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Originally published In Press as doi:10.1074/jbc.M011792200 on June 4, 2001

J. Biol. Chem., Vol. 276, Issue 35, 32597-32605, August 31, 2001
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The Binding Interaction of HMG-1 with the TATA-binding Protein/TATA Complex*

Dweepanita Das and William M. ScovellDagger

From the Department of Chemistry and Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403

Received for publication, December 29, 2000, and in revised form, May 29, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

High mobility protein-1 (HMG-1) has been shown to regulate transcription by RNA polymerase II. In the context that it acts as a transcriptional repressor, it binds to the TATA-binding protein (TBP) to form the HMG-1/TBP/TATA complex, which is proposed to inhibit the assembly of the preinitiation complex. By using electrophoretic mobility shift assays, we show that the acidic C-terminal domain of HMG-1 and the N terminus of human TBP are the domains that are essential for the formation of a stable HMG-1/TBP/TATA complex. HMG-1 binding increases the affinity of TBP for the TATA element by 20-fold, which is reflected in a significant stimulation of the rate of TBP binding, with little effect on the dissociation rate constant. In support of the binding target of HMG-1 being the N terminus of hTBP, the N-terminal polypeptide of human TBP competes with and inhibits HMG-1/TBP/TATA complex formation. Deletion of segments of the N terminus of human TBP was used to map the region(s) where HMG-1 binds. These findings indicate that interaction of HMG-1 with the Q-tract (amino acids 55-95) in hTBP is primarily responsible for stable complex formation. In addition, HMG-1 and the monoclonal antibody, 1C2, specific to the Q-tract, compete for the same site. Furthermore, calf thymus HMG-1 forms a stable complex with the TBP/TATA complex that contains TBP from either human or Drosophila but not yeast. This is again consistent with the importance of the Q-tract for this stable interaction and shows that the interaction extends over many species but does not include yeast TBP.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The TATA-binding protein is a universal transcription factor that is essential for eukaryotic transcription by all three RNA polymerases (1-4). For RNA polymerase II transcription, the regulation of TBP1 binding to the TATA element is considered a principal determinant in promoter activity and therefore a primary target for regulatory factors. TBP can be considered modular in nature, with its highly conserved C terminus being necessary and sufficient for both binding to the TATA box and basal level transcription (5, 6). In addition all activators and repressors that bind to human TBP (hTBP) are reported to bind to the C terminus (2-4). On the other hand, the interactions of regulatory factors with the 159-residue N terminus in hTBP appear much more limited, and its role in transcriptional regulation is not understood. In the only case that is characterized, it was shown that the N terminus down-regulates hTBP binding to the U6 TATA box, mediates cooperative binding with SNAPc to the U6 promoter, and facilitates an enhanced level of RNA polymerase III transcription of the U6 gene (7, 8). In addition, a monoclonal antibody specific for the Q-tract of the N terminus of hTBP was shown to inhibit selectively in vitro transcription from TATA-containing, but not TATA-less, promoters that were transcribed by RNA pol II or III. This antibody interaction did not affect TBP binding to the TATA box or inhibit the formation of the TFIIA/TFIIB/TBP/TATA complex, which suggests that the N terminus may be available for protein-protein interactions associated with subsequent assembly of the preinitiation complex (9).

HMG-1 is a ubiquitous and highly conserved nuclear protein that has been reported to serve as a transcriptional repressor (10, 11) in some systems, while functioning as a coactivator for RNA polymerase II in other contexts (12-17). Fig. 1A shows that HMG-1 is likewise modular in nature, consisting of three domains. The A- and B-domains, each containing about 80 residues with a high percentage of arginines and lysines, are homologous and structurally comparable and have been shown to bind nonspecifically to DNA (18-21). The C terminus is quite different, being polyanionic, with the last 30 residues being a stretch of exclusively aspartic or glutamic acid residues. This segment reduces binding affinity to DNA and is not required for protein stability (22) but has defied a more definitive functional role.

In this work, we show that the C terminus of HMG-1 and the Q-tract in the N terminus of human TBP are essential for stable HMG-1/TBP/TATA complex formation. HMG-1 increases the affinity of TBP for the TATA element 20-fold, which is reflected in a significant increase in the rate of TBP binding, while having little effect on the lifetime of the complex. This interaction provides a broader spectrum of regulatory controls for TBP binding and promoter activity.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Isolation, Purification, and Characterization of Proteins-- Calf thymus HMG-1 and HMG-2 proteins were purified in non-denaturing conditions using salt extraction, selective precipitation with ammonium sulfate, and further purification by high pressure liquid chromatography using a MonoQ column (23, 24).

The expression vector for HMG-1(A-B) didomain (residues 1-176), obtained from M. Bianchi, was transfected into BL21(DE3) cells, and the expressed protein was purified using published protocols (25).

The expression vectors, pET-his6hTBP, pET11d-his6180hcTBP (from F. Pugh), pAR3038dTBP (from R. Tjian), and pET11d-his6yTBP (from R. Roeder), were transfected into BL21(DE3) cells, and the expressed proteins were purified using phosphocellulose chromatography and ammonium sulfate precipitation, as described by Pugh (26).

The GST fusion proteins with full-length TBP or TBP with N-terminal deletions (Delta N) (from N. Hernandez) were obtained as pET11c expression vectors (7), transfected into BL21(DE3) cells, and expressed and purified using glutathione-agarose (Sigma) (7). The GST-nTBP-(1-158) (from T. Kouzarides) was purified according to standard procedures and was used in competition experiments. The purity of all proteins isolated was >90% as evidenced by a single Coomassie-stained band on SDS-PAGE.

