©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Studies on Transcription Activation by the Multimeric CCAAT-binding Factor CBF (*)

(Received for publication, April 14, 1994)

Françoise Coustry (§) Sankar N. Maity (¶) Benoit de Crombrugghe (**)

From the Department of Molecular Genetics, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The CCAAT-binding factor CBF is a heteromeric transcription factor that specifically binds to CCAAT sequences in many eukaryotic genes. CBF consists of three subunits, CBF-A, CBF-B, and CBF-C, all three of which are necessary for DNA binding. In this study we examined the transcription activation function of CBF by two different approaches. We first used a heterologous system in which a series of deletion mutations of CBF-B, fused to the bacterial LexA DNA binding domain, were transfected into HeLa cells together with a reporter gene driven by a minimal promoter containing LexA binding sites. These experiments showed that CBF-B needed both a glutamine-rich domain and an adjacent serine/threonine-rich domain to activate the reporter gene optimally. The glutamine-rich domain by itself activated transcription only modestly. We also set up an in vitro transcription reconstituted system in which trans-activation by CBF occurred through a physiological CCAAT motif. Nuclear extracts from NIH 3T3 cells were first depleted of CBF and then complemented with recombinant CBF-B and a highly purified fraction containing native CBF-A and CBF-C. Recombinant full-length CBF-B together with CBF-A and CBF-C activated transcription of several alpha2(I) collagen gene promoter constructs. We then tested whether in this system the glutamine- and serine/threonine-rich domains of CBF-B were needed for trans-activation by CBF. We generated a truncated form of CBF-B that was still able to bind DNA in the presence of CBF-A and CBF-C. Even in the absence of the glutamine- and serine/threonine-rich domains of CBF-B, reconstituted CBF did activate transcription, suggesting that CBF transcriptional activation can also be mediated by the other subunits of CBF or by another transcription factor present in the nuclear extracts that interacts with CBF. Taken together our results suggest a model in which CBF has the potential to activate transcription either through the glutamine- and serine/threonine-rich domains of CBF-B or through the other subunits of CBF or through another component recruited by CBF.


INTRODUCTION

The CCAAT-binding factor CBF, (^1)also named nuclear factor Y(1) , is a transcription factor that specifically binds to CCAAT sequences in many eukaryotic genes including those for alpha1(I) and alpha2(I) collagen, albumin, major histocompatibility complex class II, and beta-actin(2) . We have shown previously that CBF is a heteromeric factor consisting of three subunits, CBF-A, CBF-B, and CBF-C, all three of which are necessary for DNA binding(3) .

Analysis of CBF-A and CBF-B sequences showed remarkable similarity with segments of, respectively, the HAP3 and HAP2 subunits of the yeast heteromeric CCAAT-binding factor HAP2bulletHAP3bulletHAP4(4, 5) . The third subunit, HAP4, although associated with HAP2bulletHAP3, is not needed for DNA binding(6) . HAP4 contains an acidic domain that strongly activates transcription when fused to the DNA binding domain of LexA and is required for transactivation by the HAP2bulletHAP3bulletHAP4 complex(6) . By contrast to this factor, little is known about the domains of CBF which are needed for transcription activation.

Transcriptional activation functions of DNA-binding factors are specified by domains outside of the DNA binding domain. Several apparently unrelated structural motifs have been identified which confer transcriptional activation(7, 8, 9) . Moreover, transcription factors often have more that one activation domain(9, 10, 11) . The amino-terminal portion of CBF-B from residue 14 to 161 is very rich in glutamine residues (35%) and is flanked on its carboxyl-terminal side by a small serine/threonine-rich segment(5) . The carboxyl-terminal part of CBF-B, which is very hydrophilic and rich in basic residues, contains a subunit interaction domain and a DNA binding domain that are separate from each other(12) .

In this study we used two approaches to examine transcriptional activation by CBF. We tested a series of fusion polypeptides containing segments of CBF-B fused to the LexA-DNA binding domain to determine whether the glutamine- and serine/threonine-rich domains in CBF-B were needed for transcriptional activation of a minimal promoter containing LexA binding sites. We also used a reconstituted in vitro transcription system to study the role of CBF-B in the activation function of CBF with promoters containing a physiological CCAAT motif. By using a truncated CBF-B, in which the glutamine- and serine/threonine-rich domains were deleted, we asked whether in this system transcription activation still took place.


