(Received for publication, September 20, 1995; and in revised form, November 16, 1995)
From the
CCAAT/enhancer-binding protein (C/EBP) transcription factor
family members are related by a high degree of amino acid sequence
identity to the basic leucine zipper DNA-binding domain and show
distinct but overlapping patterns of tissue- and stage-restricted
expression. Although C/EBP and C/EBP
have been shown to
recognize a consensus sequence derived from regulatory elements in
virus and acute-phase response genes, the potential for more subtle
differences in the binding preference of the C/EBP family has not been
previously addressed. The consensus sequence of C/EBP
has not been
reported. By using the method of polymerase chain reaction-mediated
random site selection to assess the DNA binding specificity of the
C/EBP family in an unbiased manner, we demonstrated the sequence
preferences for C/EBP family members. With small variations, these
C/EBP family members showed similar sequence preferences, and the
consensus sequence was identified as RTTGCGYAAY (R = A or G, and Y = C or T). The
phosphorylation of C/EBP
by casein kinase II increased the binding
activity, but did not affect the binding specificity, whereas it was
reported that the phosphorylation of C/EBP
and C/EBP
decreased the binding affinity. The specificity of action of C/EBP
family members may be derived from the characteristics of each factor,
including the expression profiles, the DNA binding affinities, the
cofactors, and so on, in addition to the DNA binding specificities.
Many transcription factors have been found to be members of
highly related multifactor families, and thus, their specificity of
action must be addressed in order to ascertain their respective
functions. CCAAT/enhancer-binding protein (C/EBP) ()family
members are among the basic leucine zipper transcription factors, and
they bind to specific DNA sequences as dimers. Six C/EBP proteins
(designated C/EBP
, C/EBP
, C/EBP
, C/EBP
, C/EBP
,
and CHOP 10) have been identified, and C/EBP
, C/EBP
, and
C/EBP
have been studied in
detail(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) .
C/EBP, previously termed C/EBP, was identified originally as a
heat-stable protein present in soluble extracts of rat liver nuclei and
having sequence-specific DNA binding activity. Purified C/EBP
selectively recognized CCAAT homologies and enhancer core sequences,
implying that it might be a transcriptional regulatory protein.
Alignment of the avian retroviral binding sequences with the published
binding sites for C/EBP
in two CCAAT boxes and in the simian virus
40, polyoma, and murine sarcoma virus enhancers suggested
TKNNGYAAK (K = T or G, and Y = C or T) as a consensus sequence for binding of
C/EBP
(13) . Several studies showed that C/EBP
trans-activates liver- and adipose-specific genes as well as
virus genes, while C/EBP
and C/EBP
were cloned as nuclear
factors that regulate acute-phase response genes and liver-specific
genes(2, 3, 4, 7) . Akira et al.(2) reported that competition analysis of the binding of
NF-IL6 (human C/EBP
) with the published sequences to which
C/EBP-like proteins bound revealed that NF-IL6 and C/EBP
recognized the same nucleotide sequences and that the best fit was the
consensus TKNNGNAAK for NF-IL6. The consensus
sequence of C/EBP
has not been reported. The basic leucine zipper
regions of C/EBP isoforms show high similarity, and some cis-elements are recognized by each of the C/EBP
isoforms(4, 6, 7, 8) . Each isoform,
however, shows distinct but overlapping patterns of tissue- or
stage-restricted
expression(2, 3, 4, 6, 7, 8, 9, 10) .
Therefore, it is important to determine the specificity of the C/EBP
family. The polymerase chain reaction (PCR)-mediated random site
selection method has been adopted to distinguish the binding sites of
the transcription factor family
members(14, 15, 16, 17, 18, 19) .
Modification of a transcription factor, such as by phosphorylation,
glycosylation, and reduction-oxidation, affects its binding activity
and function. Phosphorylation of C/EBP by protein kinase C results
in an attenuation of binding, and modification of C/EBP
by protein
kinases A and C results in an inhibition of DNA
binding(20, 21) . In the case of C/EBP
,
dephosphorylation severely decreases the DNA binding ability in
vitro, although neither kinase nor phosphatase for this protein
has been identified yet in vivo(22) .
Utilizing a
binding site selection procedure, we show here that the bacterially
expressed C/EBP proteins have virtually identical binding specificity
and that the phosphorylation of C/EBP by casein kinase II
increases the binding activity, but does not affect the binding
specificity.
The nucleotide
sequence of C/EBP is identical to that of Silencer Factor B
previously reported(5) . For production of the C/EBP
DNA-binding domain, we used Y1089 lysogen, which contains the
DNA-binding domain of C/EBP
/Silencer Factor B cDNA, but lacks 84
amino-terminal amino acids. Bacterial C/EBP
was produced and
purified as described previously(5) .