Electrophoretic Mobility Shift Assays (EMSA)-- The individual oligonucleotides of the adenovirus major late promoter (AdMLP; -40 to -1 and -1 to -40) were purchased from National Biosystems and 32P-end-labeled by standard procedures (27). The EMSA procedures have been described (24) but involved reacting the DNA probe with the proteins of interest in binding buffer (24 mM Tris acetate, pH 8.0, 10% glycerol, 4 mM magnesium acetate, 50 mM potassium glutamate, 0.1 mM EDTA, 1 mM dithiothreitol, 0.01% Nonidet P-40, 4 mM spermidine, 5 µg/ml poly(dG-dC), and 115 µg/µl bovine serum albumin) for 30 min at 30 °C. All samples were electrophoresed in 0.35× TBE buffer (TBE, Tris borate-EDTA buffer) at 200 V for ~1.5 h in 4-6% nondenaturing polyacrylamide gels at 4 °C. After completion of electrophoresis, the gels were dried and exposed to x-ray film.

Antibodies used in the supershift experiments were obtained from R. Roeder (anti-HMG-1) and P. Chambon (monoclonal TBP antibodies (1C2 and 3G3)).

The Kd values were obtained by titration of 100 pM DNA over a range of TBP concentrations, with equilibrium established after 60 min at 30 °C. Experiments for the HMG-1/TBP/TATA complex were done with the HMG-1 concentration at 120 nM. The band intensities for the complex and free DNA in these studies and those in the kinetic determinations (below) were measured by exposing the dried gels to a PhosphorImager screen, which were scanned using the Molecular Dynamics PhosphorImager system. The ImageQuant software program was used to measure accurately the band intensities. The Kd value for the TBP/TATA complex is equal to the free TBP concentration at which there are equal concentrations of [DNA] and [TBP/DNA] (i.e. Kd = [TBP][DNA]/[[TBP/DNA]). At the very low TBP concentrations, in which the TBP concentration was not in large excess relative to DNA, the [TBP]free was calculated by standard procedures. The fraction of complex was plotted versus the concentration of TBP to generate the binding curves. The best fit of the data was derived (using Sigma Plot for PC) using over 50 data points from five independent determinations.

The off-rate constants for the complexes were determined by establishing the complex for 60 min and challenging the complex with 20 ng/µl poly(dI-dC)·poly(dI-dC). At the time points indicated, the samples were loaded on the gel. The same level of poly(dI-dC)·poly(dI-dC) was added to the controls at the corresponding time points. Reactions were initiated at staggered intervals so that all samples could be loaded on the gel at the same time. DNA concentrations were at 100 pM, with TBP concentrations being at least 15-fold in excess. The HMG-1 concentration was 120 nM. The tau 1/2 values were derived from the linear plot of ln[c/co] versus time, with the koff obtained from the relationship, koff = ln 2/tau 1/2. The relative on-rates were estimated from a plot of the fraction complex formed versus time. These plots were also derived from more than 50 points from five independent runs, with the average values used to obtain the final plot. The gel electrophoresis for both the thermodynamic and kinetic studies was run for only 20 min to minimize any dissociation of the complex during electrophoresis.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Acidic C Terminus of HMG-1 Is Essential for Stable HMG-1/TBP/TATA Complex Formation-- HMG-1 binds to TBP/TATA to form an EMSA-stable HMG-1/TBP/TATA complex, exhibiting a significantly greater stability than the TBP/TATA complex (24). To investigate the extent to which the acidic C terminus of HMG-1 contributes to the stability of the complex, the binding of HMG-1-(1-215) to TBP was compared with that for the didomain HMG-1(A-B)-(1-176) that lacks the C terminus. The EMSA profile in Fig. 1B compares the binding of HMG-1 (lanes 3-7) and HMG-1(A-B) (lanes 9-14) to the TBP/TATA complex. HMG-1 binding produces a complex with an increased mobility, as reported previously (24). The HMG-1/TBP/TATA complex is evident at an (HMG-1/TBP) molar ratio of 4, whereas it is the sole species at a molar ratio of 40. On the other hand, no complex is detectable with HMG-1(A-B) at a molar ratio as high as 640. A strong new band of increased mobility becomes apparent at these increasingly higher HMG-1(A-B) levels (lanes 11-14). Lanes 15-18 confirm that this major band observed in lanes 11-14 is also observed when HMG-1(A-B) is reacted with the DNA probe in the absence of TBP. This indicates that at HMG-1(A-B) molar ratios of 80 or greater, the highly charged HMG-1(A-B) binds directly and preferentially to DNA and not to the TBP. This is consistent with the much higher DNA binding affinity expected for HMG-1(A-B) compared with HMG-1 (28-30). We conclude that the C terminus is essential for the stability of the HMG-1/TBP/TATA complex.


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  		Fig. 1.   A, domain structure of HMG-1. HMG-1 contains three domains. The A and B boxes are basic in nature and extend from residues 1 to 89 and 91 to 176, respectively. The acidic C terminus includes residues 185-215. The HMG-1(A-B) didomain contains residues 1-176. B, comparative binding of HMG-1 and HMG-1(A-B) to TBP/TATA complex. Human TBP (1 nM, lanes 2-7 and 8-14) and AdMLP DNA (all lanes) are reacted with increasing levels of HMG-1 (lanes 3-7) and HMG-1(A-B) (lanes 9-14). HMG-1 concentrations in lanes 3-7 are 4, 13, 40, 80, and 160 nM, whereas HMG-1(A-B) concentrations in lanes 9-14 are 13, 40, 80, 160, 320, and 640 nM, respectively. Lanes 15-18 are control lanes without TBP, containing 80, 160, 320, and 640 nM HMG-1(A-B) box, respectively.