MATERIALS AND METHODS

LexA-CBF-B Fusion Constructs

LexA-CBF-B fusion proteins were obtained by inserting the DNAs coding for specific segments of CBF-B into a unique BsteII site of the pCMV-Lex-BsteII expression plasmid (a generous gift from M. G. Rosenfeld)(13) . Various segments of CBF-B cDNA were generated by PCR as follows. CBF-B DNA cloned into pBluescript, oligonucleotides corresponding to regions of CBF-B DNA to be amplified, and Taq polymerase were incubated together for 15 cycles of PCR at 94 °C for 1 min, 52 °C for 2 min, and 72 °C for 3 min. DNAs for CBF-B fragments were ligated in-frame with the DNA coding for residues 1-87 of LexA. The sequences of all constructs generated by PCR were verified by DNA sequencing.

Reporter Gene

The LexA reporter plasmid was constructed by ligating two copies of the LexA operator site 5`-GATCCAATTCTACTGTATGTACATACAGTATTCCAAA-3` in a head-to-tail orientation and inserting them into the unique SmaI site of plasmid pH39, in which a minimal mouse alpha2(I) collagen promoter (-41 to +54) drives the firefly luciferase gene(14) .

Transient Transfection Assays

One day before transfection, HeLa cells were plated at a density of 0.75 times 10^6/100-mm plate in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal calf serum (Life Technologies, Inc.). The cells were transfected with 12 µg of expression plasmid and 4 µg of reporter plasmid using the DNA-calcium phosphate coprecipitation method (15) . Cells were harvested 48 h after transfection, and the luciferase activity was assayed as described (16) using a Monolight 2010 Luminometer. A plasmid containing the beta-galactosidase gene (17) under the control of the SV40 early promoter-enhancer served as internal control for transfection efficiency. beta-Galactosidase activity was measured with a resorufin-beta-D-galactopyranoside substrate (Boehringer Mannheim).

Western Blotting

Forty-eight hours after transfection, the HeLa cell monolayers were washed once with phosphate-buffered saline and then incubated on ice for 30 min in 0.5 ml of lysis buffer (150 mM NaCl, 1% Nonidet P-40, 50 mM Tris, pH 8). Cells were then scraped in the same buffer and centrifuged for 10 min at 10,000 times g at 4 °C. The supernatants were used for Western blotting (ECL, Amersham Corp.). Extracts were fractionated on 14% SDS-polyacrylamide gel and transferred to nitrocellulose. After addition of a blocking mixture, the membrane was incubated with a 1/2,000 dilution of rabbit anti-LexA antibody (a generous gift from R. Brent, Harvard Medical School) and then incubated with a 1/1,000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit antibody. The LexA fusion proteins were detected by autoradiography using ECL.

DNA Templates for in Vitro Transcription

pFC1 was constructed by inserting four copies of a double-stranded oligonucleotide corresponding to the sequence between -105 and -65 of the mouse alpha2(I) collagen gene promoter in pH39(14) . Oligonucleotides corresponding to both strands of this sequence were synthesized with a BamHI site at the 5` end of the upper strand and a BglII site at the 5` end of the lower strand. After gel purification, these oligonucleotides were phosphorylated at their 5` end, annealed, ligated, and digested with BamHI and BglII to ensure a head-to-tail orientation of the multimers. The ends of the double-stranded multimers were blunt-ended by Klenow enzyme before insertion into the SmaI site of pH39. pFC2 was constructed in the same way as pFC1 using an oligonucleotide corresponding to the sequence between -105 and -65 of the mouse alpha2(I) collagen gene promoter in which the CCAAT box had been mutated (CCAAT CCAAA). Plasmids pH10, pH5, and pH6 contained sequences of the mouse alpha2(I) collagen gene promoter between -108 to +54, -2000 to +54 and -350 to +54, respectively(14) .

Plasmid p120 contained a segment of the mouse alpha1(III) collagen gene promoter between -80 and +16(18) .

