A protein interacting with an A-T-rich region
that is a positive control element within the SP6
promoter was
purified and identified as CArG-box binding factor-A. The purified
protein was shown to interact specifically with the coding strand of
single-stranded DNA and, with lower affinity, with double-stranded DNA.
A mutation that inhibited binding of the protein to the A-T-rich region
also aborted the transcriptional stimulatory effect of the region. Two
Ets proteins, PU.1 and elf-1, that have previously been shown to bind
to an adjacent DNA element were shown to physically interact with
CArG-box binding factor-A. An antiserum raised against the protein
recognized two different forms indicating either that different
splice-forms of CArG-box binding factor-A are expressed, or that the
protein is subject to post-translational modification.
 |
INTRODUCTION |
The control of transcriptional initiation in eucaryotes is a
multilayered process. At one end of the spectrum are the very specific
interactions between distinct sequence motifs and defined transcription
factors that can interact with a specific sequence only. Secondary
protein-protein interactions between such DNA-binding factors and
so-called transcriptional adapter molecules also show molecular
specificity (1-4) and can easily be envisioned to have a defined role
in the control of expression of a given gene. The mechanism of action
and the specificity of other control elements pertinent to gene
expression are of more general nature. Here, one may distinguish
between control mechanisms of gene expression from a given locus and
those that are acting downstream of this decisive event to facilitate
efficient transcriptional initiation and elongation induced by the
sequence-specific transcription factors mentioned above. Among the
former are locus control regions (5), matrix attachment regions (6),
histone acetylation (7), and the action of the SWI/SNF complex (8). The
latter include specific DNA bending proteins (9), topoisomerases (10), stress induction by the matrix (10) as well as specific single-strand binding proteins (11-13). Additional biochemical events that modulate the efficacy of gene expression are hairpin extrusion (14), DNA melting
(15-17), Z-DNA formation (18), and supercoiling due to torsional
stress induced by the transcription machinery (19, 20).
During recent years, several proteins have been described that interact
with single-stranded promoter elements and that appear to influence
transcriptional initiation (11, 12, 21-27). These proteins usually
show sequence specificity with regard to binding, although they are
probably not as restricted as double-stranded DNA binding transcription
factors (28). Several of these single-stranded DNA-binding proteins are
identical or related to proteins that are found within the
heterogeneous nuclear ribonucleoprotein
(hnRNP)1 complex (11, 12,
23-25) that is formed on heterogeneous nuclear RNA and is thought to
be important for protection, splicing, and transport of RNA (29). Thus,
it is feasible that these proteins have dual functions, as have been
proposed for other DNA/RNA-binding proteins in prokaryotes and in
polymerase III genes (30, 31).
The transcriptional regulation of the immunoglobulin (Ig) genes is
complex. Although each rearranged gene has a distinct promoter, distal
enhancers are found in introns and 3' of the genes (32). Within these
enhancers, a variety of DNA elements involved in the transcriptional
control have been defined (32). Furthermore, close to the intron
enhancers, matrix attachment regions are found, and recent evidence
indicates that these function as locus control regions (33, 34).
Ig
promoters show sequence conservation within but not between V
gene subgroups (35, 36). Detailed functional studies have shown that
promoters contain several DNA elements involved in transcriptional
regulation (37-45). The octamer, which is a binding site for the
ubiquitous OCT1 and the B cell-specific OCT2 transcription factors, is
a key control element in
promoters and when mutated the promoter is
inactivated (37). However, the promoter is dependent on other,
octamer-dependent, transcriptional control elements for
proper function (37). Such elements have been identified both 5' and 3'
of the octamer and examples thereof are the
-Y element (44), the
pentadecamer (pd) element containing an E-box of the E2A type
(-CAGNTG-) (38, 41), and the CCCT element (39, 42). All three elements
interact with distinct proteins in electrophoretic mobility shift assay
(EMSA) and interact functionally with the octamer (42, 43). So far,
only the proteins that interact with the
-Y element have been
identified, PU.1 and elf-1, both of which belong to the Ets family of
transcription factors (43, 45).
In this study, we purified a protein that interacts with a functionally
important A-T-rich region within the SP6
promoter pd element and
identified it as CArG-box binding factor-A (23).
 |
EXPERIMENTAL PROCEDURES |
Purification of the pdLMW Activity--
J558 plasmacytoma cells
were grown in large cell culture flasks that were harvested every 2-3
days, and nuclear extracts were prepared according to a modification of
the Dignam method using buffer C' (46, 47). The nuclear extracts were
pooled and precipitated sequentially with increasing amounts of
ammonium sulfate, and the pellets were dissolved in buffer Z (25 mM HEPES (pH 7.6), 100 mM KCl, 12.5 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20% glycerol, and 0.1%
Nonidet P-40) (48). The material that precipitated between 70 and 90%
ammonium sulfate saturation was dialyzed against buffer Z and was
loaded onto a native DNA cellulose column (Amersham Pharmacia Biotech)
equilibrated in buffer Z. After washing, bound proteins were eluted
stepwise in 100 mM steps up to 1 M KCl in
buffer Z. Fractions containing the pdLMW binding activity (41) (500 mM and 600 mM KCl) were pooled and dialyzed
against buffer A (20 mM Tris (pH 8.0), 25 mM
KCl, 5 mM MgCl2, 1 mM
dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride)
and loaded onto a MonoQTM (Amersham Pharmacia Biotech) column
equilibrated in buffer A. After washing with buffer A, bound proteins
were eluted with a continuos gradient of buffer B (as A but 500 mM KCl).
