in part, to decreasing the stability of the T-A-DNA ternary complex.


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Fig. 3.   TFIIA mutations compromise T-A complex stability and protease resistance. A, preformed (20 min) T-A complexes with TFIIA wt, gamma  Y65F, or gamma  W72A and the Ad E1B TATA box 30-mer were challenged with 100-fold molar excess of TATA oligonucleotide for the times indicated above. Time at which samples were loaded onto the gel are indicated above each set. B, T-A complexes with TFIIA-gamma wt, gamma  Y65F, gamma  W72A, or gamma  W72F were preformed for 20 min on the Ad E1B TATA box and then were challenged with the indicated concentrations of protease K for 5 min before loading. Proteolyzed T-A complexes are indicated as core T-A.

Substitutions at Trp-72 Increase Protease Sensitivity of T-A-- Mutations at residue Trp-72 produce a stable T-A complex but often give rise to a species of slower mobility. To explore the possibility that TFIIA-gamma W72A and gamma W72F may produce alternative conformations in the ternary complex with TBP and DNA, we used limited proteolysis as a probe of protein structure and stability. Limited proteolysis has been used previously to map a core domain of TBP and TFIIA and for evidence that TFIIA induces a conformational change in TBP (54, 56, 57). Treatment of TBP with protease K resulted in a slight stimulation of DNA binding and a faster mobility in acrylamide EMSA (Fig. 3B, lane 1 to lanes 6, 11, and 16). Treatment of T-A complexes formed with wt TFIIA resulted in a stable faster migrating complex (Fig. 3B, compare lane 2 to lanes 7, 12, and 17). Interestingly, complexes formed with either gamma W72A or gamma  W72F resulted in nearly complete digestion of the T-A complex, with an 8-fold reduction of complex formation compared with wild-type T-A (Fig. 3B). In contrast, the complex formed with gamma  Y65F was as protease-resistant as wt TFIIA (Fig. 3B). Digestion of TBP before DNA binding resulted in the complete loss of binding activity, similar to what was seen for the gamma Trp-72 mutants (Ref. 54 and data not shown). These results show that both TBP and TFIIA-TBP assume a stable and protease-resistant conformation once bound to DNA. The gamma Trp-72 mutants failed to form the protease-resistant complex and also failed to stimulate transcriptional activation, suggesting that the protease-resistant conformation is important for transcription activation function. We suggest that gamma Trp-72 mutants trap TBP in an inactive conformation and that this conformation may occur under natural conditions in vivo. Conformational changes in TBP may be the mechanistic basis for some transcriptional regulatory pathways.

Proteolysis Stimulates TFIID-DNA and TFIID-IIA Binding-- To further explore the effect of TAFIIs on TFIIA-TBP and TBP-DNA binding, we tested the effects of limited proteolysis on the behavior of TFIID in DNA binding reactions with or without TFIIA (Fig. 4A). Despite the large molecular mass of the TFIID complex, TFIID-DNA complexes could be partially resolved in Mg2+-acrylamide EMSA (Fig. 4A, lane 2). The addition of protease K (40 ng) for 20 min resulted in a striking stimulation of DNA binding activity in this assay (lane 3). Some of this stimulation may result from the degradation of TAFIIs that prevent TFIID-DNA from entering acrylamide gels. In contrast, recombinant TBP bound to similar amounts of DNA (lane 6) but was only modestly stimulated by treatment with protease K (lane 7). The addition of TFIIA to TFIID resulted in a significant stimulation of DNA binding (compare lanes 2 and 4). Interestingly, the addition of protease further stimulated the TFIIA-TFIID complex as well as increased the electrophoretic mobility of the complex (lane 5). The addition of TFIIA to TBP stimulated TBP-DNA binding (compare lanes 6 and 8). Protease K treatment increased the mobility of the TBP-TFIIA complex but had only a minor stimulatory effect on the amount of complex formed (lane 9). One interpretation of this result is that protease degrades TAFIIs, which inhibit TFIIA and DNA interactions with TFIID. Curiously, TFIID- and TBP- containing complexes had identical mobilities both before and after protease treatment. Previous work has also found similar mobilities between TBP and TFIID in EMSA, which may be accounted for by the unusual conformational constraints imposed on DNA bound by TBP (41, 53).