Generation of Recombinant CBF-B-GST Fusion Proteins

Full-length CBF-B and a deleted form of CBF-B were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (respectively, CBF-B1-GST and CBF-B3-GST)(19) . Full-length CBF-B cDNA was generated by PCR with EcoRI sites both before the codon for amino acid 3 and after the stop codon and then inserted into the EcoRI site of the bacterial vector pGEX-2T (Pharmacia Biotech Inc.) in-frame with the glutathione S-transferase gene. The cDNA for the truncated form of CBF-B, which codes for a polypeptide from amino acid 225 to the carboxyl terminus of CBF-B, was constructed with the same approach. The sequences of these cDNAs were verified by sequencing.

After transformation of E. coli strain HB101, synthesis of fusion proteins was induced by adding 1 mM isopropyl-beta-D-thiogalactopyranoside for 2 h at the midlog phase of bacterial growth. After centrifugation, the cell pellet was resuspended in lysis buffer containing 10 mM Tris, pH 7.9, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, and 1% Triton X-100. Bacteria were lysed by sonication, and the lysates centrifuged at 10,000 rpm for 20 min at 4 °C. The supernatant was loaded over a column containing 2 ml of glutathione-agarose (Sigma), which was equilibrated with buffer containing 50 mM Tris, pH 7.9, 100 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA, and 10% glycerol. After being washed with the same buffer, the bound protein was eluted with 5 mM glutathione in this buffer. The amount of protein was determined by the Bio-Rad protein assay, and the purity of the eluted proteins was examined by 10% SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining. Prestained marker proteins were used as molecular weight standards.

Nuclear Extracts

Mouse NIH 3T3 fibroblasts, maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, were harvested at 80% confluence, and nuclear extracts were prepared as described (20) except that leupeptin (10 µg/ml) and pepstatin (10 µg/ml) were added to all buffers.

Nuclear extracts were depleted of CBF by mixing with a DNA affinity resin in which the DNA sequence of the mouse alpha2(I) collagen gene promoter from -105 to -64 was covalently linked to Sepharose. Extracts (450 µl) and the affinity resin (90 µl) were incubated at 4 °C with gentle rocking for 30 min. The supernatant was spun at 4,000 rpm, recovered, distributed in small aliquots, and stored at -80 °C.

DNA Binding Assay

DNA binding was performed in a final volume of 10 µl by incubating 5 fmol of P end-labeled oligonucleotides, with either 1 µl of nuclear extract or various amounts of recombinant CBF-B (CBF-B1-GST or CBF-B3-GST), eventually complemented by a Mono Q fraction containing highly purified rat CBF-A and CBF-C(4) . Incubations were carried out at room temperature for 15 min. All binding reaction mixtures contained 25 mM Hepes, pH 7.9, 75 mM KCl, 10% glycerol, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 0.05% Nonidet P-40, 1 mg/ml bovine serum albumin, and 2 µg poly[d(I-C)bulletd(I-C)]. The reaction mixtures were fractionated by electrophoresis on a 5% polyacrylamide gel in 0.5 times TBE (0.5 times TBE: 45 mM Tris, 45 mM boric acid, 1 mM EDTA), and the dried gels were examined by autoradiography.

In Vitro Transcription Assays

In vitro transcription reactions were performed in a 25-µl volume including 10 µl of nuclear extract (final protein concentration in the reaction, 1 mg/ml), 300 ng of DNA template, 200 ng of p120 DNA as an internal control template, 8 mM Hepes, 8% glycerol, 40 mM KCl, 0.08 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM dithiothreitol, 5 mM MgCl(2), 2 mM spermidine, and 1 mM NTPs.

Incubations were carried out at 30 °C for 1 h and reactions stopped by adding 75 µl of a solution containing 0.4 M NaOAc, pH 5.2, 1% SDS. After extraction of protein by phenol/chloroform, the RNAs were precipitated by ethanol. To detect the newly synthesized transcripts, antisense oligonucleotide primers corresponding to a sequence in the luciferase gene and to a sequence in the chloramphenicol acetyltransferase gene were generated. These primers were labeled with [-P]ATP using T4 polynucleotide kinase, hybridized to the RNAs, and extended using avian myeloblastosis virus reverse transcriptase(21) . Primer extension products were fractionated by electrophoresis in 7 M urea, 8% polyacrylamide gel in 1 times TBE buffer, analyzed by autoradiography, and quantitated with a Betagen 603 betascope. Labeled marker nucleic acids were used as molecular weight standards.


