(Received for publication, April 14, 1994)
From the
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 2(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.
The CCAAT-binding factor CBF, ()also named nuclear
factor Y(1) , is a transcription factor that specifically binds
to CCAAT sequences in many eukaryotic genes including those for
1(I) and
2(I) collagen, albumin, major histocompatibility
complex class II, and
-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
HAP2HAP3
HAP4(4, 5) . The third subunit,
HAP4, although associated with HAP2
HAP3, 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 HAP2
HAP3
HAP4
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.
Plasmid p120
contained a segment of the mouse 1(III) collagen gene promoter
between -80 and +16(18) .
After
transformation of E. coli strain HB101, synthesis of fusion
proteins was induced by adding 1 mM isopropyl--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 were depleted of CBF by mixing with a DNA
affinity resin in which the DNA sequence of the mouse 2(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.
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
TBE buffer, analyzed by
autoradiography, and quantitated with a Betagen 603 betascope. Labeled
marker nucleic acids were used as molecular weight standards.
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 -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.
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 2(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
2(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
1(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 2(I) collagen gene promoter placed upstream of the
luciferase gene. Plasmid p120, containing the indicated sequence of the
mouse
1(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
2(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
1(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 2(I) collagen CBF binding site had
been covalently linked were almost completely depleted of CBF. Indeed,
binding either to an oligonucleotide containing the
2(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 2(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 2(I) collagen promoters were also
used as templates: pH5, which contains the -2000 to +54
sequence of the
2(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 2(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
1(III) collagen gene promoter was added as internal
control in all reactions. Labeled marker nucleic acids were used as
molecular weight standards.
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 1(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.
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. ()
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 HAP2HAP3
HAP4 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.