SDS-PAGE, Western Blots, Trypsin Digestion, Amino Acid
Sequencing, and Northern Blots--
SDS-PAGE and electroblotting were
performed according to standard procedures (49), and blotted proteins
were immunodetected by chemiluminescence (50). For blocking of the
antisera, 3 µl of protein G purified sera was preincubated with 50 µg of recombinant protein for 30 min on ice before it was added to
the membranes. For amino acid sequencing, the protein band was
identified in the gel after Coomassie Blue staining, excised, and sent
to SLU (Bo Ek, Uppsala, Sweden) for trypsin digestion, peptide
purification by reversed phase high performance liquid chromatography,
and amino acid sequencing. Northern blots were performed according to
standard procedures using 0.5 µg of poly(A) RNA per lane (49). The
RNA was purified using a QuickPrepTM micro mRNA purification kit
(Amersham Pharmacia Biotech).
Cloning of CArG-box Binding Factor-A, Expression in Bacteria, and
Production of Polyclonal Serum--
CArG-box binding factor-A was
cloned from a HybriZAP cDNA library made from murine B cells that
had been stimulated with lipopolysaccharide for 72 h, according to
the manufacturer's instructions (Stratagene), using an
end-labeled oligonucleotide (5'-ATCAAGGTTGCCCAGCCCAAAGAG-3') according to standard procedures (49). A clone that contained the
complete 5' sequence but lacking a part of the 3' untranslated region
was cloned (23). The cDNA was cut with NcoI,
blunt-ended, and then cut with SacI and inserted between the
SmaI and the SacI site in the pGEM3Z plasmid
(Promega). This plasmid was completely digested with EcoRI
and then partially digested with BamHI, and the CArG-box
binding factor-A fragment derived in this manner was inserted in frame
with the glutathione S-transferase gene between the
BamHI and the EcoRI sites in the pGEX-2 vector
(Amersham Pharmacia Biotech). The protein was expressed and purified on glutathione-Sepharose according to the manufacturer's instructions (Amersham Pharmacia Biotech), whereafter it was further purified on a
MonoQTM column (Amersham Pharmacia Biotech). The protein was cleaved by
incubating with 0.5 units of thrombin (Sigma) in 200 µl of
digestion buffer (20 mM Tris (pH 8.0), 150 mM
NaCl, 2.5 mM CaCl2, and 10% glycerol) at room
temperature for 2-3 h.
The polyclonal antiserum was raised in rabbits according to standard
procedures using the GST-CArG-box binding factor-A fusion protein. The
IgG fraction was purified on a Protein G HiTrapTM column (Amersham
Pharmacia Biotech) according to the manufacturer's instructions and
dialyzed against phosphate-buffered saline.
The Deletion Mutants--
Variants 3, 7, 8, 9, and 10 were
generated by PCR (15 cycles) using primers 1 and 5, 2 and 5, 3 and 5, 2 and 4, 3 and 4, respectively, and the cloned CBF-A cDNA as a
template. Variants 4, 5, and 6 were generated by primary PCRs using
primer 5 and 6, 7, or 8, respectively, or primer 1 and 4 for 15 cycles
to generate fragment 1-4. The fragments were gel-purified, pre-PCR in
the absence of primers was performed with fragment 4 and 1, 2, or 3 for
20 cycles, new Taq enzyme and primers 1 and 5 were added to
the reactions, and 15 cycles more were performed. After digestion with
BglII and EcoRI, the fragments were then cloned
between the BamHI and EcoRI sites in the GST
vector pGEX-2 (Amersham Pharmacia Biotech). All constructs were
verified by sequencing of the first 250 base pairs using the pGEX 5'
sequencing primer (Amersham Pharmacia Biotech). The sequence of
the primers used were: 1) 5'-GAGAGATCTCCGAGCGGGAACCAGA-3'; 2)
5'-GAGAGATCTGCGGGAAAAATGTTCGTT-3'; 3) 5'-GCTAGATCTATGAAGAAGGACCCTGTG 3'; 4) 5'-CCTCTGAATTCTAGGCAACCTTGATTTC 3'; 5)
5'-TCCGAATTCCTCTCAGTATGGCTTGTA-3'; 6)
5'-CGCCAGCAAGAACGAGGAGGACGCGGGAAAAATCTTTGTGGGAGGTCTAAACCCTG-3'; 7)
5'-GGCTATGGCTATGAAGAAGGACCCTGTGCAGCCCAAAGAGGTGTATCAGCAACAGC-3'; 8)
5'-CGCCAGCAAGAACGAGGAGGACGCGGGACAGCCCAAAGAGGTGTATCAGCAACAGC-3'.
The deletion mutants were expressed in bacteria and purified on
glutathione beads as described by the manufacturer (Amersham Pharmacia
Biotech), and subsequently dialyzed against phosphate-buffered saline
for 8 h with one buffer exchange. The amounts of the different variants were estimated by A280 measurement.