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Fig. 4.   A, stimulation of TFIID-DNA binding by limited proteolysis. Affinity-purified TFIID (IID) (lanes 2 and 3), TFIID plus TFIIA (lanes 4 and 5), TBP (lanes 6 and 7), or TBP plus TFIIA (lanes 8 and 9) were bound to the Ad E4 core promoter for 30 min. Samples in lanes 3, 5, 7, and 9 were then treated with 40 ng of protease K for 20 min. Samples were electrophoresed on 6% polyacrylamide gels containing 5 mM magnesium acetate. B, TAFII250 is highly sensitive to limited proteolysis. The TFIID fraction was incubated with increasing concentrations (1, 10, or 100 ng) of protease K as indicated above. The integrity of TAFII250 was analyzed by Western blotting and probing with monoclonal antibody directed against TAFII250 (Santa Cruz Biotechnology).

Limited proteolysis of TFIID may affect one or more components in the TFIID multiprotein complex. To determine whether at least one TAF was affected by limited proteolysis, we assayed proteolyzed TFIID by Western blotting with antibody specific for TAFII250 (Fig. 4B). TFIID was treated with protease K under conditions that were previously found to stimulate DNA binding activity. At the lowest concentration of protease K treatment (1 ng), we found that TAFII250 was partially degraded, indicating it is highly sensitive to proteolytic cleavage. With 10 ng of protease, TAFII250 was no longer detectable, indicating that recognizable epitopes had been completely degraded. Stimulation of DNA binding was observed with 40 ng of protease K (Fig. 4A), suggesting that TAFII250 was mostly degraded in those reactions. These results indicate that TAFII250 is a candidate target of proteolysis in reactions where TBP-DNA binding is stimulated.

To further characterize the protease stimulation of TFIID-DNA and TFIIA-TFIID-DNA binding, we assayed partially proteolyzed complexes by DNase I footprinting assay. The DNase I footprinting assay should eliminate destabilizing effects imposed by gel electrophoresis, a phenomenon previously observed for TBP-DNA interactions (54). Using similar protein-DNA binding and proteolysis conditions, we found that proteolysis had a modest stimulatory effect (~2.4-fold) on TFIID protection (Fig. 5A, compare lanes 2 and 3). In contrast, proteolysis had a much larger stimulatory effect (~7.7-fold) on TFIIA-TFIID binding to the TATA element (Fig. 5A, lanes 4 and 5). Proteolysis had only a slight stimulatory effect on TBP (~1.3-fold) and TFIIA-TBP (~1.4-fold) binding to the TATA box (Fig. 5B). Although these results support the observations presented in Fig. 4, they suggest that the stimulation of TFIID binding to TATA is partly dependent upon the gel assay, suggesting that proteolysis stabilizes the protein-DNA interaction against the disruptive effects of gel electrophoresis observed in Fig. 4. On the other hand, the DNase I assay also supports the observation that proteolysis stimulates TFIIA-TFIID-DNA binding in solution. The DNase I assay also indicates that proteolysis had little effect on TBP-DNA or TFIIA-TBP-DNA interactions, suggesting that TAFIIs were the likely target of proteolysis-mediated stimulation of DNA binding.


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Fig. 5.   Limited proteolysis stimulates TFIID-TFIIA DNase I footprinting activity. A, affinity-purified TFIID (hIID) (lanes 2 and 3) or TFIID plus TFIIA (lanes 4 and 5) were incubated with the Ad E4T core promoter for 30 min. Samples were then incubated for an additional 20 min in the presence (lanes 3 and 5) or the absence (lanes 2 and 4) of 40 ng of protease K followed by DNase I footprinting analysis. B, recombinant TBP (10 ng for + and 20 ng for ++) was incubated with or without TFIIA and the Ad E4T promoter as indicated above each lane. Samples were then incubated for an additional 20 min with (+) or without (-) 40 ng of protease K as indicated above each lane.

