©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of the COOH-terminal Transactivation Domain of p65 NF-B with TATA-binding Protein, Transcription Factor IIB, and Coactivators (*)

(Received for publication, October 21, 1994; and in revised form, December 27, 1994)

M. Lienhard Schmitz (§) Gertraud Stelzer (1) Herbert Altmann (2) Michael Meisterernst (1) Patrick A. Baeuerle

From the  (1)Institute of Biochemistry, Albert Ludwigs University, Hermann-Herder-Strasse 7, D-79104 Freiburg, the Laboratory for Molecular Biology/Gene Center, Würmtalstrasse 221, D-81375 Munich, and the (2)Institute of Biochemistry, Ludwigs Maximilians University, Am Klopferspitz 18a, D-82152 Martinsried, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We show that the transactivating COOH terminus of the p65 subunit of human transcription factor NF-kappaB directly binds the general transcription factors TFIIB and TATA-binding protein (TBP) in vitro. Interaction of p65 with TFIIB required the most COOH-terminal sequence repeat within TFIIB. A functional interaction of TFIIB with p65 was evident from assays in yeast cells. Cotransfection experiments in COS cells revealed that only overexpression of TBP was able to further stimulate p65-dependent transactivation of a reporter gene. The coexpression of neither TBP nor TFIIB was able to relieve squelching, indicating the involvement of additional factors in transactivation by p65. A cell-free assay using highly purified factors revealed a specific transcriptional stimulation through the COOH-terminal activation domain of NF-kappaB by at least one cofactor, PC1, isolated from HeLa cells. These data show that the potent acidic transactivation domains in the COOH terminus of p65 are able to functionally recruit various components of the basic transcription machinery as well as coactivators.


INTRODUCTION

The accurate and regulated transcription by RNA polymerase II requires dynamic interactions between several classes of transcriptional regulatory proteins. One class is constituted by sequence-specific DNA-binding proteins. These are typically composed of several discrete domains minimally mediating DNA binding, nuclear translocation, and transactivation (reviewed by Ptashne and Gann (1990)). In the physiological situation, the template for transcription is contained in the condensed chromatin structure. Some transcription factors are found to counteract the chromatin-mediated repression (``antirepression''), whereas all transcription factors are able to facilitate the inherent transcription reaction, which is designated as ``true activation'' (reviewed by Croston and Kadonaga(1993)). This process requires a second class of proteins, which are the general transcription factors. Those include TFIIA, (^1)TFIIB, TFIID, TFIIE, TFIIF, TFIIH, TFIIG/TFIIJ, and RNA polymerase II (reviewed by Roeder(1991)). A key step in transcription initiation is the binding of the multisubunit complex TFIID to the TATA element close to the transcription start site. A component of the TFIID complex is the TATA-binding protein (TBP), which is also required for transcription of RNA polymerase I and III promoters (reviewed by White and Jackson(1992)). TFIID contains numerous additional factors referred to as TBP-associated factors (TAFs), from which many of the corresponding cDNAs from Drosophila and human were recently cloned (reviewed by Goodrich and Tjian(1994)). Recombinant TBP protein can substitute for TFIID fractions for basal RNA polymerase II transcription from TATA-containing promoters. However, recombinant TBP fails to mediate activated transcription induced by transcriptional activators (Pugh and Tjian, 1990; Hoffmann et al., 1990).

Activated transcription requires a third class of transcriptional regulatory proteins, the so-called coactivators. Many of the TAFs in TFIID were found to have this coactivating function (reviewed by Goodrich and Tjian(1994)). But also non-TAF proteins were found to have such an activity. Examples are ADA2, ADA,3, SW1, SW2, and SW3, which have been identified in yeast by biochemical and genetic approaches (Berger et al., 1992; Peterson and Herskowitz, 1992; Piña et al., 1993; Kim et al., 1994). Among coactivators isolated from mammalian cells are the B cell-derived factor Oca-B (Luo et al., 1992) as well as the positive cofactors (PCs) PC1, PC2, PC3/Dr2, and p15/PC4 (Meisterernst et al., 1991; Kretzschmar et al., 1993, 1994a, 1994b; Merino et al., 1993). All of these coactivators stimulate activator-dependent transcription. The specific protein/protein interactions required for activated transcription involve interactions between transcription activation domains of sequence-specific activators with coactivators (Goodrich et al., 1993; Ferreri et al., 1994; Ge and Roeder, 1994) and general transcription factors (reviewed by Tjian and Maniatis(1994)).

A well studied paradigm for an inducible transcriptional activator is nuclear factor kappaB (NF-kappaB) (reviewed by Grilli et al. (1993) and Baeuerle and Henkel(1994)). NF-kappaB is retained in the cytoplasm by the inhibitory IkappaB subunits in an inactive form (Baeuerle and Baltimore(1988); reviewed by Schmitz et al. (1991)). The factor becomes readily activated when cells are treated with inflammatory cytokines, tumor promoters, viruses, and lipopolysaccharides (reviewed by Baeuerle and Henkel(1994)). All these agents lead to phosphorylation and subsequent proteolytic degradation of IkappaB, thereby allowing a DNA-binding NF-kappaB dimer to enter the cell nucleus. The DNA-binding form of NF-kappaB activates transcription of numerous target genes encoding proteins involved in inflammatory, immune, and acute phase response (reviewed by Grilli et al.(1993)). NF-kappaB DNA-binding subunits comprise p50 (Kieran et al., 1990; Ghosh et al., 1990; Meyer et al., 1990), p65 (Baeuerle and Baltimore, 1989; Ruben et al., 1991; Nolan et al., 1991; Ballard et al., 1992), p52 (Schmid et al., 1991), RelB (Rysek et al., 1992; Ruben et al., 1992b), and c-Rel (Wilhelmsen et al., 1984). These proteins share a conserved 300-amino acid sequence in the NH(2)-terminal portion, which is also present in the oncogene v-rel from the avian retrovirus REV-T (Stephens et al., 1983) and the morphogen dorsal from Drosophila (Steward, 1987). We refer to this conserved region as the NRD (NF-kappaB/Rel/dorsal) domain, which contains the subdomains important for DNA-binding, dimerization, nuclear localization, and protein/protein interactions. The p65 subunit is frequently detected in NF-kappaB complexes and has a strong transcription activation potential (Schmitz and Baeuerle, 1991; Ruben et al., 1992a; Fujita et al., 1992; Ballard et al., 1992; Schmitz et al., 1994). It contains at least two independent transcription activation domains (TADs) in its COOH-terminal 120 amino acids. One of these domains, TA(1), is contained in the COOH-terminal 30 amino acids (amino acids 521-551) of p65. Both TADs of p65 belong to the class of acidic activation domains and are very similar to the TAD of herpes simplex virus protein VP16 (Schmitz et al., 1994).