RESULTS

Trans-activation by CBF-B in a Heterologous System

To identify the location of potential trans-activating sequences in CBF-B, different segments of this subunit were fused in-frame with a heterologous DNA binding domain consisting of the amino-terminal 87 residues of the bacterial LexA polypeptide (Fig. 1). The fusion proteins were cloned into a mammalian expression vector and then transfected into HeLa cells together with a reporter plasmid containing two LexA binding sites. A construct containing the glutamine-rich domain, the serine/threonine-rich domain, and the subunit interaction domain (CBF-B 6-287) stimulated the promoter activity of the reporter gene about 40-fold more than the construct containing only the LexA DNA binding domain (LexA-DBD) (Fig. 1). When the subunit interaction domain was deleted (CBF-B 6-247), the activation reached more than 80-fold control levels. The construct containing the glutamine-rich domain but lacking the serine/threonine-rich domain (CBF-B 6-178) produced reporter activation that was 5-fold control levels, suggesting that the serine/threonine-rich segment plays an important role in transcriptional activation by CBF-B. A construction containing the serine/threonine-rich domain but lacking the glutamine-rich domain (CBF-B 158-247) resulted in little or no activity. A construction with only the subunit interaction domain did not stimulate transcription of the reporter gene. Taken together the results of Fig. 1indicate that both the glutamine-rich domain and the serine/threonine-rich domain were needed for optimal trans-activation.


Figure 1: Representation of LexA-CBF-B constructs and mapping of the trans-activation domain of CBF-B. LexA-CBF-B fusion proteins containing various segments of CBF-B, as schematized, were obtained as described under ``Materials and Methods.'' CBF-B is subdivided into four domains as follows: A, glutamine-rich domain (amino acids 14-161); B, serine/threonine-rich domain (amino acids 162-259); C, subunit interaction domain (amino acids 260-281); D, DNA binding domain (amino acids 282-312). HeLa cells were cotransfected with the indicated LexA-CBF-B expression vectors, the luciferase reporter plasmid containing LexA binding sites, and the beta-galactosidase internal control plasmid. Luciferase activities generated by each construct were normalized for transfection efficiency and are given relative to the activity obtained with the LexA-DNA binding domain (LexA-DBD) only. n represents the number of experiments.



To verify whether the chimeric proteins encoded by the different constructs of Fig. 1showed similar levels of expression, we used Western blotting with anti-LexA antibodies to examine extracts of cells that had been transfected with the various constructions. Fig. 2shows that cells transfected with plasmids coding for CBF-B 6-287, CBF-B 6-247, CBF-B 6-178, CBF-B 158-247, and CBF-B 253-326 contain comparable levels of CBF-B-LexA fusion proteins. Hence, the differences in activity of these chimeras were not due to differences in protein stability.


Figure 2: Western blot of LexA-CBF-B fusion proteins synthesized in HeLa cells. HeLa cells were transfected with various LexA-CBF-B constructs as indicated. Cell extracts were fractionated on SDS-polyacrylamide electrophoretic gel and submitted to Western blotting as described under ``Materials and Methods.'' Fusion proteins indicated by arrows were visualized by autoradiography. A low level of background bands is present in all extracts. Numbers on the right correspond to the molecular masses of prestained marker proteins.



Transcriptional Activation by CBF in an in Vitro Reconstituted System

To examine the transactivation function of CBF-B in a system in which the three subunits of CBF could be present and bind to a physiological CCAAT element, an in vitro transcription reconstituted system was established.