The N Terminus of hTBP Is Required for Stable HMG-1/TBP/TATA Complex Formation-- The role of the N terminus of hTBP was examined by comparing the relative binding of HMG-1 to both the full-length hTBP-(1-339) and the C-terminal TBP fragment (residues 159-339). Lanes 1-6 in Fig. 2, like the data in Fig. 1, show the strong binding profile for HMG-1 to TBP/TATA, with complete complex formation at a (HMG-1/TBP) molar ratio of 40. However, incubation of HMG-1 with cTBP/TATA complex does not lead to any detectable complex formation, as evident in lanes 9-14. Complexation of HMG-1 with cTBP/TATA could not be detected at (HMG-1/cTBP) molar ratios as high as 640. In fact, at ratios of about 200 and higher, HMG-1 inhibits cTBP binding to the TATA-containing probe. Together with this, a band of greater mobility is observed again, which results from HMG-1 binding directly and nonspecifically to the DNA probe. This interpretation is verified by reacting HMG-1 with the DNA probe in the absence of cTBP (lanes 15-17) which produces the same band profile.


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  		Fig. 2.   Comparative binding of HMG-1 to TBP/TATA and cTBP/TATA complex. The AdMLP DNA (all lanes) and human TBP (1 nM, lanes 2-6) or cTBP (7 nM, lanes 8-14) are reacted with increasing levels of HMG-1 (lanes 3-6 and 9-14). Lanes 3-6 contain 13, 40, 80, and 160 nM HMG-1, respectively. Lanes 9-14 contain 152, 300, 600, 1200, 2400, and 4800 nM HMG-1, respectively. Lanes 15-17 are controls for HMG-1 (1200, 2400, and 4800 nM, respectively) without cTBP.

HMG-1 Binding Increases the Affinity of hTBP for the TATA Element-- The relative stabilities of the TBP/TATA and HMG-1/TBP/TATA complexes were quantitatively compared by titrating the TATA-containing oligonucleotide with TBP, in the absence and presence of saturating levels of HMG-1. The EMSA binding profiles for the two complexes are shown in Fig. 3A. Qualitative examination of the binding at low TBP levels (compare lanes 2 and 11) shows more complex formed in the presence of HMG-1. The band intensity data were used to plot the fraction of each complex formed as a function of TBP concentration (Fig. 3B), from which the corresponding Kd values were determined. The Kd value for the TBP/TATA complex was 1.5 nM, which is comparable to values reported previously (31-34). The corresponding plot for TBP binding in the presence of saturating levels of HMG-1 shows stimulated binding of TBP to the TATA element. Complex formation is observed at significantly lower TBP levels than required for the binding of only TBP to the TATA element. This complexation reduced the Kd value by about 20-fold, with 50% HMG-1/TBP/TATA complex formation occurring at about 70 pM TBP.


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  		Fig. 3.   A, EMSA titration of AdMLP TATA with TBP in the presence and absence of HMG-1 protein. TBP concentrations were 0.24 (lanes 2 and 11), 0.42 (lanes 3 and 12), 0.53 (lanes 4 and 13), 1 (lanes 5 and 14), 1.5 (lanes 6 and 15), 3.1 (lanes 7 and 16), 5.3 (lanes 8 and 17), and 9 nM TBP (lanes 9 and 18), with AdMLP TATA at 100 pM. The concentration of HMG-1 in lanes 11-18 was maintained at 120 nM. Reactions were incubated for 60 min at 30 °C. B, equilibrium binding profile for TBP binding to TATA in the presence and absence of HMG-1. The fraction of HMG-1/TBP/TATA () and TBP/TATA (open circle ) complexes at each TBP concentration were obtained from the EMSA profiles and plotted as a function of TBP concentration. The data represent the average of points from five independent titrations. The Kd values are 1.5 nM for TBP/TATA and 70 pM for HMG-1/TBP/TATA.

HMG-2 protein is similar to HMG-1 in size and exhibits a high degree of homology, with both proteins being implicated in the regulation of transcription (18-21). However, HMG-2 has eight fewer acidic residues (22 versus 30 in HMG-1) in the C-terminal acidic tract (35, 36). We determined that HMG-2 also stimulates TBP binding, with both proteins exhibiting comparable effects in enhancing TBP binding (data not shown).

Since the Kd value is a reflection of the ratio of kd/kon, the impact of HMG-1 on the complex dissociation rate constant was determined. The EMSA profile for dissociation for each complex is shown in Fig. 4A, with the data plotted in Fig. 4B. The dissociation profiles for the complexes differ only slightly, indicating that the presence of HMG-1 has little effect on the half-life of the complex. The values of tau 1/2 are 160 and 130 min, respectively, for the TBP/TATA and the HMG-1/TBP/TATA complexes. The corresponding values for koff are 7.2 × 10-5 and 8.9 × 10-5 s-1, respectively. The value for the TBP/TATA complex is comparable to previously reported values (33) obtained for TBP dissociation.


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  		Fig. 4.   A, the EMSA profile for the dissociation of TBP/TATA and HMG-1/TBP/TATA. TBP (1.5 nM) was incubated with Ad MLP TATA DNA (100 pM) for 1 h to establish equilibrium. The HMG-1 concentration in the 2nd panel was at 120 nM. The samples were challenged with poly(dI-dC)·poly(dI-dC) at the indicated times. B, dissociation kinetics for the TBP/TATA and HMG-1/TBP/TATA complexes. The plot of the fraction of complex remaining was plotted as a function of time. The bands in A were quantified as noted, and the fraction of complex remaining was plotted as a function of time. The best fit of the points was done utilizing the Sigma Plot. The average values from five independent runs is shown for HMG-1/TBP/TATA complex () and TBP/TATA complex (open circle ). C, the EMSA profile for the formation of TBP/TATA and HMG-1/TBP/TATA complexes. The TBP (1.5 nM) and probe (100 pM) were added and incubated for the indicated times. The HMG-1 concentration in the upper panel was 120 nM. The profiles are shown in the respective panels for formation of the TBP/TATA and HMG-1/TBP/TATA complexes. D, the time course for TBP binding in TBP/TATA and HMG-1/TBP/TATA complex formation. The bands in C were quantified, and the fraction of complex formed was plotted as a function of time. The best fit was obtained utilizing the Sigma Plot. The average values from five independent runs are shown in the plot for HMG-1/TBP/TATA complex (open circle ) and TBP/TATA complex ().