TFIIA Can Prevent TAFII250 Inhibition of TBP-DNA Binding-- Evidence from proteolysis experiments suggests that degradation of TAFIIs may stimulate TFIIA-TFIID-DNA complex formation. To determine if a single TAFII may confer an inhibitory effect on TFIIA association with TBP, we examined the properties of affinity-purified recombinant human TAFII250 in DNA binding reactions with or without TFIIA (Fig. 6). Others have shown that the addition of recombinant TAFII250 to recombinant TBP results in an inhibition of TATA binding activity (28, 32). TFIIA binds directly to TBP and can preclude the interaction of TBP with several transcriptional inhibitors (44-47). To determine if TFIIA could prevent the TAFII250 inhibition of TBP-TATA binding, we initially determined whether addition of TAFII250 could inhibit DNA binding of preformed T-A complexes (Fig. 6). Incubation of TBP with TFIIA resulted in a strong stimulation of DNA binding (Fig. 6A, lane 3). When TFIIA was preincubated with TBP for 15 min followed by the addition of TAFII250, TFIIA-TBP-DNA complex formation was reduced only slightly, indicating that this complex was largely resistant to TAFII250-mediated repression (lane 4). In contrast, if TAFII250 was preincubated with TBP for 15 min followed by the addition of TFIIA, DNA binding was abolished (Fig. 6A, lane 5). A time course of preincubation with TFIIA-TBP-DNA was performed to determine how much time was required to establish a stable TAFII250-resistant TFIIA-TBP-DNA complex (Fig. 6B). We found that simultaneous addition of TAFII250 and TFIIA to TBP and DNA resulted in a significant inhibition of DNA binding (Fig. 6B, lane 4). However, after 15 min of preincubation with TFIIA, TBP, and DNA, a stable complex formed that was resistant to TAFII250 inhibition (lane 6). Thus, TFIIA competes with TAFII250 for interaction with TBP, and the resulting complexes have opposite effects on the DNA binding properties of TBP.


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Fig. 6.   TFIIA can prevent TAFII250 inhibition of TBP-DNA binding. A, the E4T core promoter was incubated with TBP (lane 2), TBP plus TFIIA (lanes 3 and 4), or TBP plus TAFII250 (lane 5). After a 15-min incubation at 30 °C, TAFII250 was added to the TBP-TFIIA containing reaction (lane 4) or TFIIA was added to the TBP-TAFII250 containing reaction (lane 5). Reactions were incubated for an additional 45 min before electrophoresis on 6% polyacrylamide gels containing 5 mM magnesium acetate. t, time. B, TBP and TFIIA were preincubated for 0, 5, 15, or 30 min before the addition of TAFII250. The time at which TAFII250 was added is indicated above (tau ). TBP-TFIIA complex is indicated by the arrow (T-A).

Mutant TAFII250 Enhances Transcription By an Activator in Vivo-- To determine what effect TAFII250 inhibition of TBP-DNA or TBP-TFIIA complexes have on transcription activator function, we assayed Zta-mediated transcriptional activation in the ts13 cell line at permissive and nonpermissive temperatures (Fig. 7). The ts13 cell line contains a mutated TAFII250 gene, which results in G1 cell cycle arrest at nonpermissive temperatures (39.5 °C). Transfection of Zta resulted in ~15-fold level of activation in the ts13 cell line at the permissive temperature (34 °C) on the Z5E4TCAT reporter plasmid. In contrast, transfection of Zta resulted in a ~42-fold level of activation in ts13 cells at the nonpermissive temperature (Fig. 7A). Activator levels were not effected by temperature shift in ts13 cells, and a similar temperature shift had no effect on transactivation levels in control baby hamster kidney cell lines (data not shown). Furthermore, a temperature shift resulted in a slight decrease in basal level transcription, indicating that temperature shift by itself did not produce a generalized increase in CAT activity (data not shown). Similar increased transcriptional effects were observed with TAFII250 inactivation in ts13 cells using a different reporter promoter (BHLF1) with Zta or a different activator (GAL4-VP16) with the G5E4TCAT reporter (data not shown). This suggests that the transcriptional effect is not specific for the Zta activator or the template. To determine if this transcriptional enhancement was a result of the mutated TAFII250 gene, wild-type TAFII250 was cotransfected with Zta and reporter plasmids in ts13 cells (Fig. 7B). Cotransfection of wild-type TAFII250 reduced Zta activation levels at the nonpermissive temperature to levels similar to that observed at the permissive temperature. Thus, introduction of wild-type TAFII250 partially reversed the enhancement of activation caused by temperature shift, suggesting that mutant TAFII250 is responsible for the Zta-dependent transcriptional enhancement. Although these transfection experiments do not address the direct role of TAFII250 in transcription function, they do provide indirect evidence to suggest that TAFII250 can inhibit transcription function by an activator in vivo.