In this study we have addressed the question of which proteins interact with the transactivating COOH terminus of human p65 to mediate its activating potential. The general transcription factors TBP and TFIIB were found to specifically bind to the p65 TADs. The binding of TFIIB to NF-kappaB p65 required the most COOH-terminal sequence repeat of TFIIB. TBP was able to stimulate p65-dependent expression in intact cells. Under cell-free conditions, activation of transcription by the p65 COOH terminus was found to depend on both the TFIID complex and on at least one coactivator fraction, called PC1 (Meisterernst et al., 1991).


MATERIALS AND METHODS

Eucaryotic Cell Culture, Transfections, and Gene Expression Assays

Monkey COS-7 cells were grown at 37 °C in Dulbecco's modified Eagle's medium containing 1% penicillin/streptomycin and 10% fetal calf serum (all from Life Technologies, Inc.). Approximately 5-10 times 10^6 cells were transfected with 2 pmol of the reporter construct, 0.5 pmol of the p65 expression plasmid, and 1 pmol of the expression plasmids for TBP and/or TFIIB. All plasmids used for transfections were purified on CsCl gradients. Cells were transfected in solution as described (Lopata et al., 1984) and plated after transfection onto 10-cm dishes. After 36-48 h cells were harvested by scraping with a rubber policeman. The protein concentration was determined by the method of Bradford(1976), and equal amounts of proteins were assayed for chloramphenicol acetyltransferase (CAT) activity. Acetylated and non-acetylated forms of [^14C]chloramphenicol were separated by thin-layer chromatography, and the incubation conditions were chosen to result in conversion of [^14C]chloramphenicol not exceeding 60%. Transfections were performed at least in duplicate, and the results were quantified by liquid scintillation counting. For squelching experiments, 2 pmol of the Gal4-dependent reporter plasmid, 0.25 pmol of the Gal4 fusion protein construct, and 3 pmol of the expression plasmid or control vector (RcCMV, Invitrogen) were used. In the course of the experiments dealing with the relief of squelching, between 0.5 and 3 pmol of the expression vectors for TBP and TFIIB were cotransfected. The analysis of the in vivo interaction between p65 and TFIIB was carried out in the yeast strain EGY48. This strain contains four LexA binding sites in the promoter controlling the expression of a Gal1-LacZ fusion protein and has been described elsewhere (Brent and Ptashne, 1985). Yeast transformations were conducted as described (Schiestl and Gietz, 1989), and beta-galactosidase assays were performed according to standard protocols (Altmann et al., 1994).

Plasmids

The vector pBS-TBP contains the TBP cDNA in a Bluescript vector and was a kind gift of Dr. T. Wirth (Heidelberg). Subcloning of the full-length cDNA from TFIIB into a Bluescript vector after PCR amplification from pHIIB (Ha et al., 1991) resulted in the plasmid pBS-IIB. The COOH-terminal repeat of TFIIB was deleted by removing the HindIII/Eco47III-fragment from the parental vector pBS-IIB. Eucaryotic expression vectors for TBP and TFIIB were made by inserting the appropriate portions from pBS-TBP and pBS-IIB into the RcCMV vector (Invitrogen). The LexA-TFIIB fusion protein construct was made by inserting the EcoRI/BamHI-fragment from pBS-IIB into pEG202 (Golemis and Brent, 1992) digested with the same enzymes. The Gal-inducible yeast expression vector for p65 was constructed by inserting the BamHI fragment from pBS-p65Delta (Narayanan et al., 1992) into pMB221T (Bröker et al., 1991). The vectors pHisGal, pHisGalp65 (Schmitz and Baeuerle, 1994), RcCMV-relADeltaDNA, and RcCMV-relADeltaDNADeltaTA (Schmitz et al., 1994) have been described. The clone p65DeltaC contains amino acids 1-442 of p65 and was generated by inserting the HindIII/PvuII fragment from RcCMV-p65 into RcCMV, which was opened with XbaI, filled in using Klenow polymerase, and redigested with HindIII. The vector pHisGal-p65 was constructed by inserting the XhoI/HindIII fragment from Gal4-p65 (Schmitz and Baeuerle, 1991) into pHisGal. The kappaB-dependent reporter plasmid J16 contained the two NF-kappaB binding sites 5`-GGGACTTTCC-3` in the promoter region controlling the expression of a CAT reporter gene (Pierce et al., 1988). The Gal4-dependent reporter plasmid had two Gal4 binding sites upstream from a truncated thymidine kinase promoter and a CAT gene (Baniahmad et al., 1992).

Analysis of Protein/Protein Interactions

The DNA-binding domain of the Gal4 protein (amino acids 1-147) and the Gal4-p65 fusion proteins were expressed in bacteria and purified to homogeneity as described (Schmitz and Baeuerle, 1994). These purified proteins, as well as bovine serum albumin (BSA) as an additional control protein, were coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.) according to the instructions of the manufacturer. After the coupling procedure, the columns were equilibrated with binding buffer (BB) consisting of 12 mM Hepes/KOH, pH 8, 12% glycerol, 100 mM KCl, 5 mM MgCl(2), 1 mM beta-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 10 µM ZnCl(2), and 1 mM spermidine. Proteins were labeled with [S]methionine (Amersham Corp.) using the coupled TnT in vitro transcription/translation system from rabbit reticulocytes (Promega Inc.). Equal amounts of S-labeled proteins were incubated with 50 µl of protein-coupled Sepharose beads in BB. Binding reactions were allowed at room temperature for 40 min. The beads were subsequently washed several times in BB, and the salt concentration of the binding buffer was increased stepwise to 300 mM KCl in the course of the washing steps. The bound proteins were eluted by boiling the Sepharose beads in 1 times SDS sample buffer and loading the samples on 12% SDS-polyacrylamide gels. Gels were subsequently dried and autoradiographed.

Proteins tested in immunoprecipitation assays were labeled with [S]methionine (Amersham Corp.) by in vitro transcription/translation. The respective proteins were incubated in a total volume of 50 µl in IP buffer consisting of 12 mM Hepes/KOH, pH 8, 12% (v/v) glycerol, 400 mM KCl, 5 mM MgCl(2),1 mM phenylmethylsulfonyl fluoride, 10 µM ZnCl(2), 1 mM spermidine, and 0.5% (v/v) Tween 20 for 30 min at room temperature. Subsequently, 0.5 µl of a polyclonal antibody (Santa Cruz Biotechnology) directed against the NRD region from p65 was added and the sample was incubated on a rotating wheel for 3 h at 4 °C. After addition of 50 µl of Protein A-Sepharose beads (Pharmacia Biotech Inc.) preswollen for 20 min in IP buffer and 10 µg of BSA (Sigma), the mix was incubated again on a rotating wheel for 1 h at 4 °C. The beads were washed six times with IP buffer. Finally, the beads were boiled for 5 min in 20 µl of 1 times SDS sample buffer and proteins separated on a reducing 10% SDS-polyacrylamide gel. The gel was then dried and exposed to x-ray film at -80 °C.