This finding would indicate that the effect of HMG-1 on the Kd value should be associated predominantly with an increase in the on-rate for TBP. To obtain an estimate of the relative on-rates and determine if this was generally consistent with the Kd data, the comparative time course of TBP binding was monitored for the two complexes and is shown in Fig. 4C. Comparison of the relative band intensities at the same time points (e.g. 5 min) shows that, qualitatively, the presence of HMG-1 stimulates the rate of TBP binding. As shown in Fig. 4D, quantitative measurements indicate that HMG-1 clearly stimulates the rate of TBP binding, enhancing the initial rate by about 10-fold. The initial slope for the formation of the HMG-1/TBP/TATA complex represents only an estimate or lower limit value due to the high rate of reaction and difficulty of obtaining consistent data at times less than 1 min. These kinetic data are, however, consistent with the thermodynamic data and indicate that HMG-1 decreases the Kd value by primarily increasing the on-rate kinetics, while having little discernible effect on the dissociation kinetics.

The N-terminal hTBP Polypeptide Inhibits hTBP Binding to HMG-1-- It was of interest to provide additional support for the role of the N terminus of TBP as the target for HMG-1. If HMG-1 interacts directly with the N terminus of hTBP and this provides the primary stability for the complex, then the presence of the exogenous N-terminal polypeptide would be expected to inhibit the formation of the HMG-1/TBP/TATA complex. Fig. 5 shows the effect of increasing levels of GST-nTBP-(1-159), 34-260 nM (lanes 2-6), when it is preincubated with 80 nM HMG-1 for 20 min on ice, followed by 30 min of incubation with TBP/TATA. Little inhibition is observed at the lower level of nTBP (molar ratio (nTBP/HMG-1) of 0.5; (nTBP/TBP) of 34) (lane 2), whereas progressive inhibition occurs at the higher nTBP levels, with complete inhibition of complex formation observed at about the 225 nM level (molar ratio (nTBP/HMG-1) of 3; (nTBP/TBP) of 225) (lane 4-5). The presence of nTBP exhibited no detectable effect on TBP/TATA complex formation (data not shown). These data provide additional support for the N terminus of hTBP as the principal target for HMG-1 binding and for interaction with this region providing the primary stability in complex formation.


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  		Fig. 5.   N terminus (residues 1-159) of TBP prevents HMG-1/TBP/TATA complexation. GST-nTBP (lane 2, 34 nM; lane 3, 105 nM; lane 4, 170 nM; lane 5, 225 nM; lane 6, 260 nM) or GST (lane 7, 260 nM) were preincubated with 80 nM HMG-1 (all lanes) for 20 min on ice, after which hTBP (1 nM) and DNA were added to all reactions, and incubation was continued for another 30 min at 30 °C.

Comparative Binding of HMG-1 with N-terminal Deletion Mutants of hTBP-- In order to determine if a particular segment of the N terminus of hTBP might play a predominant role in the HMG-1 interaction, the effect that HMG-1 has on increasing the stability of the TBP/TATA complex was examined using hTBP deletion mutants. The schematic of hTBP and the five N-terminal deletion mutants used are shown in Fig. 6A. The non-conserved 159-residue N terminus can be conveniently divided into three regions as follows: the segment containing the initial residues from 1 to 54 (fragment I); the central region, inclusive of residues 55-95 and containing the Q-tract, which is made up of 34 consecutive glutamine residues (fragment II); and the segment from 96 to 158 (fragment III), which lies between the Q-tract and the conserved C terminus (residues 159-335). The Delta N nomenclature for the mutants is from Mittal and Hernandez (7). The pairs of adjacent lanes in Fig. 6B show the relative stability of the TBP/TATA and the corresponding HMG-1/TBP/TATA complexes. Comparison of lanes 1 and 2 shows that the extent of GST-TBP binding to TATA (lane 1) is significantly increased by complexation with HMG-1. This is essentially the same value obtained when TBP (not in the GST fusion) is used. This indicates that the presence of the GST does not change the HMG-1/TBP binding, which is also what was observed previously with the SNAPc/TBP binding (7, 8). The relative increases for the mutant-TBPs were determined using a PhosphorImager (data not shown) and were compared with this value. The mutants were found to fall into two different groups. HMG-1 increases the stability of the complex formed for both Delta N + I + II (lanes 3 and 4) and Delta N + II (lanes 5 and 6), with the stability being comparable, but slightly less, than that for hTBP itself. This suggests that segment II (the Q-tract) plays the major role in stabilizing the interaction with HMG-1, with segment I providing little additional stability. Fragment Delta N + I (lanes 11 and 12) has a reduced TATA binding affinity, in agreement with previous reports (7, 8) and the addition of HMG-1 has no effect on stability (extended exposure in lanes 11' and 12' is shown in the right panel). To ensure that Delta N + I remained capable of binding to the TATA element and was not simply inactivated during the purification procedure, TFIIB was reacted with TBP/TATA to form a stable TFIIB/TBP/TATA complex (data not shown). There is no mobility shift and insignificant intensity change on reaction of HMG-1 with either Delta N + III or Delta N (lanes 7-10), indicating that HMG-1 has little or no interaction with them. The band intensity for the TBP/TATA complex with these latter two TBP mutants is greater than for the others, in agreement with previous findings that showed that the N terminus within hTBP reduces the binding affinity of TBP to the TATA element (7). The latter three deletion mutants, Delta N, Delta N + I, and Delta N + III, represent the second group, all of which exhibit no significant interaction with HMG-1. These findings indicate that the Q-tract in segment II is a major target for HMG-1 binding.