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Fig. 7.   Inactivation of TAFII250 in ts 13 cells enhances transcription activation by Zta. A, the Z5E4TCAT reporter plasmid was cotransfected with Zta or vector DNA in the ts 13 cell line as indicated. Transfected cells were incubated at the permissive (p) (34 °C) or at the nonpermissive (n) temperature (39.5 °C) for 36-48 h post transfection. B, wild-type TAFII250 partially restores transcription inhibition. CMV-TAFII250 was cotransfected with Z5E4TCAT and Zta or vector DNA as indicated. Samples were incubated at permissive or nonpermissive temperatures, as in A. Quantitation for the average of three independent experiments is presented below each representative experiment as a ratio of p/n. Each experiment varied by less the 24%.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Although the interaction of TFIIA with TBP has been well characterized, relatively little is understood of how TAFIIs may alter the interaction of TFIIA with TBP in the TFIID complex. In this work, we have investigated the effect of TAFIIs on the interaction of TFIIA with TBP. Several different experimental approaches were used to demonstrate that TAFIIs can inhibit the binding of TBP with TFIIA and DNA. We have shown that a subset of TFIIA mutations is more defective for interaction with TFIID than with TBP, suggesting that TAFIIs can destabilize interactions between TBP and TFIIA (Figs. 1 and 2). Limited proteolysis of TFIID markedly stimulated DNA binding which suggests that degradation of TAFIIs may alleviate repression of TFIID-DNA binding activity (Figs. 4 and 5). Limited proteolysis also stimulated the amount of D-A complex formed, further suggesting that TAFIIs may also inhibit TFIIA from binding TBP. Purified human TAFII250 was found to inhibit T-A complex formation, demonstrating that at least one TAFII precludes the interaction between TFIIA and TBP (Fig. 6). Finally, we show that transcription activation by Zta can be enhanced in vivo by the inactivation of TAFII250 in ts13 cell lines by shifting to the nonpermissive temperature (Fig. 7). Taken together, these results suggest that TAFIIs can regulate transcription by inhibiting TBP interactions with TFIIA and DNA.

We have found that a subset of TFIIA mutations are more defective for interaction with TFIID than with TBP (Figs. 1 and 2). Phenylalanine substitution mutations at gamma Tyr-65 and Trp-72 were capable of forming a stable T-A complex in both Mg2+-agarose and polyacrylamide EMSA but were defective in forming a stable EMSA complex with TFIID (Fig. 2). These mutants were also incapable of supporting activated transcription in vitro, indicating that TFIIA must associate with TFIID to function in transcription (Fig. 2). Further examination of these mutations revealed that they formed an aberrant T-A complex, with either an increased dissociation rate (gamma Y65F) or increased protease sensitivity (gamma W72F and W72A) (Fig. 3). Thus, subtle changes in the stability or conformation of the T-A complex were sufficient to disrupt association with TFIID and inhibit transcription activation function. The TAFIIs further destabilize the association of these TFIIA mutants with TBP, thus revealing an inhibitory effect on TFIIA assembly. Additionally, these TFIIA mutants may fail to induce changes in the TAFII configuration necessary for TFIIA-TFIID-DNA complex formation to occur. In either case, a stable interaction between TFIIA and TBP appears essential to overcome TAFII inhibition of TFIID-DNA binding.

The potential inhibitory role of TAFIIs on TFIID binding was further supported by the stimulation of TFIID-DNA binding by limited proteolysis (Figs. 4 and 5). We found limited proteolysis stimulated D-A complex formation significantly better than T-A complex formation. Earlier work has shown that limited proteolysis could stimulate TBP-DNA binding, and this was largely a result of the degradation of the nonconserved amino-terminal domain of TBP (54, 56, 58). Moreover, the addition of TFIIA was shown to bypass the need for the proteolysis of TBP, suggesting that TFIIA stimulated a conformational change in the amino-terminal domain of TBP (56). Our results do not exclude the possibility that stimulation of DNA binding by TFIIA involves a conformational change in the TBP amino-terminal domain. Recent findings indicate that the inhibition of DNA binding by the amino-terminal domain can be relieved by interaction with the SNAP complex, which is required for activation of the U6 gene (58). The configuration of the amino-terminal domain in the TFIID complex is unknown, and it is possible that TAFIIs may increase its inhibitory effect on TBP-DNA binding.