In Vitro Transcription Assays

Transcription templates, which contain sequences of the adenovirus major late or the human immunodeficiency virus-1 core promoters carrying five upstream-located Gal4 recognition sites and a 380-base pair G-less cassette, have been described elsewhere (Kretzschmar et al., 1994a). Standard transcription reactions included 20 ng of each template, 5-10 ng of recombinant TFIIB, 1.0 µl of TFIID (DE52 fraction, 0.5 mg/ml), 20 ng of recombinant TFIIEalpha, 5-10 ng of recombinant TFIIEbeta, 1.5 µl of TFIIF/TFIIH fractions (phenyl-Superose; Stelzer et al., 1994), and 0.1 µl of RNA Polymerase II (DE52 fraction; Stelzer et al., 1994). The PC1 coactivator fraction was purified up to the heparin-Sepharose step as described by Meisterernst et al.(1991). Subsequently, PC1 was further enriched on Mono S in a linear gradient from 0.1 M to 0.5 M sodium phosphate (pH 7.0) in a buffer containing 10% glycerol, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.001% Nonidet P-40. PC2 fractions (second DE52 column) were separated from PC1 on DEAE-cellulose (DE52) as described previously (Kretzschmar et al., 1994a). Transcription reactions were incubated at 28 °C for 1 h and subsequently processed and analyzed as described previously (Meisterernst and Roeder, 1991).


RESULTS

The Transactivating COOH Terminus of NF-kappaB p65 Binds TFIIB and TBP

The TADs of p65 belong to the group of acidic activators (Schmitz et al., 1994). Since a variety of acidic activation domains were shown to interact with the general transcription factors TBP and TFIIB, we investigated whether they also interact with the TADs of NF-kappaB p65. A His-tagged fusion protein between Gal4(1-147) and the p65 COOH terminus(471-551) and as a control the Gal4(1-147) protein were expressed in Escherichia coli and purified to homogeneity (Schmitz and Baeuerle, 1994). The purified proteins and BSA as an additional control were coupled to CNBr-activated Sepharose 4B. Equal amounts of in vitro translated [S]methionine-labeled TBP and TFIIB proteins were passed over Sepharose beads coupled with the acidic protein BSA, Gal4, or Gal4-p65. After extensive washing of the columns with 300 mM KCl, bound proteins were eluted with 1 times SDS sample buffer and analyzed by electrophoresis on a SDS gel. The result of such an experiment is shown in Fig. 1A. Most of the loaded general transcription factors were found to be specifically retained by the Gal-p65 column (lanes1 and 4), whereas they bound only weakly to the control column with Gal4 alone (lanes2 and 5). The weak binding to Gal4 is most probably due to the presence of a cryptic transcription activation domain between amino acids 90 and 147 of Gal4 (Lin et al., 1988). Virtually no TBP and TFIIB could be eluted with SDS sample buffer from a column with coupled BSA as control for an acidic protein (lanes3 and 6). Several other proteins were tested for their ability to bind to the Gal-p65 protein. None of the histone proteins H1, H2A, H2B, H3, and H4 were retained on the column (data not shown). An interaction between p65 and TFIIB and TBP was also evident from coimmunoprecipitation experiments (Fig. 1B). Only in the presence of the full-length p65 protein could an antiserum against p65 immunoprecipitate in vitro translated [S]methionine-labeled TBP and TFIIB (lanes2 and 3). A COOH-terminally truncated p65 (amino acids 1-442) could not precipitate significant amounts of TBP and TFIID (lanes5 and 6), showing that the two basal factors predominantly interacted with the COOH-terminal portion of p65.


Figure 1: Specific association of TBP and TFIIB with the transactivation domain of human NF-kappaB p65 in vitro. A, TBP and TFIIB bind to the transactivating COOH terminus of human p65. Equal amounts of in vitro translated [S]methionine-labeled TBP (lanes 1-3) and TFIIB (lanes4-6) were incubated with Gal4(1-147), Gal-p65, and BSA, which were covalently coupled to Sepharose beads as indicated at top of the figure. The beads were extensively washed and eluted with SDS, and proteins were separated by 12% SDS-PAGE. An autoradiogram of a representative experiment is shown. The position of prestained molecular weight markers is indicated. The arrows point to the positions of TFIIB and TBP. B, the COOH terminus of p65 is required for binding of TBP and TFIIB. Rabbit reticulocyte lysates were programmed in the presence of [S]methionine with cDNAs for the p65, TBP, TFIIB, and p65DeltaC (lacking the 109 COOH-terminal amino acids of p65) proteins. The labeled proteins were incubated in the indicated combinations and subsequently precipitated with an anti-p65 antibody. An autoradiogram of a 12% SDS-polyacrylamide gel is shown and the positions of the marker proteins indicated at the left. The arrows mark the positions of the respective proteins. C, the COOH-terminal sequence repeat of TFIIB is necessary for the specific interaction with the p65 COOH terminus. The structure of the wild-type (TFIIB) and the COOH-terminally truncated TFIIB (TFIIBDeltaC) molecule is drawn schematically at the top. The location of the repeated domains is shown by arrows, and their amino acid positions within the molecule are given. +, positions of two regions that are highly enriched in basic amino acids. Lanes1 and 2 show the input proteins TFIIBDeltaC and TFIIB, respectively. Each of the proteins was incubated with Gal-p65 protein coupled to Sepharose, and the experiment was further conducted as described for A. Lanes3 and 4 show an autoradiogram of the eluates after SDS-PAGE. Details of the figure legend are as explained in A.



TFIIB carries in its COOH-terminal half two imperfect direct repeated sequences, the first of which contains a potential positively charged amphipathic alpha-helix at its COOH terminus (see Fig. 1B). Binding experiments with a TFIIB mutant retaining the complete first repeat but lacking the more COOH-terminal repeat were performed in order to assess the role of this conserved structure. In contrast to the full-length TFIIB protein, the COOH-terminally truncated version of TFIIB was not able to efficiently bind to the Gal-p65 protein (Fig. 1B, compare lanes3 and 4). This shows that the first repeat structure within TFIIB, including the positively charged amphipathic alpha-helix, is not sufficient to mediate the specific contact to p65. Alternatively, the truncation might have altered the structure of TFIIB to preclude p65 binding. In conclusion, the transactivating p65 COOH terminus can directly interact with the basal transcription factors TFIIB and TBP, as was found for various other acidic domains.