To ensure that the observed transcriptional activity was due mainly to the presence of CBF and did not reflect synergistic effects of other factors binding to upstream promoter elements, we designed a reporter gene (pFC1) containing a minimal promoter composed of the -41 to +54 sequence of the mouse alpha2(I) collagen gene linked to four tandem copies of the sequence from -105 to -65 in this gene (Fig. 3). The -41 to +54 segment essentially consists of a TATA element and the transcription start site. The -105 to -65 sequence contains a CCAAT motif, which is a functional CBF binding site. A mutant template (pFC2), containing four tandem copies of the mutant CBF binding site in which a CCAAA sequence replaced the wild type CCAAT motif, was also constructed (Fig. 3). This mutation strongly decreased the binding of CBF(2) . We used two plasmids for comparison with pFC1: pH39 contains only the minimal promoter of the alpha2(I) collagen gene (-41 to +54), whereas pH10 contains the sequence between -108 and +54 of this gene and includes a single copy of the CBF binding site. All in vitro transcription reactions included an internal control plasmid containing the alpha1(III) collagen gene promoter, which does not contain a CBF binding site. These different templates were transcribed with nuclear extracts from NIH 3T3 fibroblasts (Fig. 4). The transcriptional activity of pFC1 was approximately four times higher than that of the pH10 promoter and about 20 times higher than that of the minimal promoter pH39 (Fig. 4A). The activity of the pFC2 promoter was about one-fourth that of the wild type pFC1 promoter (Fig. 4B). It is possible that the residual activity of the pFC2 promoter above the pH39 could be due to low CBF binding to this mutant promoter. Our results are consistent with the notion that CBF activates transcription of the wild type template pFC1.


Figure 3: Schematic representation of the DNA templates used in in vitro transcription assays. Plasmids pH39, pH10, pFC1, pFC2, pH6, and pH5 contained the indicated sequences of the mouse alpha2(I) collagen gene promoter placed upstream of the luciferase gene. Plasmid p120, containing the indicated sequence of the mouse alpha1(III) collagen gene promoter placed upstream of the chloramphenicol acetyltransferase gene (CAT), represented the DNA used as an internal control promoter. Closed boxes represent the wild type CBF binding site, and open boxes the mutated CBF binding site.




Figure 4: Comparison of the transcriptional activity of different promoter templates. Several templates derived from the alpha2(I) collagen gene promoter (pH39, pH10, and pFC1 in panel A; pFC1 and pFC2 in panel B) were transcribed with NIH 3T3 nuclear extract. The newly synthesized RNAs were reverse transcribed by primer extension and the products analyzed by electrophoresis on polyacrylamide gel. All transcription reaction mixtures included plasmid p120 containing the alpha1(III) collagen gene promoter as internal control. Labeled marker nucleic acids were used as molecular weight standards.



Transcriptional activation by CBF was then examined by adding reconstituted CBF to an NIH 3T3 cell nuclear extract that was depleted of endogenous CBF. To reconstitute CBF activity, purified recombinant CBF-B, in which the full-length CBF-B was fused to GST (CBF-B1-GST), was added to a chromatographic fraction highly enriched for native CBF-A and CBF-C, which was obtained after a multistep purification protocol(4) . Nuclear extracts that had been incubated with an affinity resin to which the alpha2(I) collagen CBF binding site had been covalently linked were almost completely depleted of CBF. Indeed, binding either to an oligonucleotide containing the alpha2(I) collagen promoter containing the CBF binding site or to an oligonucleotide containing the CBF binding site in the mouse major histocompatibility complex class II promoter was severely reduced (Fig. 5B, lanes 2 and 4). Removal of CBF was specific since binding to an oligonucleotide containing a binding site for CTF/NF1, another CCAAT-binding protein, was practically unchanged (Fig. 5B, lanes 5 and 6).


Figure 5: Comparison of the binding of NIH 3T3 nuclear extracts to CCAAT-containing oligonucleotides before and after depletion of CBF. Panel A, sequence of the CCAAT-containing oligonucleotides used in DNA binding assay. CBF-COL corresponds to the sequence from -105 to -65 of the mouse alpha2(I) collagen gene; CBF-MHC corresponds to the sequence from -81 to -35 of the mouse major histocompatibility complex class II Ea gene; NF1 corresponds to a segment from +15 to +46 of the adenovirus major late promoter. Panel B, undepleted and CBF-B-depleted NIH 3T3 nuclear extracts were incubated with end-labeled oligonucleotides shown in panel A. Reaction mixtures were fractionated on a polyacrylamide gel, and DNA-protein complexes were detected by autoradiography.