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  		Fig. 6.   A, schematic representation of the N-terminal deletion mutants of hTBP. The positions of segments I (residues 1-54), II (residues 55-95), and III (residues 96-158) in hTBP are shown, with schematic descriptions of the TBP deletion mutants of interest. B, comparative binding of HMG-1 with N-terminal deletions of hTBP. All lanes contain 16-22 nM of a GST-TBP fusion protein and AdMLP DNA, in addition to a 40 molar excess of HMG-1 in the even-numbered lanes (+). The N-terminal deletion mutants of hTBP used are indicated above the lanes. Lanes 1 and 2 have full-length TBP, in addition to 640 nM HMG-1 in lane 2. Lanes 3 and 4 have fragment Delta N + I + II, in addition to 720 nM HMG-1 in lane 4. Lanes 5 and 6 contain fragment Delta N + II, with 800 nM HMG-1 added in lane 6. Lanes 7 and 8 contain Delta N(c-TBP), in addition to 880 nM HMG-1 in lane 8. Lanes 9 and 10 contain fragment Delta N + III, in addition to 800 nM HMG-1 in lane 10. Lanes 11 and 12 contain fragment Delta N + I, in addition to 800 nM HMG-1 in lane 12. Lanes 13 and 14 are control lanes, which have 38 nM GST, in addition to 1520 nM HMG-1 also in lane 14. Lanes containing fragment Delta N + I, with and without HMG-1 (lanes 11' and 12'), were overexposed and are shown to the right. The asterisk represents fragment (Delta N + I)/DNA complex.

Monoclonal Antibody 1C2 Inhibits HMG-1 Binding to hTBP-- If the Q-tract is important for the HMG-1 interaction, HMG-1 should compete with an antibody specific to the Q-tract region and reduce or eliminate the formation of a supershifted complex. Antibodies that are targeted to epitopes that are not directly involved in the HMG-1 binding should correspondingly yield a supershifted complex in the presence or absence of HMG-1. Fig. 7A shows the sequence for the first 95 residues in the N terminus of hTBP, highlighting the location of the epitopes for two monoclonal antibodies (mAb). mAb1C2 was originally reported to be specific for residues 53-62 (shown in parenthesis), which lies at the junction of segment I and II and within the Q-tract. Recently, it was shown to be specific for the Q-tract (37). On the other hand, mAb3G3 targets residues 1-10 in hTBP.


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  		Fig. 7.   A, binding sites for monoclonal antibodies 3G3 and 1C2 in the N-terminal domain of hTBP. The partial sequence for the N-terminal domain of hTBP is shown, with segments I (residues 1-54) and II (residues 55-95) underlined. The minimal epitopes for mAb3G3 (residues 1-10) and mAb1C2 (residues 53-62 and Q-tract) are shown in bold type and with a dashed line above them. B, HMG-1 competitive binding for preestablished mAb/TBP/TATA complex. Human TBP (1 nM, present in all lanes) was preincubated for 15 min with AdMLP TATA, and then 2 ng of purified mAbs was added followed by an additional 15-min incubation, after which increasing amounts of HMG-1 were added, with incubation continued for another 15 min. HMG-1 is present in lane 1, 160 nM; lanes 4-8 and 12-16, at 13, 20, 40, 80, and 160 nM, respectively. 2 ng of mAb1C2 is added in lanes 3-9, and 2 ng of mAb3G3 is added in lanes 11-17. Lanes 9 and 17 are control lanes with 300 nM of bovine serum albumin in lieu of HMG-1. The positions of TBP/TATA and HMG-1/TBP/TATA are indicated. The asterisk indicates the supershifted band, and NS denotes a nonspecific complex. C, mAb competitive binding for preestablished HMG-1/TBP/TATA complex. Human TBP (1 nM, present in all lanes) and HMG-1 were preincubated for 15 min with AdMLP TATA, after which increasing amounts of mAbs were added and then the incubation was continued for an additional 15 min. HMG-1 (80 nM) is present in lanes 1-4 and 6-10. mAb1C2 is present in lanes 1-4 (0.8, 2, 4, and 20 ng, respectively). mAb1C2 is present in lane 5 (20 ng) without HMG-1. mAb3G3 is present in lanes 7-10 (0.8, 2, 4, and 20 ng, respectively). mAb3G3 is present in lane 11 (20 ng) without HMG-1. Band positions are as indicated in B.

In the first set of experiments, TBP, TATA, and the antibody (1C2 or 3G3) were incubated, followed by addition of increasing amounts of HMG-1. Lane 3 in Fig. 7B shows that mAb1C2 produces a supershifted complex in the absence of HMG-1. The addition of increasing levels of HMG-1 produces the HMG-1/TBP/TATA complex as seen in the characteristic band for the complex (lanes 4-8). As seen in lane 5, the presence of HMG-1, at as low as 5 ng, competed effectively with mAb1C2 for binding to TBP and disrupted its binding to TBP/TATA, resulting in the loss of a supershifted complex. In contrast, the parallel experiments that used mAb3G3 (lanes 10-16) showed that HMG-1 binding did not disrupt antibody binding to TBP, as evident by the continued presence of the supershifted complex. These data indicate that HMG-1 and mAb1C2 compete for the same or overlapping sites and that the HMG-1 binding to TBP is stronger than that for mAb1C2 to TBP. On the other hand, HMG-1 and mAb3G3 do not compete for the same site(s) and bind simultaneously to different and non-overlapping sites.