TFIIB Can Associate with p65 in Intact Cells

The interaction between TFIIB and p65 was functionally tested in yeast cells. A fusion protein between the DNA-binding domain of the bacterial repressor LexA and the full-length TFIIB protein was introduced into the yeast strain EGY48. This yeast strain bears internally a lacZ indicator gene controlled by four LexA binding sites. This yeast strain was additionally transformed with a vector containing the NF-kappaB p65 cDNA under the control of the yeast Gal1 promoter. To prevent p65 from binding to a cryptic kappaB site and from homodimerizing, a p65 mutant (p65Delta) deleted in the DNA binding/dimerization domain (amino acids 222-231) was used (Narayanan et al., 1992). The Gal1 promoter is inactive in the presence of glucose as carbon source and allows the inducible transcription of p65Delta only in the presence of galactose. No LacZ activity could be detected in colonies grown on media containing glucose, showing that the LexA-TFIIB protein was transcriptionally inactive (Fig. 2, upperpanel). Induction of p65Delta expression in the presence of galactose caused lacZ expression in yeast cells (lowerpanel), suggesting that the p65Delta protein entered the nucleus and associated with LexA-TFIIB. The transactivating COOH terminus of p65 was apparently tethered via the LexA-TFIIB fusion protein to the promoter controlling the expression of the lacZ reporter gene. Yeast cells transformed with the expression plasmid for p65Delta and a plasmid encoding only the DNA-binding domain of LexA (amino acids 1-202) showed no increase of lacZ expression after induction of p65Delta with galactose (data not shown). These experiments demonstrate that p65 binds to the TFIIB portion of the LexA-TFIIB fusion protein.


Figure 2: TFIIB and p65 interact in vivo. The experiment is displayed in a schematic drawing at left. Yeast cells bearing an integrated lacZ reporter gene controlled by LexA binding sites were cotransformed with two plasmids: a plasmid constitutively expressing a fusion protein between the DNA-binding domain of LexA and TFIIB, and a plasmid encoding NF-kappaB p65Delta controlled by a Gal1 promoter, which is shown as an arrow. Induction of p65Delta expression with galactose leads to intracellular association of p65Delta with the TFIIB portion. The resulting transcriptional activation of the lacZ reporter gene is symbolized by an arrow. The right part of this figure shows yeast colonies grown on Ura, Leu, His plates containing the LacZ substrate X-Gal either in the presence of glucose (top) or galactose (lower).



Cooperation of p65 and TBP in Vivo

The functional implications of the interaction of p65 with both TBP and TFIIB were investigated by studying the effect of transient overexpression of TBP and TFIIB on p65-mediated transcription. COS7 cells were transfected with a plasmid containing the CAT reporter gene under the control of two NF-kappaB binding sites and different combinations of expression vectors for p65, TBP and TFIIB. As expected, expression of p65 alone led to a significant increase in kappaB-dependent transcription (Fig. 3, lane2). Coexpression of TBP resulted in a 2-3-fold stimulation of p65-dependent transactivation (Fig. 3, lane 3). In contrast to the stimulatory effect seen with TBP, no significant effect could be detected upon coexpression of TFIIB. Expression of both general transcription factors also failed to display a statistically significant stimulatory effect on p65-dependent gene expression, even when the amount of expression vectors was varied over a wide concentration range (data not shown). No enhancement of kappaB-dependent transactivation was seen upon expression of TBP or TFIIB alone (Fig. 3, lanes6 and 7), showing that TBP could exert its stimulatory effect only in conjunction with p65. The finding that TBP enhances p65-dependent transcription supports that the physical interaction between the two proteins is of biological significance.


Figure 3: Functional interaction of p65 and TBP in transcription activation. COS7 cells were cotransfected with the kappaB-dependent CAT reporter plasmid J16 (Pierce et al., 1988) and the expression vectors for p65, TBP, and TFIIB as indicated. Gene activation was measured as percent acetylation of [^14C]chloramphenicol, and the activity of the reporter plasmid alone was set to 1. The standard deviations are indicated by bars and were obtained from four independent experiments.



Additional Molecules Are Required for Transcription by p65

In squelching experiments the activity of a given TAD can be influenced by the simultaneous overexpression of homologous or heterologous TADs (for a review, see Ptashne and Gann(1990)). The reduced transcriptional activity of the activator under squelching conditions is explained by the exhaustion of a limiting factor necessary for transcription. On the assumption that TBP and TFIIB are the only proteins contacting and influencing the activity of p65 their cotransfection should relieve squelching. As seen in Fig. 4, the Gal-p65 protein is able to stimulate the expression of a CAT reporter gene, which is controlled by two Gal4 binding sites (Fig. 4, compare lanes1 and 2). Overexpression of relADeltaDNA, which gives rise to a p65 incapable of binding to DNA (Schmitz et al., 1994), significantly reduced transactivation by Gal-p65 (Fig. 4, lane4). The control vector relADeltaDNADeltaTA, a derivative of relADeltaDNA lacking the transactivating COOH terminus, showed no squelching effect (Fig. 4, lane3). The observed effects were specific because the Gal4 DNA binding activity did not change under squelching conditions (data not shown). Furthermore, a cotransfected RSV-lacZ control plasmid showed comparable activity under normal and squelching conditions and coexpression of the unrelated transcription factor GHF (growth hormone factor) did not alter the transcriptional activity of Gal-p65 (data not shown). However, neither the coexpression of TBP or TFIIB nor their simultaneous expression could relieve the observed squelching effect (Fig. 4, lanes 5-7). This failure to relieve the squelching effect was independent of the amount of expression vector used (data not shown). These results indicate that the transactivating p65 COOH terminus requires additional proteins in order to mediate its transactivating effects.


Figure 4: Activated transcription by p65 requires additional proteins. COS7 cells were transfected with a Gal4-dependent CAT reporter gene and the indicated expression vectors. The negative and positive controls in lanes 1 and 2 received the same amount of DNA as ``empty'' expression vector (RcCMV). The squelching plasmid relADeltaDNA is a p65 mutant incapable of binding to DNA (Schmitz et al., 1994). relADeltaDNADeltaTA is a derivative thereof lacking both transactivation domains. The figure shows the result from a representative CAT assay in which the squelching construct was present at 12-fold molar excess over the Gal activator plasmid. The presence (+) or absence(-) of the respective expression plasmids is indicated in the lower part of the figure. In the typical experiment shown, 2 pmol of expression vectors for TBP and TFIIB were used. The weak increase in lane7 was not reproducible in three independent experiments.



Coactivators Stimulate p65-dependent Transcription Activation in Vitro

In order to identify further protein components involved in p65-dependent transcription cell-free transcription, experiments were performed both in crude nuclear extracts from HeLa cells and in purified class II transcription systems. Initially, the purified Gal4(1-147) and Gal4(1-147)-p65 proteins were tested for their transcriptional activity in crude HeLa nuclear extracts, which are thought to be more physiological than assays consisting of highly purified components. In contrast to Gal4(1-147), the Gal4-p65 protein stimulated the activity of the adenovirus 2 major late promoter, which contains five upstream-located Gal4 recognition sites (Fig. 5, compare lanes4 and 5). While the effects of the TA(1) domain are weak in vitro as compared to the in vivo activity, these experiments suggest that transcriptional activation can at least in part be reconstituted in vitro. However, the Gal4(1-147) protein can also display significant transcriptional activity when added in saturating concentrations (Fig. 5, compare lanes1 and 2).