EMSA, Transfections, and CAT Assays--
EMSAs, transfections,
and CAT assays were performed as described (43), but for the EMSAs
single-stranded probe was used unless stated different in the figure
legends. Preimmune or immune serum was added before the addition of the
probe, and the reactions were then preincubated for 30 min at room
temperature. The sequences of the probes can be found in the
figures.
For the EMSA in Fig. 5B, the promoter region of the SP6
gene was cloned between the PstI and the HindIII
sites of the pGEM3Z vector (Promega). An end-labeled promoter probe was
generated by Klenow fill-in labeling after XbaI digestion
followed by digestion with HindIII. Native PAGE purified
probe in bind buffer was denatured at 95 °C for 5 min and then
rapidly cooled on ice. 6000 cpm of native or denatured probe was added
to 25 µl of footprint binding buffer with different amounts of
protein as indicated. The reactions were incubated for 5 min at
37 °C, whereafter an aliquot of 10 µl was separated on a 5%
native PAGE gel in 1× TBE. Stop buffer (0.67% SDS, 30% glycerol, 0.3 mg/ml tRNA, 0.3 mg/ml proteinase K, and 0.3% bromphenol blue) (51) was
then added, and the reaction was continued for 5 min. The reactions
were then separated by native PAGE as above.
DNase Footprinting Assays and Methylation Interference--
The
footprint assays were performed according to standard procedures, and
single-stranded or double-stranded probes (20 000 cpm) were used as
indicated. After preincubation for 30 min in 50 µl of bind buffer (10 mM HEPES (pH 7.9), 50 mM KCl, 5 mM
MgCl2, 0.5 mM EDTA, 0.5 mM
spermidine, 0.5 mM dithiothreitol, and 10% glycerol) on
ice, 50 µl of 5 mM CaCl2 and 10 mM MgCl2 was added and finally 0.25 µg of
DNase was added. After incubation for 1 min the reaction was stopped by
the addition of 100 µl of DNase stop buffer (200 mM NaCl,
20 mM EDTA, 1% SDS, 250 µg/ml yeast tRNA), whereafter
the reaction was phenol-extracted and ethanol-precipitated. The
reactions were separated on 20% denaturing PAGE gels. The ladders were
made according to the rapid protocol for G/A Maxam-Gilbert sequencing
(49). The methylation interference assay was performed essentially as
described in Ref. 41, although a single-stranded probe was used and
that the probe was gel-purified after methylation. Recombinant
thrombin-cut CBF was used in EMSA with methylated probe (100,000 cpm),
and about 50% of the probe was shifted. The bound and free probe was
identified after autoradiography, excised and eluted over night. The
reaction was separated on a 15% denaturing PAGE gel.
CArG-box Binding Factor A Pull-down Assay--
Recombinant GST
or GST/CArG-box binding factor fusion protein was bound to
glutathione-Sepharose (Amersham Pharmacia Biotech) in NETN buffer (20 mM Tris (pH 8.0), 100 mM NaCl, 1 mM
EDTA, 0.5% Nonidet P-40, and 0.1% bovine serum albumin), and the
beads were then washed in NETN. After incubating 10 µl of beads with
1 µl of in vitro translated protein (43) labeled with
[35S]methionine (Amersham Pharmacia Biotech) in 300 µl
of NETN for 2 h at 4 °C, the beads were washed five times in
NETN at room temperature, 10 µl of SDS load was added to the beads,
and bound proteins were separated on a 10% SDS-PAGE after heating to
95 °C. The gels were either fixed (45% methanol and 10% HAc) and
dried or transferred to nitrocellulose filters, and then
autoradiographed. The pull-downs using the different deletion mutants
were made as described above, and 0.006 OD units were loaded of each
mutant to 10 µl of beads as described above. Quantifications were
made in a phosphorimager (Fuji Bio-Imaging Analyzer BAS2000).
 |
RESULTS |
Functional Activity and Protein Interactions with an A-T-rich
Region in the SP6
Promoter pd Element--
The region 5' of the
octamer in the murine SP6
promoter has two sequence elements, the
pd and
-Y elements, that stimulate transcription by synergistic
interactions with the octamer (41, 43-45). The pd element can be
further divided into two sites, a 5' E-box of the E2A type and a 3'
A-T-rich region (41). The individual function of these three sites was
tested by transient transfections of CAT reporter constructs into
lipopolysaccharide-stimulated B cells (41). A promoter fragment
containing the pd, the
-Y, and the octamer element stimulated
transcriptional initiation when placed upstream from a TATA-box, as
compared with the TATA-box alone (Fig.
1A; compare the first
two lanes). When the octamer was mutated only limited
transcriptional stimulation was observed (third lane); this
confirmed earlier studies showing that the octamer is obligate for
promoter function (37). The individual function of the three sites 5'
of the octamer was tested by introducing mutations in the reporter
construct. Deletion of the 5' E-box of the pd element, or mutations in
either the A-T-rich region or the
-Y element all diminished the
costimulatory function (fourth through sixth
lanes). Thus, the interdependence of the three 5' elements for
octamer costimulation was confirmed (43).

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Fig. 1.