The template pFC1 was then transcribed with the undepleted and depleted nuclear extracts complemented or not by reconstituted CBF. Two other alpha2(I) collagen promoters were also used as templates: pH5, which contains the -2000 to +54 sequence of the alpha2(I) collagen promoter, and pH6, which contains the -350 to +54 sequence (see Fig. 3). Removal of CBF from the nuclear extracts resulted in a substantial decrease in transcription with all three templates (Fig. 6A, compare lanes 1, 4, and 7, with, respectively, lanes 2, 5, and 8). Addition of recombinant CBF-B (CBF-B1-GST) plus the fraction containing purified CBF-A and CBF-C to the depleted extracts increased transcription from the three templates to levels comparable to those observed with undepleted nuclear extracts (Fig. 6A, lanes 3, 6, and 9). Thus reconstituted CBF was able to activate the transcription of these templates. The failure of reconstituted CBF to increase transcription of the mutant pFC2 template (Fig. 6B) demonstrated that the activation of transcription by recombinant CBF-B and native CBF-A and CBF-C occurred through an intact CBF binding site.


Figure 6: Transcriptional activation by recombinant CBF-B complemented with CBF-A/CBF-C fraction. Several templates derived from the alpha2(I) collagen gene promoter (pFC1, pH5, and pH6 in panel A; pFC1 and pFC2 in panel B) were transcribed using NIH 3T3 undepleted or CBF-depleted nuclear extracts supplemented or not with 450 ng of recombinant CBF-B and 200 ng of the purified CBF-A/CBF-C fraction as indicated. The newly synthesized RNAs were reverse transcribed by primer extension and the products analyzed by autoradiography after electrophoresis on polyacrylamide gel. p120 containing alpha1(III) collagen gene promoter was added as internal control in all reactions. Labeled marker nucleic acids were used as molecular weight standards.



Transcriptional Activation by CBF Containing Recombinant Truncated CBF-B

We asked the question whether in the absence of the glutamine-rich and serine/threonine-rich activation domains of CBF-B, transcriptional activation by the combined subunits of CBF would take place in the in vitro transcription system. We, therefore, generated a recombinant truncated form of CBF-B (CBF-B3-GST) from which these two domains were deleted (Fig. 7A). After purification by affinity chromatography through a glutathione-agarose column, the recombinant fusion protein showed an apparent molecular mass of 34 kDa compared with 66 kDa for the full-length CBF-B1-GST fusion polypeptide (Fig. 7B). This CBF-B3-GST fusion polypeptide together with purified CBF-A and CBF-C formed a DNA-protein complex with an oligonucleotide containing the CBF binding site at an efficiency similar to that of the parent CBF-B1-GST polypeptide. Parallel increases in DNA binding were observed when increasing concentrations of either the full-length or truncated CBF-B were used in DNA binding assays (Fig. 7C).


Figure 7: Schematic representation, expression, and DNA binding of recombinant CBF-B1-GST and CBF-B3-GST. Panel A, domain structure of recombinant full-length CBF-B1-GST (amino acids 3-341) and truncated CBF-B3-GST (amino acids 225-341). Panel B, SDS-polyacrylamide gel electrophoretic analysis of purified CBF-B1-GST and CBF-B3-GST. Recombinant proteins expressed in E. coli and purified by glutathione-agarose affinity chromatography were fractionated on polyacrylamide gel and visualized by Coomassie Blue staining. Prestained marker proteins were used as molecular mass standards. Panel C, comparison of DNA binding by recombinant CBF-B1-GST and CBF-B3-GST. The indicated amounts (shown in ng) of recombinant proteins were incubated with end-labeled CBF-COL oligonucleotide (see Fig. 5A) in the presence or not of 200 ng of a CBF-A/CBF-C fraction, as indicated. Reaction mixtures were fractionated on a polyacrylamide gel, and the DNA-protein complexes were detected by autoradiography.