Figure 5: In vitro transcription activity of the Gal4-TA(1) protein in crude nuclear extracts. The indicated amounts of purified Gal4(1-147) and Gal4-p65 proteins were tested for their influence on the activity of the Gal4-dependent Ad2ML promoter. An arrow points to the specific transcript from the G-less cassette. The autoradiogram shows the typical result from a transcription reaction performed for 1 h at 28 °C.



In contrast to crude transcription systems, both Gal4-p65 and a Gal4-AH (amphipathic helix) protein were completely inactive (Fig. 6A, lanes2 and 4) in transcription systems reconstituted of highly purified general transcription factors (Kretzschmar et al., 1994a). Both proteins were transcriptionally active only in the presence of coactivators, as exemplified here for the coactivator fraction PC1 (Fig. 6A, lanes6 and 8).


Figure 6: Activated transcription by Gal4-p65 is specifically stimulated by the PC1 coactivator fraction. A, effects of the PC1 fraction on transcriptional activation by Gal4 derivatives as indicated in a transcription system consisting of general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II. B, comparison of PC1 and PC2 effects on Gal4 derivatives as indicated. An autoradiogram is shown with the upperarrow (Gal) pointing to the Gal-dependent transcript and the lowerarrow (MLDelta) pointing to the internal control template without Gal4 binding sites.



Several positive cofactors, originating from the upstream stimulatory activity fraction (Meisterernst et al., 1991), which had been shown previously to stimulate activator-dependent transcription in vitro, were subsequently analyzed for their ability to allow activation of transcription by Gal4 derivatives. All four cofactors tested (PC1 to p15/PC4; reviewed by Kretzschmar et al. (1994b)) stimulated transcriptional activation by the Gal4-p65 protein (data not shown). Remarkably, as exemplified for PC2 (Fig. 6B, lane8versuslane9), the coactivating potential of cofactor fractions PC2, PC3/Dr2, and p15/PC4 was mostly dependent on the Gal4(1-147) protein, while the domain responsible for transcriptional activation in vivo (the TA(1) domain) had very little or no additional effect on transcription. However, in the presence of a partially purified PC1 fraction, the Gal4p65 protein could stimulate transcription above levels observed with a Gal4(1-147) protein (Fig. 6B, lane2versuslane1). Conversely, and in agreement with previous studies (Kretzschmar et al., 1994a), Gal4-AH did stimulate transcription above levels observed with a Gal4(1-147) protein in the presence of a partially purified PC2 fraction (Fig. 6B, lane6versuslane8) but not in the presence of a PC1 fraction (lane2versuslane4). Although specific effects on transcription of activation domains AH and TA(1) were weak (approximately 2-fold above levels observed with Gal4(1-147)), these differential effects of activators and cofactors, if analyzed in parallel (Fig. 6B), were reproducibly observed in experiments that were conducted with optimal activator concentrations (Kretzschmar et al., 1994a). These experiments suggested that PC1, in contrast to other presently defined cofactors (PC2, PC3/Dr2, and p15/PC4), displays specificity for the COOH-terminal transcriptional activation domain of p65, while the cofactor PC2 acts preferentially through the AH domain of Gal4-AH. They further indicate structural differences in the activation domains AH and TA(1), although both have been classified previously as acidic activators (Ptashne and Gann, 1990; Schmitz et al., 1994). It therefore seems possible that distinct cofactors bind activation domains with different affinity.


DISCUSSION

In this study, we demonstrate a direct interaction of the acidic activation domain of NF-kappaB p65 with two components of the basal transcription machinery: TBP and TFIIB. The interaction with TFIIB was of sufficient avidity to allow assembly of p65 with a LexA-TFIIB fusion protein and subsequent LexA-dependent transcriptional activation in yeast cells. The overexpression of TBP did significantly stimulate p65-dependent gene expression. Similar enhancing effects of TBP were detected for gene activation by the NRD protein family member c-Rel (Kerr et al., 1993; Xu et al., 1993) and the human T-cell leukemia virus type 1 transactivator Tax (Caron et al., 1993). The stimulatory effect of TBP may be explained by the fact that it can be rate-limiting in cells (Colgan and Manley, 1992). This could be caused by the absorption of most cellular TBP to the associated TAF proteins, which may not allow a direct interaction of TBP with the acidic domain of p65. Alternatively, TBP overexpression could titrate negative regulators of RNA polymerase II transcription such as NC2/DR1, which directly contact TBP (Meisterernst and Roeder, 1991; Yeung et al., 1994). The failure of TFIIB to enhance p65-dependent transactivation can be explained by squelching effects mediated by TFIIB or, alternatively, by the fact that it is not present in limiting concentrations. TFIIB and TBP contact next to each other the promoter DNA, and binding of p65 to them is likely to occur simultaneously in the cell. This redundant binding to both general transcription factors would have the advantage of increasing the overall affinity of p65 to the transcription initiation complex. The stimulatory effect of TBP did never exceed a factor of 3, indicating that transactivation by p65 requires additional coactivators. This is consistent with squelching and in vitro transcription experiments, showing that TFIID consisting of TBP and TAFs, but not TBP alone, mediates the response of regulatory factors.

The finding that transcriptional activation domains can directly bind general transcription factors is not without precedent. The general transcription factors TFIIB, TBP, TFIIJ, and TFIIH were all found to have the potential to interact with certain activation domains (Xiao et al., 1994) (reviewed by Tjian and Maniatis(1994)). General transcription factor TFIIB was found to specifically interact with the acidic activation domain of VP16 (Lin et al., 1991; Lin and Green, 1991), the glutamine-rich activation domain of Fushi tarazu (Colgan et al., 1993), and the human thyroid hormone receptor beta (Baniahmad et al., 1993). The TBP protein is also specifically contacted by various types of transactivation domains, such as that of p53 (Seto et al., 1992), E2F-1 (Hagemeier et al., 1993a), c-Fos and c-Jun (Ransone et al., 1993), PU-1 (Hagemeier et al., 1993b), Sp1 (Emili et al., 1994), and the c-Myc transcription factor (Maheswaran et al., 1994). The TATA box-binding protein TBP is also contacting viral activator proteins that are devoid of intrinsic DNA binding activity such as VP16 (Stringer et al., 1990), the Tat protein of human immunodeficiency virus (Kashanchi et al., 1994), the human T-cell leukemia virus type 1 activator protein Tax1 (Caron et al., 1993), the adenovirus activator E1A (Horikoshi et al., 1991; Lee et al., 1991), and the Epstein-Barr virus proteins Zta (Lieberman and Berk, 1991) and R (Manet et al., 1993). Transcription activation domains from some transcription factors bind to both TFIIB and TBP, as seen for ICP4, a transcriptional regulatory protein from herpes simplex virus (Smith et al., 1993), the immediate early protein 2 from human cytomegalovirus (Caswell et al., 1993), v-Rel, chicken and mouse c-Rel (Kerr et al., 1993; Xu et al., 1993), as well as p65 (this study).