A, CAT assays with different promoter
constructs. At the top is shown the autoradiograph of a
representative experiment, in the middle a schematic figure of the SP6
promoter and at the bottom schematic figures of the
different promoter constructs. B, An EMSA with nuclear
extract from J558 cells and the wild type pd element, an pd element
with a mutation of the A-T-rich sequence or a mutation of the E-box as
probe. To the left of the panel are indicated the mobility
of the pdLMW and pdMMW complexes. The pdMMW complex overlaps with an
unspecific complex labeled with an asterisk (see text).
Under the EMSA are shown the sequences of the probes used.
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|
Next, the pd element was used as probe in an EMSA with nuclear extract
from the plasmacytoma cell line J558L as protein source (Fig.
1B). As described previously, two complexes were formed (pdMMW and pdLMW; Ref. 41). When the A-T-rich region was mutated, no
pdLMW complex was formed while the pdMMW complex was unaffected. A
mutation of the E-box did not affect the pdLMW complex but the pdMMW
complex was reduced in intensity. The binding of the pdMMW complex
merits some comment, as it may seem to be in contrast to an earlier
report where mutations of the E-box disrupted binding (41). However,
different methods were used to generate the nuclear extracts; the
method of Schreiber et al. (52) was used in the earlier
report, whereas a modification of the Dignam method (47) was used here.
Thus, the pdMMW shift here appears to be composed of two overlapping
complexes: a specific E-box binding complex that does not bind to the
mutated E-box and a nonspecific binding complex that also interacts
with single-stranded DNA (data not shown).
Purification and Amino Acid Sequencing of the pd A-T Region
Interacting Protein--
To identify the A-T region interacting
protein, we developed a three-step method that was used for
purification (Fig. 2A). Nuclear extract was made from the J558L plasmacytoma cell line according to a modification of the original Dignam method (46, 47), and
the extract was sequentially precipitated with ammonium sulfate in 10%
steps. The pdLMW activity precipitated mainly in the 70-80% and
80-90% ammonium sulfate saturation fractions, while the pdMMW
activity (and most other transcription factors) precipitated at
concentrations below 40% saturation (data not shown). The 70-80% and
80-90% fractions were pooled and loaded onto a native DNA cellulose
column, and while most contaminating proteins passed through the column
at 100 mM salt concentration, the pdLMW activity was eluted
in the 500 and 600 mM fractions (data not shown). These two
fractions were pooled, loaded onto a MonoQTM ion exchange column, and
eluted with a continuos gradient of KCl. Every second fraction was
tested for protein content by SDS-PAGE and for pdLMW binding activity
in EMSA (Fig. 2B, left panels). The activity
eluted in one peak in fractions 24-26, as did an approximately 35-kDa
protein, a molecular mass in agreement with a preliminary
characterization of the pdLMW activity (41). A contaminating protein of
higher molecular mass eluted earlier than and overlapping with the
35-kDa protein. The purification steps were monitored by SDS-PAGE (Fig. 2B, right panels). Although some contaminating
proteins were evident after the DNA-cellulose step, only the higher
molecular mass protein contaminated the purified protein after the
MonoQTM step. Hence, we conclude that a 35-kDa protein co-purified with
the pdLMW forming activity during the three purification steps.

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Fig. 2.
A, purification scheme for the pdLMW
binding activity. The activity was followed in EMSA with a probe
containing the pd element from the murine SP6 promoter (data not
shown). B, to the left are shown the fractions
from the ion exchange step tested in Coomassie Blue-stained SDS-PAGE
and for pdLMW complex formation as indicated; and to the
right are the different purification steps analyzed in the
same way. To the left of the SDS-PAGEs are indicated the
mobilities of a molecular weight marker and to the right are
arrows indicating the purified protein.
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To identify the protein, fraction 25 was acetone-precipitated and
separated on SDS-PAGE. After staining with Coomassie Blue to identify
the position of the protein, the band was excised from the gel, trypsin
digested, and the resulting peptides were separated using reverse phase
high performance liquid chromatography. Two peptides were amino acid
sequenced, and the sequences were used to search the EMBL data base
using the FASTA algorithm. CArG-box binding factor-A (CBF-A; Ref. 23)
was the highest scoring candidate for both peptides (Table
I; only one mismatch was observed at an
ambiguous amino acid position), and this identification was further
strengthened by the fact that CBF-A has potential trypsin digestion
sites just upstream from either of the sequenced peptides.
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Table I
Protein/peptide sequences
The obtained amino acid sequences for two sequenced peptides are shown
aligned to the sequence of CBF-A. Unambiguous sequence positions are
indicated by uppercase letters, while positions with some ambiguity are
indicated by lowercase letters.
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CBF-A Is Part of the pdLMW Complex--
The cDNA for CBF-A was
cloned from a
library derived from murine B cells that had been
stimulated with lipopolysaccharide for 72 h, using an
oligonucleotide probe corresponding to nucleotides 883-907 in the
published sequence (23). A GST fusion protein was expressed in
Escherichia coli by inserting the coding region from amino
acid 10 to the stop codon in the pGEX-2T expression vector and purified
as described under "Experimental Procedures." Subsequently, the
purified protein was tested in EMSA for interactions with the pd probe.
Although purified GST did not form a complex with the probe in EMSA,
purified GST-CBF fusion formed two complexes, a stronger with lower
mobility and a weaker with somewhat higher mobility (Fig.