The transcriptional activation properties of the full-length and truncated CBF-B-GST fusion proteins were then compared after complementation of a depleted NIH 3T3 nuclear extract with purified CBF-A and CBF-C. Fig. 8shows that both full-length CBF-B1-GST (panel A) and the truncated CBF-B3-GST (panel B) stimulated transcription of the pFC1 template in the presence of purified CBF-A and CBF-C. Increasing concentrations of either the full-length CBF-B1-GST or the truncated CBF-B3-GST resulted in a similar degree of activation (Fig. 8, A, B, and C). The highest concentration of CBF-B1-GST induced a 6-7-fold activation of transcription. Based on the experiments presented in Fig. 7C it is very likely that the increase in transcriptional activation which is observed with increasing concentrations of the recombinant forms of CBF-B is directly related to the increase in DNA binding of the multimeric CBF protein. The lack of activation by the two forms of recombinant CBF-B alone (Fig. 8, A and B, lanes 2-5) or by CBF-A + CBF-C alone (Fig. 8, A and B, lanes 1) is due to the absence of binding by the multimeric CBF. These in vitro transcription experiments indicate that transcription activation by CBF could occur even in the absence of the glutamine-rich and serine/threonine-rich transcriptional activation domain of CBF-B. The DNA binding and the subunit interaction domains of CBF-B are needed, however, to allow binding of CBF to the CCAAT motif.


Figure 8: Transcriptional activation by recombinant CBF-B1-GST and CBF-B3-GST complemented with CBF-A/CBF-C fraction. The DNA template pFC1 was transcribed using CBF-depleted nuclear extracts in the presence of the indicated amounts (shown in ng) of CBF-B1-GST (panel A) or CBF-B3-GST (panel B) and 200 ng of a fraction containing purified CBF-A and CBF-C as indicated. The newly synthesized RNAs were reverse transcribed by primer extension and the products analyzed by autoradiography after electrophoresis on polyacrylamide gel. p120 containing alpha1(III) collagen gene promoter was added as internal control in all reactions. Labeled marker nucleic acids were used as molecular weight standards. Panel C, the transcriptional activity of pFC1, in the presence of either CBF-B1-GST or CBF-B3-GST, was quantified by betascope and plotted as a function of the amount of recombinant proteins.




DISCUSSION

We have used two different approaches to examine the transcriptional activation properties of the multisubunit transcription factor CBF. Because the CBF-B subunit contains a glutamine-rich domain that is similar to the transcription activation domains of other transcription factors(22, 23, 24, 25, 26) , we first examined the function of this domain in DNA transfection experiments using LexA fusion polypeptides. The heterologous system, which consisted of LexA-CBF-B fusion polypeptides in conjunction with a promoter carrying a LexA binding site, differs from the physiological situation in which the multisubunit CBF activates transcription at CCAAT-containing promoters; nonetheless it provides an opportunity to examine the transcriptional potential of the CBF-B subunit. Our experiments showed that both the glutamine-rich domain and the adjacent serine/threonine-rich domain of CBF-B were needed for optimal activation. These results are somewhat different from those of another laboratory in which CBF-B fusion polypeptides were generated with the DNA binding domain of the yeast transcription factor GAL4(27) . In this study activation was less pronounced, and no significant differences were observed whether or not the serine/threonine domain was present in the construction. Since we had previously noted that the GAL4 DNA binding domain by itself caused a relatively high level of expression of the reporter gene compared with the LexA DNA binding domain, which has practically no effect by itself, we chose to use LexA fusion polypeptides for our experiments.

Cooperativity between two activation domains in the same protein has been shown for several transcription factors(9, 10, 11) . Moreover, regions of high serine/threonine content could be substrates for phosphorylation. Although the serine/threonine-rich sequence of CBF-B contains no known phosphorylation motifs, it is possible that phosphorylation of the serine/threonine domain of CBF-B could modulate CBF-B activity. Phosphorylation of serine/threonine-rich sequences has previously been observed to control the activity of other transcription factors(10, 28) .

In Sp1 the glutamine-rich transcription activation domain was recently shown to interact directly with one of the subunits of the general transcription factor TFIID, the 110-kDa-polypeptide TATA-binding protein-associated factor TAF110(29) . This subunit of TFIID is a coactivator of transcription and presumably mediates the transcriptional stimulation produced by Sp1. Our recent experiments have indicated that the glutamine-rich domain of CBF-B can also interact with TAF110. (^2)