One study (Kerr et al., 1993) mapped the region within mouse c-Rel that is responsible for the interaction with TBP to the first 50 amino acids within the NRD domain. Contradictory to that, Xu et al.(1993) mapped the COOH-terminal transactivation domain of c-Rel as TBP- and TFIIB-binding domain (Xu et al., 1993). Our data are consistent with a COOH-terminal location of this domain in the c-Rel-related protein p65/RelA. The interaction of general transcription factors with transactivating domains rather than DNA binding domains is in agreement with the notion that transactivating domains are used to contact the basal transcription machinery (reviewed by Hahn(1993)).

Within TFIIB the more COOH-terminal repeat was found to be required for the interaction with p65. Accordingly, deletion of this repeat also abrogated binding to VP16 (Roberts et al., 1993) and to the human thyroid hormone receptor beta (Baniahmad et al., 1993). The functional importance of this repeat structure was also seen in experiments where cotransfection of a COOH-terminally truncated TFIIB inhibited activation by a Gal4-Fushi tarazu protein in Drosophila Schneider cells (Colgan et al., 1993).

The direct interactions between a transactivating domain and components of the basic transcription machinery are thought to bring an activation domain over large distances into close proximity of the initiation complex close to the transcription start site. Here an activation domain may serve multiple functions. It can stabilize the interaction of TFIID with promoter DNA, as is seen in the case of the Zta protein (Lieberman and Berk, 1991). After binding of TBP to the promoter DNA, TFIIB enters the complex through interaction with TBP. Using an immobilized DNA template assay, it was possible to show that this recruitment of TFIIB to the initiation complex is enhanced by the acidic activation domain of VP16 (Roberts et al., 1993), which is highly related to that of p65 (Schmitz et al., 1994). In a subsequent TAF-dependent step, the activation domain helps the general transcription factors TFIIF, TFIIE, and RNA polymerase II to enter the transcription initiation complex (Choy and Green, 1993). Activation domains were further proposed to stabilize conformation-specific TBPbullet TFIIBbulletDNA complexes, thus reducing the amount of non-productive initiation complexes (reviewed by Hahn (1993)). Finally, activation domains were found to exert their effect in enhancing the rate of transcription elongation.

The cell-free reconstitution of transcription activation with the TA(1) domain of p65 revealed a requirement for cofactors in addition to general transcription factors and TAFs. The activity of Gal4-TA(1) was not stimulated by the coactivator fractions PC2, PC3/Dr2, and p15/PC4 above levels seen with Gal4(1-147) alone. It has been shown previously that the DNA-binding portion of Gal4 (amino acids 1-147), although almost inactive in intact cells, exerts transcriptional activity in in vitro assays (Lin et al., 1988; Ge and Roeder, 1994; Kretzschmar et al., 1994a), while effects by the acidic activation domains of VP16 were moderate. This discrepancy may be caused by the absence of a bona fide chromatin structure in the test tube (and, e.g. the failure of Gal4(1-147) to mediate antirepression). Alternatively or in addition it may indicate the absence of additional functional cofactors in the in vitro-reconstituted transcription systems. Specific stimulation of transcription by the Gal4-TA(1) above levels obtained with Gal4(1-147) was seen only in the presence of the PC1 coactivator fraction. Moreover, at least one additional cofactor fraction has been identified in fractionated HeLa nuclear extracts, which also specifically supported transcriptional activation through the acidic activation domains of both VP16 and NF-kappaB. (^2)However, it is presently not clear by which mechanism PC1 supports p65-dependent activation of transcription. It might be possible that PC1 works in a manner similar to that of PC2, which was proposed to act by increasing the activity of the preinitiation complex in a TAF-dependent process (Kretzschmar et al., 1994a). Coactivators were also found to act as ``bridging factors,'' such as the recently cloned p15/PC4, which binds to TFIIA as well as to VP16 (Kretzschmar et al., 1994b; Ge and Roeder, 1994). Here we have presented evidence that PC1 preferentially activates through the acidic activation domain of p65, while a second acidic activation domain, the AH peptide, is more efficient with PC2. The distinct responsiveness of these two activation domains to coactivators may add a further level of regulation to the transcription process. Future studies are necessary to study the interplay of activators, coactivators, and general transcription factors to elucidate the mechanism by which acidic activation domains influence the activity of the basal transcription machinery.


FOOTNOTES

*
This work was supported by grants from the Bundesministerium für Forschung und Technologie, the Deutsche Forschungsgemeinschaft (SFB190), and the European Community (Biotechnology Programme) (to P. A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-761-203-5221; Fax: 49-761-203-5257.

(^1)
The abbreviations used are: TF, transcription factor; AH, amphipathic helix; BSA, bovine serum albumin; CAT, chloramphenicol acetyltransferase; NRD, NF-kappaB/Rel/dorsal; PC, positive cofactor; TA(1), transactivation domain 1; TAD, transcription activation domain; TAF, TBP-associated factor; TBP, TATA-binding protein.

(^2)
A. Goppelt and M. Meisterernst, unpublished observation.


ACKNOWLEDGEMENTS

We are extremely grateful to Heike Klein and Susanne Kunz for superb technical assistance, Klaus Paal and Dr. Georg Arnold for synthesis of oligonucleotides, Dr. Roger Brent for the gift of yeast strain EGY48 and plasmid pEG202, Dr. Danny Reinberg for the plasmid pHIIB, Dr. T. Wirth for the gift of a plasmid containing the TBP cDNA, and Dr. Kathy Tamai for comments on the manuscript.