3A, left panel,
lanes 1 and 2). The same complexes were formed
even after MonoQTM purification (lane 3), and the complex with higher mobility appears to be a breakdown product of the GST-CBF
fusion protein, as a minor protein band is evident in SDS-PAGE with a
slightly lower molecular weight than the major band (data not shown).
When the fusion protein was cleaved by thrombin the formation of both
complexes were diminished and a novel strong complex was formed with
approximately the same mobility as the purified protein (Fig.
3A, right panel). As the same amount of protein
was loaded in lanes 1 and 2, it appeared that the
GST part of the fusion protein disturbed binding. We conclude that recombinant CBF-A interacts with the pd probe in EMSA and has a
mobility similar to that for the purified pdLMW protein.

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Fig. 3.
A, an EMSA with the pd element as probe
and purified GST, glutathione-purified GST-CBF-A fusion protein, or ion
exchange column-purified fusion protein as source of protein is shown
to the left and a similar EMSA with ion exchange-purified
protein, thrombin-cleaved purified protein, thrombin only, and purified
pdLMW forming activity as a source of protein is shown to the
right. To the left of the autoradiographs are
marked the mobility of the two complexes formed with the GST fusion
protein and the complex formed after thrombin cleavage, and to the
right the mobility of the purified pdLMW complex.
F indicates free probe. B, EMSAs are shown where
the purified protein or nuclear extract were incubated with the pd
probe after preincubation with either preimmune serum or immune serum
as indicated.
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The GST-CBF fusion protein was used to generate a polyclonal antiserum.
The specificity was confirmed by Western blotting using recombinant
protein and either preimmune or immune serum and blocking with the
recombinant protein (Fig. 4; see below). Neither the preimmune nor the immune serum affected the pd probe alone
in EMSA (Fig. 3B, left panel, lanes
1-5), and while the preimmune serum did not affect the binding of
the purified protein in EMSA, the immune serum did (Fig. 3B,
middle panel). The same result was obtained using the
recombinant protein in EMSA (data not shown). Thus, the purified
protein is immunologically cross-reactive with CBF-A. Next, we tested
if the immune serum did interfere with the formation of the pdLMW
complex in EMSA using nuclear extract as a source of protein. This was
the case; although no effect on the pdLMW complex formation was evident
when the nuclear extract was preincubated with the preimmune serum, it
was diminished when immune serum was used (Fig. 3B,
right panel). However, some residual complex formation was
observed that migrated at the slower front of the pdLMW complex. Note
that the nuclear extract used in this experiment was made according to
Schreiber et al. (52), since the pdLMW complex was first
described using this extract (41), and hence the pdMMW like complex
that was observed using the modified Dignam nuclear extract did not
form as discussed above. However, the residual pd complex in the
presence of the immune serum was also evident when a nuclear extract
according to Dignam (47) was used as a source of protein (data not
shown). Thus, although CArG-box binding factor-A is the main protein
involved in the formation of the pdLMW complex, another minor protein
complex with similar mobility can interact with the pd element.

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Fig. 4.
A, Western lots were performed with
preimmune or immune sera using uninduced bacterial extract (left
lanes), expressed GST (middle lanes), or GST-CBF
(right lane). B, Western blots were
performed as in A but the immune serum was blocked with GST
(middle panel) or GST-CBF (right panel) before it
was incubated with the blot. C, Western blots were performed
with J558 extract, S194 extract or purified CBF using either preimmune
serum (left panel) or immune serum (middle
panels). To the right the preimmune serum was blocked
as in B before it was used. D, Northern blots are
shown where poly(A)+ mRNA from the indicated B cell
lines were probed with a CBF-A probe and a parallel blot where the same
mRNA samples were probed with a GADPH probe.
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Two Forms of CBF-A Can Be Detected in Nuclear Extracts--
The
specificity of the antiserum was tested in Western blots (Fig.
4A). The antiserum, but not the preimmune serum, did
recognize either recombinant GST-CBF fusion protein or GST alone. No
other bacterial proteins where detected in the assay, and while the interaction to GST could be blocked by either GST or GST-CBF, the
interaction to GST-CBF could only be blocked by recombinant GST-CBF
(Fig. 4B).
Next, a Western blot with nuclear extract from J558 cells was probed
with antiserum or preimmune serum (Fig. 4C). Although no
interactions were evident using the preimmune serum, two protein bands
were detected using the CBF antiserum. The interaction with the two
protein bands could not be blocked by recombinant GST, whereas it was
by the GST-CBF fusion protein (Fig. 4C, right
panels). Surprisingly, two novel bands reproducibly appeared in
the assay after blocking with recombinant GST-CBF. Our interpretation
of this finding is that the CBF used as competitor interacted directly with other proteins in the nuclear extract, and that these interactions were subsequently detected by the anti-CBF serum. The mobilities of the
two bands detected in nuclear extracts were subsequently compared with
the mobility of purified CBF-A (Fig. 4C, left
panel). The purified protein was detected as a single band that
corresponded in mobility to the faster moving of the two bands in the
nuclear extract. To test if the slower mobility protein was specific to the J558 cell line, the S194 myeloma cell line was also tested. As
shown in Fig. 4C (middle panel), both bands were
present also in this cell line. The same was also true for a panel of
different murine B cell lines (data not shown).