Fig. 7C shows the titration in which the HMG-1/TBP/TATA complex was preestablished, and increasing levels of antibody were added in an attempt to compete with HMG-1 binding. The addition of increasing amounts of mAb1C2 to the complex did not displace HMG-1 from the complex. This antibody produced only a marginal band for a supershifted complex at very high levels of antibody, in contrast to that observed in the control lane 5, in which HMG-1 was absent. On the other hand, the comparable experiment in which the preestablished complex was titrated with mAb3G3 showed a consistent band for the supershifted complex in the presence of HMG-1, indicating again that mAb3G3 and HMG-1 bind simultaneously in the complex. Both of these findings are consistent with HMG-1 binding to the Q-tract of the N terminus of TBP.

HMG-1 Binds Strongly to TATA-binding Proteins That Contain Q-tracts-- Since the data indicate that the Q-tract is the primary target for HMG-1, TBP proteins from other species were investigated to determine if HMG-1 binding correlated with the presence of a Q-tract in the N terminus of TBP and whether this interaction extended more broadly to other species. We examined the HMG-1 interaction with human, Drosophila, and yeast TBP, which are shown schematically in Fig. 8A. Both human and Drosophila TBP have glutamine tracts, whereas yeast TBP does not.


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  		Fig. 8.   A, comparative binding of HMG-1 to hTBP/TATA, dTBP/TATA, and yTBP/TATA. Top panel, schematic representation of the TBPs from human, Drosophila, and yeast showing the Q-tracts. Bottom panel, hTBP (1 nM, lanes 1 and 2; 2.5 nM, lanes 3 and 4) or dTBP (1 nM, lanes 5 and 6; 8.5 nM, lanes 7 and 8), or yTBP (8.7 nM, lanes 9 and 10; 17.5 nM, lanes 11 and 12) is reacted with HMG-1 (80 nM, lane 2; 200 nM, lane 4; 80 nM, lane 6; 680 nM, lane 8; 700 nM, lane 10; 1400 nM, lane 12). All even-numbered lanes contain HMG-1 at a molar excess of 80 with respect to TBP. B, HMG-1 forms a stable EMSA complex with dTBP/TATA. dTBP (1.4 nM) is reacted with AdMLP DNA and HMG-1 (200 nM) in lanes 1-4, with increasing levels of anti-HMG-1 added in lanes 2 and 3. As a negative control, anti-TFIIB was added to dTBP, AdMLP DNA, and HMG-1 in lane 4. TBP, HMG-1, and AdMLP DNA were preincubated for 15 min, and anti-HMG-1 or anti-TFIIB was then incubated for an additional 15 min.

Fig. 8A shows the EMSA for the reaction of HMG-1 at two different levels with hTBP (lanes 1-4), dTBP (lanes 5-8), and yTBP (lanes 9-12) in a TBP/TATA complex. Binding of HMG-1 with hTBP/TATA is strong and results in a bandshift and a large increase in band intensity for the HMG-1/TBP/TATA complex (compare lanes 3 and 4). Binding of HMG-1 to dTBP/TATA shifts the EMSA band only marginally, and the increase in band intensity indicates that HMG-1 binding does stabilize the complex. Judging from the relative band intensity increase, the increase in stability is considerably smaller than that produced with hTBP. To verify that the addition of HMG-1 produced a complex in which HMG-1 is stably bound, the addition of anti-HMG-1 is shown to supershift the HMG-1/dTBP/TATA complex (Fig. 8B). Reaction of HMG-1 with yTBP/TATA is notably and significantly different. There is no evidence for HMG-1 binding to the yTBP/TATA complex. In fact, at both levels of yTBP used, the extent of yTBP/TATA complex formation decreased in the presence of HMG-1.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The rate of TBP binding and the stability of the TBP/TATA interaction within class II eukaryotic promoters are both highly regulated events, of central importance in the decision to commit to and/or initiate transcription. We show that HMG-1 significantly increases the affinity of TBP for the TATA element and stimulates the rate of TBP/TATA binding. In addition, we present a number of lines of evidence that show that HMG-1/TBP/TATA complex formation requires the acidic C terminus of HMG-1 and the Q-tract within the N terminus of TBP. The presence of HMG-1 expands the spectrum of regulatory controls for TBP binding and the promoter-bound complex.

The comparative binding of TBP with HMG-1 and the HMG-1(A-B) peptide, which has the acidic C terminus deleted, demonstrates (Fig. 1) the requirement for the C terminus of HMG-1 for complex formation with TBP/TATA. Only at very high levels of HMG-1(A-B) is there any evidence for a binding interaction, and at these levels, HMG-1(A-B) does not bind to TBP but binds nonspecifically to DNA. This brings out and emphasizes two important functional roles for the C terminus of HMG-1. First, it reduces the binding affinity of HMG-1 for DNA. Second, and more importantly, the C terminus is the critical domain for targeting HMG-1 to bind to TBP and effecting this protein-protein interaction. In contrast to this functional domain, the basic A and B boxes strongly promote nonspecific binding in the minor groove of DNA (38, 39). This is consistent with the findings that the didomain HMG-1(A-B) peptide binds more strongly to DNA. This is due in large part to its high positive charge (+22) relative to HMG-1, which has a more moderate negative charge (-8). These findings, coupled with those from previous studies (10, 12-17, 24), further highlight the distinctly different character of the domains in HMG-1, which may provide the basis for understanding its reported multifunctionalities in context-dependent transcription (40).