REFERENCES

  1. Altmann, H., Wendler, W. & Winnacker, E.-L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3901-3905 [Abstract]
  2. Baeuerle, P. A. & Baltimore, D. (1988) Science 242, 540-546 [Medline] [Order article via Infotrieve]
  3. Baeuerle, P. A. & Baltimore, D. (1989) Genes & Dev. 3, 1689-1698
  4. Baeuerle, P. A. & Henkel, T. (1994) Annu. Rev. Immunol. 12, 141-179 [CrossRef][Medline] [Order article via Infotrieve]
  5. Ballard, D. W., Dixon, E. P., Pfeffer, N. J., Bogerd, H., Doerre, S., Stein, B. & Greene, W. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1875-1879 [Abstract]
  6. Baniahmad, A., Köhne, A. C. & Renkawitz, R. (1992) EMBO J. 11, 1015-1023 [Abstract]
  7. Baniahmad, A., Ha, I., Reinberg, D., Tsai, S., Tsai, M.-J. & O'Malley, B. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8832-8836 [Abstract]
  8. Berger, S. L., Piña, B., Siverman, N., Marcus, G. A., Agapite, J., Regier, J. L., Triezenberg, S. & Guarente, L. (1992) Cell 70, 251-265 [Medline] [Order article via Infotrieve]
  9. Bradford, M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  10. Brent, R. & Ptashne, M. (1985) Cell 43, 729-736 [Medline] [Order article via Infotrieve]
  11. Bröker, M., Bäume, O., Göttig, A., Ochs, J., Bodenbenner, M. & Amann, E. (1991) Appl. Microbiol. Biotechnol. 34, 756-764 [Medline] [Order article via Infotrieve]
  12. Caron, C., Rousset, R., Béraud, C., Moncollin, V., Egly, J.-M. & Jalinot, P. (1993) EMBO J. 12, 4269-4278 [Abstract]
  13. Caswell, R., Hagemeier, C., Chiou, C.-J., Hayward, G., Kouzarides, T. & Sinclair, J. (1993) J. Gen. Virol. 74, 2691-2698 [Abstract]
  14. Choy, B. & Green, M. R. (1993) Nature 366, 531-536 [CrossRef][Medline] [Order article via Infotrieve]
  15. Colgan, J. & Manley, J. L. (1992) Genes & Dev. 6, 304-315
  16. Colgan, J., Wampler, S. & Manley, J. L. (1993) Nature 362, 549-553 [CrossRef][Medline] [Order article via Infotrieve]
  17. Croston, G. E. & Kadonaga, J. T. (1993) Curr. Opin. Cell Biol. 5, 417-423 [Medline] [Order article via Infotrieve]
  18. Emili, A., Greenblatt, J. & Ingles, C. J. (1994) Mol. Cell. Biol. 14, 1582-1593 [Abstract]
  19. Ferreri, K., Gill, G. & Montminy, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1210-1213 [Abstract]
  20. Fujita, T., Nolan, G. P., Ghosh, S. & Baltimore, D. (1992) Genes & Dev. 6, 775-787
  21. Ge, H. & Roeder, R. G. (1994) Cell 78, 513-523 [Medline] [Order article via Infotrieve]
  22. Ghosh, S., Gifford, A. M., Riviere, L. R., Tempst, P., Nolan, G. P. & Baltimore, D. (1990) Cell 62, 1019-1029 [Medline] [Order article via Infotrieve]
  23. Golemis, E. & Brent, R. (1993) Mol. Cell. Biol. 12, 3006-3014 [Abstract]
  24. Goodrich, J. A. & Tjian, R. (1994) Curr. Opin. Cell Biol. 6, 403-409 [Medline] [Order article via Infotrieve]
  25. Goodrich, J. A., Hoey, T., Thut, C. J., Admon, A. & Tjian, R. (1993) Cell 75, 519-530 [Medline] [Order article via Infotrieve]
  26. Grilli, M., Chiu, J. J.-S. & Lenardo, M. J. (1993) Int. Rev. Cytol. 143, 1-62 [Medline] [Order article via Infotrieve]
  27. Ha, I., Lane, W. S. & Reinberg, D. (1991) Nature 352, 689-695 [CrossRef][Medline] [Order article via Infotrieve]
  28. Hagemeier, C., Cook, A. & Kouzarides, T. (1993a) Nucleic Acids Res. 21, 4998-5004 [Abstract]
  29. Hagemeier, C., Bannister, A. J., Cook, A. & Kouzarides, T. (1993b) Proc. Natl. Acad. Sci. U. S. A. 90, 1580-1584 [Abstract]
  30. Hahn, S. (1993) Nature 363, 672-673 [CrossRef][Medline] [Order article via Infotrieve]
  31. Hoffmann, A., Sinn, E., Yamamoto, T., Wang, J., Roy, A., Horikoshi, M. & Roeder, R. G. (1990) Nature 346, 387-390 [CrossRef][Medline] [Order article via Infotrieve]
  32. Horikoshi, N., Maguire, K., Kralli, A., Maldonado, E., Reinberg, D. & Weinmann, R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5124-5128 [Abstract]
  33. Kashanchi, F., Piras, G., Radonovich, M. F., Duvall, J. F., Fattaey, A., Chiang, C.-M., Roeder, R. G. & Brady, J. N. (1994) Nature 367, 295-299 [CrossRef][Medline] [Order article via Infotrieve]
  34. Kerr, L. D., Ransone, L. J., Wamsley, P., Schmitt, M. J., Boyer, T. G., Zhou, Q., Berk, A. J. & Verma, I. M. (1993) Nature 365, 412-419 [CrossRef][Medline] [Order article via Infotrieve]
  35. Kieran, M., Blank, V., Logeat, F., Vandekerckhove, J., Lottspeich, F., Le Bail, O., Urban, M. B., Kourilsky, P., Baeuerle, P. A. & Israel, A. (1990) Cell 62, 1007-1018 [Medline] [Order article via Infotrieve]
  36. Kim, Y.-J., Björklund, S., Li, Y., Sayre, M. H. & Kornberg, R. D. (1994) Cell 77, 599-608 [Medline] [Order article via Infotrieve]
  37. Kretzschmar, M., Meisterernst, M. & Roeder, R. G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11508-11512 [Abstract]
  38. Kretzschmar, M., Stelzer, G., Roeder, R. G. & Meisterernst, M. (1994a) Mol. Cell. Biol. 14, 3927-3937 [Abstract]
  39. Kretzschmar, M., Kaiser, K., Lottspeich, F. & Meisterernst, M. (1994b) Cell 78, 525-534 [Medline] [Order article via Infotrieve]
  40. Lee, W. S., Kao, C. C., Bryant, G. O., Liu, X. & Berk, A. J. (1991) Cell 67, 365-376 [Medline] [Order article via Infotrieve]
  41. Lieberman, P. M. & Berk, A. J. (1991) Genes & Dev. 5, 2441-2454
  42. Lin, Y.-S. & Green, M. R. (1991) Cell 64, 971-981 [Medline] [Order article via Infotrieve]
  43. Lin, Y.-S., Carey, M. F., Ptashne, M. & Green, M. R. (1988) Cell 54, 659-664 [Medline] [Order article via Infotrieve]