To investigate whether CBF-A was encoded by multiple mRNA species
we performed a Northern blot using poly(A)+ RNA from three
mouse B cell lines (Fig. 4D). All expressed CBF-A RNA, but a
weakly hybridizing RNA with lower mobility could also be detected. The
relative amount of this larger transcript, as compared with the major
hybridizing band, did not correspond to the relative amounts of the two
different proteins that were observed in Western blots. Rather, the
observed pattern is consistent with the higher molecular weight RNA
being a splicing intermediate. Hence, we prefer to interpret our data
as to indicate that CBF-A undergoes post-translational modifications.
However, the resolution of our Northern blot makes it impossible to
exclude that more than one splice variant of CBF-A were expressed if
these were of a similar size (60).
CBF-A Interacts with Either Single- or Double-stranded
DNA--
During the purification of the A-T-rich region interacting
protein, it was noted that the pdLMW activity copurified with a single-strand binding activity. Therefore, the binding of the protein
was further characterized in EMSA with the pd element labeled on either
strand, in single- or double-stranded form, as probe. The protein
interacted strongly with single-stranded probe labeled on the coding
strand, while less binding was evident using double-stranded probe
(Fig. 5A, left
panel, lanes 1 and 2). Even though the
protein interacted with the noncoding strand forming a complex of
similar mobility (lanes 3 and 4), this binding was considerably weaker than the binding to the coding strand. However,
both double-stranded DNA or either of the strands competed for CBF-A
binding to a coding strand probe, while an unrelated single-stranded
oligonucleotide did not (Fig. 5A, right
panel).

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Fig. 5.
A, left panel shows EMSAs
with purified protein and different pd element probes as indicated. The
probes were either from the coding strand (c) or non-coding
(nc) of the promoter and were single-stranded
(ss) or double-stranded (ds) as indicated. The
binding to each to the probes was tested in EMSA as indicated with the
same amount of purified protein in each lane (fraction 24) as source of
protein. To the right, the binding of the purified protein
was competed with either of the strands, double-stranded competitor or
a single-stranded, unrelated competitor. B, an EMSA with
double-stranded or heat-denatured full-length SP6 promoter as probe
and purified CBF-A as source of protein is shown at the top,
and an assay to determine of the probe is double-stranded or
single-stranded is shown at the bottom. The experimental
protocol is shown to the right.
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It could not be excluded that a significant fraction of the
double-stranded probes and competitors used were in fact
single-stranded, or that the two strands were separated before binding.
We therefore developed a method that directly addressed if the protein
could bind double-stranded DNA, as the issue whether or not CBF-A could also interact with double-stranded DNA was important for our
understanding of its biological activity. The SP6
promoter was
inserted into the pGEM3Z plasmid and end-labeled as described under
"Experimental Procedures." Then either native double-stranded probe
or heat-denatured probe was incubated with increasing amounts of
purified protein. After a 5-min incubation, half of each reaction was
loaded onto a non-denaturing PAGE, while SDS-containing stop buffer was
added to the remainder of the reactions and they were then incubated for another 5 min before loading. A mobility shift representing binding
of protein to the probe was observed with either double- or
single-stranded probe, although a smaller amount of protein was needed
to shift the single-stranded probe (Fig. 5B, upper panel). The shift of double-stranded probe was not due to the probe being denatured, as judged by the difference in mobility that was
evident between single-stranded and double-stranded probe after the
addition of SDS stop buffer (Fig. 5B, lower
panel). Rather, it appeared that the single-stranded probe was
reannealed during the incubation with protein. Thus, CBF-A is able to
interact with double-stranded DNA but with a lower efficiency than to a single-stranded template.
Mapping of the Interaction between CBF-A and DNA--
We next
determined which part of the pd element that was contacted by CBF-A. A
single-stranded probe was used that corresponded to the coding strand
of the promoter and included the pd, the
-Y and the octamer
elements. When increasing amounts of purified protein or recombinant
CBF-A were added to the probe before the addition of DNase, a fully
protected footprint that extended over the A-T-rich region of the pd
element was observed (Fig. 6,
A and B). Partial protections of the
-Y
element proximal to the pd element and of the pd E-box were also
evident, while the DNase reactivity of the rest of the
-Y element
and the octamer was enhanced. A similar footprint over the A-T-rich
region was observed when the pd element only was used as probe (data
not shown), showing that the single-stranded footprint was specific for
the A-T-rich region. The interaction between single-stranded DNA and
CBF-A was further analyzed in a methylation interference assay using recombinant CBF-A (Fig. 6C). In this assay, two A residues
were important for CBF interaction (marked with arrowheads),
and these were identical to those shown to be critical for function in
Fig. 1. A partial protection of the last G residue of the pd element E-box was also detected (marked with an asterisk; the
results of the footprints and the methylation interference assays are summarized in Fig. 7). These results were
those observed previously in a methylation interference using nuclear
extracts as a source of protein (41). We also attempted footprint and
methylation assays of double-stranded probes. However, in these assays,
we could only detect very weak protection at high protein
concentrations (Fig. 6D and data not shown). Thus, whereas a
specific interaction could be shown between single-stranded DNA and
CBF-A, the interaction between the protein and double-stranded DNA was
either unspecific or of to low affinity to be visualized by these
techniques. This conclusion is also supported by the data shown in Fig.