No activation took place after transfection of DNA for a LexA fusion that coded only for the subunit association and DNA binding domains or only for the subunit association domain of CBF-B. This situation is different from what was reported with the CBF-B homologue HAP2 in Saccharomyces cerevisiae(6) . Indeed, similar LexA fusions with either the subunit interaction domain plus the DNA binding domain or with only the subunit interaction domain of HAP2 were able to activate transcription of a reporter gene. Evidence was presented that this activation took place by recruitment of the HAP3 and HAP4 subunits of the multisubunit yeast transcription factor HAP2bulletHAP3bulletHAP4 to the LexA binding site at the promoter. Our results suggested that as fusion polypeptides with the LexA DNA binding domain, the CBF-B DNA binding and subunit interaction domains were unable to recruit either transcriptionally competent endogenous CBF-A and CBF-C or eventually other transcriptionally competent factors in HeLa cells. One possible explanation for this difference is that in mammalian cells all endogenous CBF-A and CBF-C would normally be complexed with the endogenous CBF-B and that no free CBF-A and CBF-C would exist which could interact with the LexA-CBF-B fusion polypeptides in transient expression experiments.

To obtain a system that would more closely resemble the physiological situation in which the three subunits of CBF are present together, a reconstituted in vitro transcription system was used. Interestingly, transcription activation also took place when we used a mutant recombinant CBF-B from which the glutamine-rich and serine/threonine-rich segments were deleted, leaving only the subunit association domain and the DNA binding domain of CBF-B. Activation was dependent on the presence of both this truncated recombinant CBF-B and the other two CBF subunits. This suggests that in this system in the absence of the glutamine and serine/threonine-rich trans-activation domains of CBF-B, transcription activation can be mediated either by one or the other two subunits of CBF or by another transcription factor that is recruited by CBF. It is, indeed, very unlikely that in the in vitro transcription system activation occurred through the truncated CBF-B which essentially retains only the highly conserved DNA binding and subunit interaction domains of CBF-B(5, 12) .

These experiments do not directly establish whether the glutamine-rich domain and the serine/threonine-rich domain of the E. coli synthesized recombinant CBF-B function as transcriptional activators in vitro. Indeed, the activation by full-length CBF-B was not significantly higher than the activation by the truncated CBF-B. Since a similar degree of activation was produced by increasing amounts of either full-length or truncated CBF-B, it is unlikely that our failure to clearly detect the effects of additive activations were due to limitations in one or more of the many factors that are needed to achieve transcription. It is possible that the transcriptional activation domain of recombinant CBF-B synthesized in E. coli is inactive or that the presence of a GST moiety inhibits transcriptional activation by CBF-B.

The two experimental approaches that were used here to examine transcription activation by CBF nonetheless complement each other. The transfection experiments with the LexA-CBF-B fusion polypeptides identified a complex transcription activation domain in CBF-B, whereas the in vitro transcription experiments using a truncated CBF-B, in which only the DNA binding and subunit interaction domains are preserved, suggest the presence of an additional functional transcription activation domain that is presumably carried either by one of the other two CBF subunits or by a protein in the extracts that is recruited by CBF and directly interacts with CBF.

In conclusion, our experiments suggest a model whereby transcriptional activation by CBF can be mediated through different polypeptides. These different activation domains could regulate the activities of various promoters depending on the sequence context of these promoters and the arrangement of binding sites for other activators. The different activation domains could also modulate the activity of CBF in response to different cellular regulatory signals.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant CA49515 (to B. d. C.). 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.

§
Recipient of a fellowship from the région Champagne-Ardenne (France).

Recipient of an Arthritis Foundation Investigator Award.

**
To whom correspondence should be addressed: Dept. of Molecular Genetics, The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-2590; Fax: 713-794-4295.

(^1)
The abbreviations used are: CBF, CCAAT-binding factor; PCR, polymerase chain reaction; GST, glutathione S-transferase.

(^2)
F. Coustry, S. N. Maity, and B. de Crombrugghe, unpublished data.


ACKNOWLEDGEMENTS

We acknowledge the help of Satrajit Sinha for the preparation of Mono Q fraction, Kaiyi Li for generation of recombinant CBF-B proteins, Heidi Eberspaecher for oligonucleotides synthesis, and Shirley Hall for performing DNA sequence analysis. We thank Véronique Lefebvre for critical review of the manuscript and Patricia McCauley and Janie Finch for editorial assistance.


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