  44. Lin, Y.-S., Ha, I., Maldonado, E., Reinberg, D. & Green, M. R. (1991) Nature 353, 569-571 [CrossRef][Medline] [Order article via Infotrieve]
  45. Lopata, M. A., Cleveland, D. W. & Sollner-Webb, B. (1984) Nucleic Acids Res. 12, 5707-5717 [Abstract]
  46. Luo, Y., Fujii, H., Gerster, T. & Roeder, R. G. (1992) Cell 71, 231-241 [Medline] [Order article via Infotrieve]
  47. Maheswaran, S., Lee, H. & Sonenshein, G. E. (1994) Mol. Cell. Biol. 14, 1147-1152 [Abstract]
  48. Manet, E., Allera, C., Gruffat, H., Mikaelian, I., Rigolet, A. & Sergeant, A. (1993) Gene Exp. 3, 49-59
  49. Meisterernst, M. & Roeder, R. G. (1991) Cell 67, 557-567 [Medline] [Order article via Infotrieve]
  50. Meisterernst, M., Roy, A. L., Lieu, H. M. & Roeder, R. G. (1991) Cell 66, 981-993 [Medline] [Order article via Infotrieve]
  51. Merino, A., Madden, K. R., Lane, W. S., Champoux, J. J. & Reinberg, D. (1993) Nature 365, 227-232 [CrossRef][Medline] [Order article via Infotrieve]
  52. Meyer, R., Hatada, E. N., Hohmann, H.-P., Haiker, M., Bartsch, C., Rötlisberger, U., Lahm, H.-W., Schlaeger, E. J., van Loon, A. P. & Scheidereit, C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 966-970 [Abstract]
  53. Narayanan, R., Klement, J. F., Ruben, S. M., Higgins, K. A. & Rosen, C. A. (1992) Science 256, 367-370 [Medline] [Order article via Infotrieve]
  54. Nolan, G. P., Ghosh, S., Liou, H.-C., Tempst, P. & Baltimore, D. (1991) Cell 64, 961-969 [Medline] [Order article via Infotrieve]
  55. Peterson, C. L. & Herskowitz, I. (1992) Cell 68, 573-583 [Medline] [Order article via Infotrieve]
  56. Pierce, J. W., Lenardo, M. & Baltimore, D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1482-1486 [Abstract]
  57. Piña, B., Berger, S., Marcus, G. A., Silverman, N., Agapite, J. & Guarente, L. (1993) Mol. Cell. Biol. 13, 5981-5989 [Abstract]
  58. Ptashne, M. & Gann, A. A. (1990) Nature 346, 329-331 [CrossRef][Medline] [Order article via Infotrieve]
  59. Pugh, B. F. & Tjian, R. (1990) Cell 61, 1187-1197 [Medline] [Order article via Infotrieve]
  60. Ransone, L. J., Kerr, L. D., Schmitt, M. J., Wamsley, P. & Verma, I. M. (1993) Gene Exp. 3, 37-48
  61. Rice, N. R., MacKichan, M. L. & Israel, A. (1992) Cell 71, 243-253 [Medline] [Order article via Infotrieve]
  62. Roeder, R. G. (1991) Trends Biochem. Sci. 16, 402-408 [CrossRef][Medline] [Order article via Infotrieve]
  63. Roberts, S. G., Ha, I., Maldonado, E., Reinberg, D. & Green, M. R. (1993) Nature 363, 741-744 [CrossRef][Medline] [Order article via Infotrieve]
  64. Ruben, S. M., Dillon, P. J., Schreck, R., Henkel, T., Chen, C.-H., Maher, M., Baeuerle, P. A. & Rosen, C. A. (1991) Science 251, 1490-1493 [Medline] [Order article via Infotrieve]
  65. Ruben, S. M., Narayanan, R., Klement, J. F., Chen, C.-H. & Rosen, C. A. (1992a) Mol. Cell. Biol. 12, 444-454 [Abstract]
  66. Ruben, S. M., Klement, J. F., Coleman, T. A., Maher, M., Chen, C.-H. & Rosen, C. A. (1992b) Genes & Dev. 6, 745-760
  67. Rysek, R. P., Bull, P., Takamiya, M., Bours, V., Siebenlist, U., Dobrzanski, P. & Bravo, R. (1992) Mol. Cell. Biol. 12, 674-684 [Abstract]
  68. Schiestl, R. H. & Gietz, R. D. (1989) Curr. Genet. 16, 339-346 [Medline] [Order article via Infotrieve]
  69. Schmid, R. M., Perkins, N. D., Duckett, C. S., Andrews, P. C. & Nabel, G. J (1991) Nature 352, 733-736 [CrossRef][Medline] [Order article via Infotrieve]
  70. Schmitz, M. L. & Baeuerle, P. A. (1991) EMBO J. 10, 3805-3817 [Abstract]
  71. Schmitz, M. L. & Baeuerle, P. A. (1994) BioTechniques 17, 714-718 [Medline] [Order article via Infotrieve]
  72. Schmitz, M. L., Henkel, T. & Baeuerle, P. A. (1991) Trends Cell Biol. 1, 130-137
  73. Schmitz, M. L., dos Santos Silva, M. A., Altmann, H., Czisch, M., Holak, T. & Baeuerle, P. A. (1994) J. Biol. Chem. 269, 25613-25620 [Abstract/Free Full Text]
  74. Seto, E., Usheva, A., Zambetti, P., Momand, J., Horikoshi, N, Weinmann, R. & Shenk, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 12028-12032 [Abstract]
  75. Smith, C. A., Bates, P., Rivera-Gonzales, R., Gu, B. & DeLucia, N. A. (1993) J. Virol. 67, 4676-4687 [Abstract]
  76. Stelzer, G., Goppelt, A., Lottspeich, F. & Meisterernst, M. (1994) Mol. Cell. Biol. 14, 4712-4721 [Abstract]
  77. Stephens, R. M., Rice, N. R., Hiebsch, R. R., Bose, H. R., Jr. & Gilden, R. V. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6229-6233 [Abstract]
  78. Steward, R. (1987) Science 238, 692-694 [Medline] [Order article via Infotrieve]
  79. Stringer, K. F., Ingles, C. J. & Greenblatt, J. (1990) Nature 345, 783-786 [CrossRef][Medline] [Order article via Infotrieve]
  80. Tjian, R. & Maniatis, T. (1994) Cell 77, 5-8 [Medline] [Order article via Infotrieve]
  81. White, R. J. & Jackson, S. P. (1992) Trends Genet. 8, 284-288 [Medline] [Order article via Infotrieve]
  82. Wilhelmsen, K. C., Eggleton, K. & Temin, H. M. (1984) J. Virol. 52, 172-182 [Medline] [Order article via Infotrieve]
  83. Xiao, H., Pearson, A., Coulombe, B., Truant, R., Zhang, S., Regier, J. L., Triezenberg, S. J., Reinberg, D., Flores, O., Ingles, C. J. & Greenblatt, J. (1994) Mol. Cell. Biol. 14, 7013-7024 [Abstract]
  84. Xu, X., Prorock, C., Ishikawa, H., Maldonado, E., Ito, Y. & Gélinas, C. (1993) Mol. Cell. Biol. 13, 6733-6741 [Abstract]
  85. Yeung, K. C., Inostroza, J. A., Mermelstein, F. H., Kannabiran, C. & Reinberg, D. (1994) Genes & Dev. 8, 2097-2109

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