5.

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Fig. 6.
A, a single-stranded probe that includes
the octamer and the region 5' of it was used in a DNase footprint assay
to map the interaction between recombinant CBF-A and DNA. The probe was
incubated with increasing amounts of recombinant protein before the
addition of DNase I. From the left on the autoradiograph are
a A-G ladder, undigested probe, probe digested in the absence of
protein, and probe digested after preincubation with increasing amounts
of recombinant protein. To the left of the figure is shown
the sequence of the probe with protected regions marked. B,
a DNase footprint as in A using purified CBF-A.
C, a methylation interference assay using recombinant CBF-A
protein. Two protected A residues are marked with arrowheads
and a partly protected G residue with an asterisk.
D, a DNase footprint assay using double-stranded
probe.
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Fig. 7.
A, EMSAs with different mutated probes
are shown. In the left panel purified CBF was used, and in
the right panel recombinant, thrombin-cleaved CBF was used.
B, at the top are shown the results from the
DNase footprint and methylation interference assays. Protected residues
in the methylation interference assay are indicated above the sequence.
The arrowheads indicate complete protection, the
asterisk a partial protection. The regions that were
protected in the footprint assay are underlined,
dashed when only partially protected, and a hyper-reactive
region is overlined. Below are shown the sequences for the
probes used in B.
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The specificity between CBF-A and single-stranded DNA was further
analyzed in EMSA using single-stranded probes with different mutations
as compared with the wild type sequence and recombinant or purified
CBF-A (Fig. 7A). The wild type sequence interacted with
either of the proteins (lane 1, pd), as did
probes with mutations in the E-box or in the core of the
-Y element
(lanes 3 and 6, mut1 and
mut2). Probes with mutations of the 3' part of the E-box (lane 4, mut2) or the two functionally important
A residues that were protected in methylation interference (lane
2, ATmut) did not interact with the proteins, while a
weak binding was evident using a probe with three T residues mutated
downstream of the A residues (lane 5, mut 3).
These results were in good agreement with the results of the footprint
and methylation interference analysis and are summarized in Fig.
7B.
We next expressed a set of deletion mutants of the CBF-A protein in
bacteria and purified them on glutathione-Sepharose beads as described
under "Experimental Procedures" (schematically shown in Fig.
8B). All the variants were
expressed and yielded recombinant proteins of the expected sizes, and
no extensive breakdown was evident in Western blots (Fig.
8A, right panel). The deletion mutants
were then tested for DNA interactions in EMSA using the single-stranded
pd element as a probe (Fig. 8A, left panel). All variants that had two RNP domains intact interacted strongly with DNA
(lanes 2, 3, 7, and 9), and
a weak interaction was also evident for two variants that contained one
RNP domain (lanes 4 and 5). Neither the GST tag
alone (lane 1) nor a mutant where both RNP domains had been
deleted (lane 6) showed significant DNA binding. Two of the
recombinant proteins contained one RNP domain but did not bind DNA
(lanes 8 and 10). In these mutants, the RNP
domain was directly fused to the GST tag, which might interfere with DNA binding. In conclusion, the RNP domains of CBF-A were involved in
the interaction with DNA and both of them were needed for a high
affinity interaction.

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Fig. 8.
A, EMSAs with different deletion mutants
of CBF-A and the single-stranded wild type pd element as probe are
shown to the left (the probe has been run out of the gel).
To the right is shown a Western blot of the same amounts of
the different deletion mutants as were used in the EMSA. B,
a schematic figure of the different deletion mutants used in
A and their DNA binding characteristics is shown.
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CBF-A Interacts Directly with the Ets Proteins PU.1 and
elf-1--
The close proximity between the CBF-A binding site and the
ets site in the SP6
promoter made us assay for
interactions between their ligands. As shown in Fig.
9A, recombinant GST/CBF-A
fusion protein bound to glutathione beads was able to pull down about 3.5% of in vitro translated PU.1 or elf-1, whereas GST
alone or empty beads did not. The specificity of the interaction was
demonstrated by that neither in vitro translated OCT2 nor
E47/E12 protein were pulled down by GST/CBF-A (data not shown and Fig.
9A). Thus, CBF-A can interact with the Ets proteins elf-1
and PU.1 in vitro.

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Fig. 9.
A, in vitro translated PU.1,
elf-1, or E2A proteins were tested in a pull-down assay with either GST
or GST-CBF-A bound to glutathione beads. The lanes labeled
10% contain 10% if the total amounts that were added in
the interaction assays, and the lanes labeled ,
GST, and CBF show the amounts that were pulled
down with empty beads, GST beads, or beads with GST-CBF-A fusion
protein, respectively. Below the autoradiographs are shown the relative
amounts (in %) that were pulled down by the different variants with
standard deviations (n = 3 or 4). B, an
assay similar to that in A with different deletion mutants
of CBF-A bound to the beads and PU.1. Below the autoradiographs are
shown the relative amounts (as compared with the GST-CBF fusion) that
were pulled down. C, a Western blot of proteins that were
bound to beads, incubated with reticulocyte lysate, washed as in
B, and then eluted in SDS loading buffer. D, a
schematic figure of the different deletion mutants that were used and
the interaction with PU.1 indicated.