The limited proteolysis experiments suggest that some TAFIIs in the native TFIID complex inhibit TBP-DNA binding (Figs. 4 and 5). TAFII250 has been shown by others to inhibit TBP binding to DNA (21, 28, 32). Because the addition of recombinant TAFII250 to TBP does not necessarily reconstitute the physiological properties of the entire TFIID complex, it was important to demonstrate that TAFIIs have inhibitory activities in the context of the native TFIID complex. TAFII250 was highly sensitive to proteolysis in the TFIID complex and thus qualifies as a candidate target of protease-mediated stimulation of TFIID-DNA binding (Fig. 4B). We have also shown that purified TAFII250 can inhibit T-A complex formation and that preincubation of TFIIA with TBP reverses this inhibition (Fig. 6). Because native TFIID consists of a preassembled TBP-TAFII250 complex, it is not yet clear how TFIIA overcomes TAFII250 repression in assembled TFIID. Interaction of TFIIA with other TAFs and activators may be required for TFIIA-TFIID-DNA complex formation.

Several lines of evidence indicate that TFIIA and TAFIIs interact and may be dependent upon each other for proper transcription function. TFIIA binds directly to dTAFII110 in vitro, and mutations in dTAFII110 that disrupt TFIIA binding do not function properly in transcription in vivo (59, 60). TFIIA inhibits the cross-linking of human TAFII55 and p31 to promoter sequences upstream of the TATA element and enhances the cross-linking of TAFII250 and TAFII135 to sequences downstream of the adenovirus major late promoter TATA element (40). TFIIA is required for an activator-mediated conformational change in TFIID that results in an enhanced interaction downstream of the transcriptional initiation site (20, 41). Together, these results indicate that TFIIA can bind to and alter the organization of TAFIIs in the TFIID complex. Although it is possible that TFIIA may dissociate TAFII250 from TBP, it is likely that TAFII250 remains associated with the TFIIA-TFIID-promoter complex by its association with multiple other TAFs in the TFIID complex.

The yeast homologue of TAFII250 also dissociated TBP from DNA and prevented TFIIA binding to TBP (61, 62). A partially conserved amino-terminal domain of yTAFII145 bound directly to TBP and was required for the inhibition of TBP-DNA and TBP-TFIIA binding. Interestingly, an amino-terminal deletion mutation of yTAFII145 was found to be temperature-sensitive in yeast, and this conditional lethality could be rescued by overexpression of TFIIA subunits (62). This latter result suggests that TFIIA and the amino-terminal domain of yTAFII145 function cooperatively and not antagonistically as might be predicted from the in vitro binding studies. yTAFII145 may inhibit TBP binding and TFIIA access but nevertheless stabilizes these associations once formed in vivo. These results emphasize the potential complexity of interactions between TFIID, TFIIA, and activators. Furthermore, representational display analysis of transcripts from yeast cells grown under nonpermissive conditions for yTAFII145 reveal that an equal number of transcripts were enhanced compared with the numbers that were diminished (38). This is consistent with a role of yTAFII145 in transcriptional repression as well as coactivation for some genes in yeast. Our data suggest that mammalian TAFII250 has similar activities involved in the inhibition of transcription for some activators and/or promoters in vivo. We suggest that the inhibition of TFIIA-TBP-DNA complex formation by TAFII250 may be one mechanism by which TAFII250 regulates gene expression. Activators like Zta may facilitate conformational changes and/or displacement of TAFII250 that allow TFIIA to bind TBP and promote active preinitiation complex formation.

    ACKNOWLEDGEMENT

We thank R. Tjian for baculovirus human TAFII250 and E. Wang for plasmids.

    FOOTNOTES

* This research was supported in part by American Cancer Society Grant MV-547 (to M. C.) and National Institutes of Health Grants GM54687 (to P. M. L.)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.

§ Supported by a National Institutes of Health National Research Service Award and a Veterans of Foreign Wars post-doctoral cancer fellowship during this study.

parallel Supported by United States Public Health Service National Research Service Award GM07185.

** Also supported by the Leukemia Society of America. To whom correspondence should be addressed: The Wistar Institute, 3601 Spruce Street, Rm. 351, Philadelphia, PA 19104. Tel.: 215-898-9491; Fax: 215-898-0663; E-mail: lieberman{at}wista.wistar.upenn.edu.

1 The abbreviations used are: TBP, TATA-binding protein; TAFII, TBP-associated factor; yTAF, yeast TAF; TFIIA and TFIID, transcription factors IIA and IID, respectively; wt, wild type; hIID, holo-TFIID; EMSA, electrophoresis mobility shift assay; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus.

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Abstract
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Discussion
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