Although the functions of the C terminus of hTBP in transcription have been well documented (1-6), defining a role for the N terminus of hTBP has proved to be more difficult. The findings here (Figs. 2 and 3) indicate that the N terminus of hTBP is the primary target for the HMG-1 interaction. The targeting of HMG-1 to the N terminus appears unique when compared with the domain where other regulatory factors bind to TBP. Although TBP interacts with a multitude of repressors and activators, virtually all bind to the C terminus of TBP (2-4). Only in the case of the SNAPc promoter specificity factor does binding occur in the N terminus and in the Q-tract (7).

HMG-1 binds to TBP, both free in solution (10)2 and when bound to the TATA element. This latter interaction is consistent with its role as a repressor of both basal and Gal4-AH/USA-activated RNA pol II transcription (10). Notwithstanding the present results that show that the HMG-1 binding to the N terminus of hTBP is critical for a stable complex, there is evidence that HMG-1 also has secondary binding interactions with the C terminus of TBP (41).2 The HMG-1 binding to the N terminus, or both interactions, may inhibit simultaneous binding of HMG-1 and other general factors to TBP. For example, TFIIB can compete off HMG-1 from the HMG-1/TBP/TATA complex (HMG-1 is limiting), but when there are high excesses of HMG-1, an HMG-1/TFIIB/TBP/TATA complex can form (24, 41). In contrast, it has been proposed that TFIIA can dissociate the HMG-1 from HMG-1/TBP/TATA. In this way, increasing the level of TFIIA, but not TFIIB, was shown to restore basal and activated transcription in an in vitro assay (10).

The N terminus of hTBP reduces the binding affinity of TBP for the TATA element (7, 42). Interestingly, this is the same general effect that the C terminus of HMG-1 has on its nonspecific binding to DNA. It has been reported that TFIIA binding to yeast TBP enhances TBP binding to promoter DNA by eliminating the otherwise inhibitory effect of the N terminus in TBP (42). Our data indicate that the binding of HMG-1 to the N terminus of hTBP also increases the affinity of TBP for TATA. Although the result of the binding interaction may be quite complex, the direct HMG-1/TBP interaction may provide a similar effect in reducing an energetically unfavorable interaction between the N terminus and DNA and contributing to the mechanism by which HMG-1 binding stabilizes the complex.

Interestingly, the presence of TFIIA stimulates TBP binding and stabilizes TBP/TATA and facilitates further preinitiation complex assembly that can lead to productive transcriptional initiation. HMG-1 also stimulates the rate of TBP binding and leads to an increased TBP/TATA stability. In contrast, however, HMG-1 leads to the formation of a temporal, transcriptionally inactive promoter complex. This may provide a novel mechanism by which HMG-1 can establish a reversible "poised" but transcriptionally inactive complex that can suppress basal level transcription and, in selected promoters, be in a position to facilitate subsequent activation as proposed previously (43, 44).

EMSA experiments with hTBP deletions and monoclonal antibodies to epitopes in the N terminus were instrumental, and reinforced each other, in defining the Q-tract in hTBP as the decisive segment for HMG-1 binding. Segments I and III have little or no effect on enhancing the stability of the HMG-1/TBP/TATA complex (Fig. 6), whereas segment II greatly enhances complex formation. In further support of this, mAb1C2, which was specific for the Q-tract (9), was unable to bind to TBP in the presence of HMG-1 (Fig. 7). This is consistent with the idea that HMG-1 and mAb1C2 bind to the same or overlapping sites in the Q-tract. In contrast, the binding of mAb3G3 to its epitope in the first 10 residues of the N terminus was unaffected by the presence of HMG-1, and its binding supershifted the complex at all levels of HMG-1.

In one of the first reports that suggested a direct functional role for the N terminus of hTBP in transcription, it was shown that the N terminus mediates cooperative binding with SNAPc to the U6 promoter, resulting in enhanced U6 transcription by RNA polymerase III (7, 8). In this case, segments I and II were implicated as the target for SNAPc interaction. Monoclonal antibody 1C2, which binds hTBP at the same site as does HMG-1, was shown to inhibit in vitro transcription from TATA-containing promoters, from both RNA pol II and III (9). One can speculate from our data that HMG-1 may act as a more general transcriptional repressor, not only repressing transcription carried out by RNA pol II (10), but also repressing RNA pol III transcription for this class of small nuclear RNA genes. For both HMG-1 and mAb1C2, the target is the Q-tract. This suggests that in both these cases, the interaction may obstruct or obviate the function of the Q-tract in important protein-protein or protein-DNA interactions.

The presence of HMG-1 increased the stability of the HMG-1/TBP/TATA complex by about 20-fold, relative to TBP/TATA complex. This is a similar to, but greater than, the increased stability reported for the binding of the general transcription factors TFIIB or TFIIA to TBP/TATA. In these cases, the affinity of TBP for TATA was increased by up to 10-fold (31-34). There is no universal agreement on these values which suggests that there may be a significant dependence on conditions (32, 34). In the most extensive studies, Pugh et al. (32) have shown that TFIIA significantly increases the stability of the TBP/TATA interaction, as reflected by both an increase in the on-rate for TBP binding and a decrease in the off-rate by about a factor of 4 (32).

HMG-1 increased the affinity of the TBP/TATA interaction but without any significant effect on the dissociation kinetics of the complex. The decreased Kd value was predominantly a result of HMG-1 stimulating the on-rate constant for TBP. This complexation could provide TBP with a significant kinetic advantage in extending the conditions under which it could gain access to the TATA element. It would permit TBP to bind most promoters under conditions of limiting TBP levels and/or facilitate efficient TBP binding to promoters that lack strong TBP-binding sites.