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To map the interaction domain of CBF-A with Ets proteins, the deletion
mutants that were used in Fig. 8 were tested for interactions with PU.1
(schematically shown in Fig. 9D). Fig. 9B shows a
typical experiment. All the deletion mutants interacted with PU.1 and pulled down about 50% of the amount that was pulled down by the full-length variant used in Fig. 9A, except for the last
variant (lane 10) that only contained one RNP domain. The
same result was obtained using elf-1 instead of PU.1 (data not shown).
To verify that all the GST fusion proteins bound to the glutathione beads and were not broken down during the incubation with reticulocyte lysate, we made a Western blot of recombinant protein eluted from glutathione beads after incubation with reticulocyte lysate. All the
proteins were detected in the assay, but variant 4 was partially broken
down and lower amounts were bound of variants 3, 5, and 7. If the
amount of PU.1 pulled down with these deletion mutants was corrected
for the lower amount of CBF-A recovered, it may be concluded that all
mutants except 10 were equally efficient in the assay. Thus, the
interaction between PU.1 and CBF-A is complex and involves two separate
interaction domains on CBF-A; the RNP domains and the carboxyl-terminal
region of the molecule. Whether these interactions are mutually
exclusive or act in consort is currently unclear.
 |
DISCUSSION |
The A-T-rich region of the pd element has been shown to be active
in octamer-dependent stimulation of
transcription and to interact specifically with protein (pdLMW) in EMSA (41). Here we
identify the pdLMW protein as CArG-box binding factor-A (CBF-A; Ref.
23). This protein was originally cloned from a
expression library
due to its interaction with the intron CArG-box of the human smooth
muscle
-actin gene, and it has high homology to D-box binding
factor, a chicken DNA interacting protein cloned using the same
approach (25). Both of these proteins were shown to interact mainly
with single-stranded DNA, but in neither of the reports it was possible
to exclude binding to double-stranded DNA. In this study, it was shown
that the protein can interact also with double-stranded DNA or possibly
with single-stranded structures that are formed within the
double-stranded DNA; the protein was purified on a native DNA column
and interacted with a double-stranded probe in EMSA, although with a
lower efficiency than with single-stranded DNA.
CBF-A and the above mentioned D-box binding factor have RNP domains in
common with hnRNP A and C proteins (29), a domain that is thought to be
involved in the binding to nucleic acid (53). hnRNP A and C are parts
of the hnRNP complex, which is thought to have a role in mRNA
transport and splicing (29). However, some of the proteins that are
associated with mRNA in hnRNP complexes have also been implicated
as transcription factors. These include the hnRNP K protein, which
associates with a region that is found in single-stranded form in the
c-myc promoter and has potential as a transcriptional
activator both in vitro and in vivo (11-13), and
a protein related to hnRNP C that stimulates transcription of a viral
gene upon overexpression (24). Furthermore, the TLS/FUS oncoprotein has
been found in association with the hnRNP complex (54), while recent
data have indicated a role for it in transcription as it is found
associated with TBP in a subpopulation of TFIID complexes (55). Thus,
many of the hnRNP proteins may have dual functions in the cell,
i.e. they are involved in both splicing and transporting of
RNA and in transcriptional control.
In this context, it should also be noted that pdLMW is located in the
cytoplasmic fraction of resting B lymphocytes while being translocated
to the nucleus upon mitogenic activation (41). This could indicate dual
functions, but also be a regulatory step where the need for the protein
in the nucleus is enhanced in activated cells. The antiserum that was
raised against the protein interacted with two distinct bands in a
Western blot, while only one major RNA was evident in Northern blot.
Although it cannot be formally excluded that the two forms in the
Western blot were due to cross-reactivity, or that two overlapping RNA
splice forms of CBF-A exist (60), it is to our mind more likely that
the two protein species arise due to post-translational modifications.
These modifications could regulate either the localization or the
function of the protein. The protein has several potential
phosphorylation sites, and only the lower mobility form was evident
during the purification. Thus, post-translational modifications may
interfere with the binding to nucleic acids.
CBF-A does not appear to be a classical transcription factor containing
an activation domain since a multimerized pd element is a poor
activator of transcription (41). Hence, we speculate that the protein
rather has a structural role in the promoter, distorting the DNA either
by bending or inducing other structural alterations that may facilitate
efficient transcriptional activation. Furthermore, if the interactions
with the Ets proteins PU.1 and elf-1 are to take place in
vivo, CBF-A might act by increasing the local concentration of
these transcription factors. CBF-A was originally described as a
repressor of transcription (23), while the region to which it binds in
the SP6
promoter is a positive control element. Such a phenotypic
switch has been described for other transcription factors (56-59), and
is most likely due to complex interactions with other regulatory
elements in the different transcriptional units.
In conclusion, CBF-A was purified due to its interaction with a
positive control element within the SP6
promoter. Whether the
protein is only involved in transcriptional regulation or whether it
also has other functions in the cell is currently unclear. However,
this investigation has yielded information and reagents that will
facilitate future analysis of this question.