The interaction of HMG-1 with TBP may have broader physiological importance in light of recent findings that HMG-1 was identified as a component of TFIID (41, 45). HMG-1 was found in association with a crude TFIID fraction from HeLa cells and was shown to serve as a coactivator for herpes simplex virus IPC4 in vitro (45). It was also shown that the GST-A box of HMG-1 was able to pull down TFIID in crude HeLa extract, suggesting that HMG-1 binds to TBP and/or other TFIID components (41). Both these studies suggest that the strong HMG-1 interaction with hTBP described here may not only be retained but may be further reinforced in binding to the multisubunit TFIID complex.

As a result of a lack of HMG-1 interaction with yeast TBP, it has been suggested that HMG-1 binds to TBP in a species-specific manner (41). A comparison made of calf thymus HMG-1 interacting with TBP from human, Drosophila, and yeast found that HMG-1 formed a stable EMSA complex with both human and Drosophila TBP, but not with yeast TBP (Fig. 7). In fact, HMG-1 dissociates yTBP from the TATA element, in a manner similar to that observed with its interaction with human c-TBP (Fig. 2). Although the C terminus of TBP is highly conserved over these species (81% homology) (6, 46-51, 57), the N terminus is generally considered to be highly divergent, with the exception that the N terminus is highly conserved over vertebrates (52-53). A Q-tract remains in all vertebrates, although its length varies, whereas segments I and III are 78 and 73% identical, respectively, in human (46, 47, 54, 55), mice (52), hamster (7), and two different vipers (53, 56). In addition, the N terminus of Drosophila TBP is longer than that in human TBP (Fig. 8) and is similar in that it contains primarily hydrophobic residues, with very few charged residues (5% of the residues). In addition, it contains two smaller Q-tracts (6 and 8 glutamines, respectively) that are separated by 32 residues (48). However, yeast TBP is quite different, in that the N terminus is significantly shorter, has a large fraction of charged residues (39%), and does not contain a Q-tract (6, 57). The data are consistent with HMG-1 binding to Q-tracts in the N terminus, although the enhanced stability as a result of HMG-1 binding to dTBP is significantly reduced compared with that in hTBP. This is what would be expected as a result of both the reduced sizes and the separation of the two Q-tracts in dTBP. Surprisingly, it was reported that there was no detectable interaction between HMG-1 and dTBP in GST pull-down experiments (41). From the limited findings, calf thymus HMG-1 binding to TBP clearly occurs in different species and may be quite general, extending throughout the vertebrates, to Drosophila, and perhaps to others.

These results lead to a general view for the principal interaction between HMG-1 and hTBP in the HMG-1/TBP/TATA complex. A simple working model (Fig. 9) that is consistent with the current data emphasizes a significant and direct interaction of the acidic residues in HMG-1 with the Q-tract in the N terminus of hTBP. In this interaction, one can hypothesize that the amide hydrogens in the glutamine residues may serve as hydrogen bond donors, with the negatively charged carboxylate groups of glutamate/aspartate residues in HMG-1 acting as hydrogen bond acceptors. This "zipper of electrostatic hydrogen bonds" would be expected to provide significant stability to this intermolecular interaction. In addition, the HMG-1 interaction to the N terminus may reduce an energetically unfavorable interaction with DNA. Together with the EMSA data, and the GST-TBP pull-down results that show detectable interactions between the A and B boxes and TBP (data not shown), we propose that although the primary stabilizing interaction involves the C terminus of HMG-1 and the N terminus of hTBP, additional sites in both HMG-1 and hTBP appear to be implicated in the overall complexation (22, 39).


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  		Fig. 9.   Model for HMG-1/hTBP interaction. Schematic representation of the acidic C terminus of HMG-1 binding to the Q-tract in the N terminus of hTBP in the HMG-1/TBP/TATA complex.


    ACKNOWLEDGEMENTS

We thank M. Bianchi, P. Chambon, N. Hernandez, T. Kouzarides, F. Pugh, R. Roeder, and R. Tjian for expression vectors and/or antibodies used in this study.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant R15GM54357, Ohio Cancer Associates (OCRA), and the American Cancer Society, Ohio Division.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. Tel.: 419-372-8293; Fax: 419-372-9809; E-mail: wscovel@bgnet.bgsu.edu.

Published, JBC Papers in Press, June 4, 2001, DOI 10.1074/jbc.M011792200

2 D. Das and W. M. Scovell, unpublished data.

    ABBREVIATIONS

The abbreviations used are: TBP, TATA-binding protein; AdMLP, adenovirus major late promoter; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; HMG-1, high mobility group-1 protein; hTBP, human TATA-binding protein; mAb, monoclonal antibody; Q, glutamine; SNAPc, small nuclear RNA-activating protein complex; pol, polymerase; cTBP, the C terminus of human TBP, residues 160-335; nTBP, the N terminus of human TBP, residues 1-159; dTBP, Drosophila TBP; yTBP, yeast TBP.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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S. C. Roemer, J. Adelman, M. E. A. Churchill, and D. P. Edwards
Mechanism of high-mobility group protein B enhancement of progesterone receptor sequence-specific DNA binding
Nucleic Acids Res., June 1, 2008; 36(11): 3655 - 3666.
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 J. Virol.Home page
A. Francois, M. Guilbaud, R. Awedikian, G. Chadeuf, P. Moullier, and A. Salvetti
The Cellular TATA Binding Protein Is Required for Rep-Dependent Replication of a Minimal Adeno-Associated Virus Type 2 p5 Element
J. Virol., September 1, 2005; 79(17): 11082 - 11094.
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 Mol. Cell. Biol.Home page
D. Biswas, Y. Yu, M. Prall, T. Formosa, and D. J. Stillman
The Yeast FACT Complex Has a Role in Transcriptional Initiation
Mol. Cell. Biol., July 15, 2005; 25(14): 5812 - 5822.
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 J. Virol.