C/EBP was
expressed by the histidine fusion protein system (QIAGEN Inc.). The XhoI-DraI fragment derived from the rat genomic
C/EBP
gene (
)and encoding the DNA-binding domain was
subcloned into a pQE-30 expression vector. The recombinant plasmid was
transformed into M15(pREP4). The transformant was grown under the same
conditions as for C/EBP
with a slight modification. In brief, the
protein was induced by isopropyl-
-D-thiogalactopyranoside
when A
reached 0.8. The cells were suspended in
10% of the original culture volume of buffer A (6 M guanidine
hydrochloride, 0.1 M NaH
PO
, 0.01 M Tris (pH 8.0)) and stirred for 1 h at room temperature. The
supernatant obtained by centrifugation at 13,500 rpm for 15 min was
loaded onto a Ni
-nitrilotriacetic acid column
equilibrated with buffer A, washed with the same buffer, and eluted
with buffer E (8 M urea, 0.1 M NaH
PO
, 0.01 M Tris (pH 4.5)). The
eluate was dialyzed against 0.1 M HM buffer (25 mM HEPES (pH 7.8), 1 mM dithiothreitol, 12.5 mM MgCl
, 20% glycerol, 0.1 M KCl), and the
resultant supernatant was used for DNA binding analysis.
Double-stranded molecules were generated by annealing
oligonucleotides containing 16 random nucleotides with Primers 1 and 2
and then extending by Taq DNA polymerase (Promega). Enrichment
for binding sites was performed by the filter binding method (19, 24) . The binding mixture (35 µl) contained
purified bacterially expressed protein (1 µg of C/EBP, 7
µg of C/EBP
, or 0.6 µg of C/EBP
), 0.3 µg of
double-stranded random oligonucleotide, 1.4 µg of poly(dI-dC), and
3.5 µl of 10
binding buffer (100 mM Tris-HCl (pH
7.5), 50% glycerol, 10 mM dithiothreitol, 10 mM EDTA). Each mixture was incubated for 30 min at room temperature.
Thereafter, this solution was passed slowly through a presoaked
nitrocellulose filter (Schleicher & Schuell, BA85). The filter was
washed three times with 3 ml of 1
binding buffer before the
bound oligonucleotides were eluted with 100 µl of elution buffer
containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 20
mM NaCl, and 0.1% SDS. The eluate was phenolized and then
amplified by PCR using Primers 1 and 2. The amplified products (0.3
µg) were put back into a binding reaction, and the procedure was
repeated. After five rounds (in the case of C/EBP
and C/EBP
)
or seven rounds (in the case of C/EBP
) of the enrichment, the
amplified products were end-labeled with
[
-
P]ATP by using T4 polynucleotide kinase
and used as a probe. The labeled amplified products were incubated with
bacterially expressed C/EBP proteins, and protein-DNA complexes were
separated on 6% (in the case of C/EBP
and C/EBP
) or 4% (in
the case of C/EBP
) native polyacrylamide gels. Bound DNA was
eluted with a solution of 0.5 M ammonium acetate, 1 mM EDTA, 0.1% SDS, 10% methanol, and 50 µg/ml proteinase K. The
eluate was phenolized and then amplified by PCR using Primers 3 and 4.
Amplified products were digested with BamHI and XhoI
and subcloned into pBluescript KS (Stratagene). Clones were sequenced
by the dideoxy chain termination method(25) .
Figure 1:
Compilation of sequences selected by
C/EBP, C/EBP
, and C/EBP
. Bacterially expressed proteins
were used for selection of random oligonucleotides. A, list of
pentamer sequences selected by C/EBP isoforms. The percentages of
pentamer sequences selected by each protein are shown. B,
alignment of the binding sequences selected. The frequency of
nucleotide selection at each position of a composite pentanucleotide as
well as flanking nucleotides is indicated as percent. The consensus
binding sequences for each isoform are also shown. The bases in the
consensus sequence are numbered consecutively from 1 to 5 (the values
are positive for the right half-site and negative for the left-half
site).
Figure 2: DNA binding specificity of C/EBP family members. Double-stranded oligonucleotides containing palindrome consensus sequence for C/EBP binding were incubated with bacterially expressed C/EBP proteins in the absence (lane 1) or presence (A, lanes 2-13; B, lanes 2-9) of increasing amounts of the competitor for gel shift analyses. Each competitor was present in either 50-fold excess (even-numbered lanes) or 250-fold excess (odd-numbered lanes) relative to the probe. Competitor sequences (A, double point mutants; B, position +3 mutants) are shown on top of the lanes.
The selection data revealed that sequences previously identified as binding sites for some of the proteins were rarely selected. We examined the hypothesis that C/EBP isoforms could bind to mutant oligonucleotides at position +3, one of the most important bases for binding. A palindrome sequence including 5`-ATTGCGCAAT-3` was used as a probe, and the mutant oligonucleotides were adopted as competitors (Fig. 2B). The palindrome sequence completely competed binding to the probe at low concentration. The mutant oligonucleotides also inhibited binding to the specific probe at higher concentration. The mutant +3-C showed more effective competition than the other two competitors, +3-G and +3-T. These data revealed that C/EBP isoforms could bind to the mutant oligonucleotides at position +3 (+3-G, +3-T, and +3-C).
Figure 3:
Effect of phosphorylation on the binding
ability of C/EBP by casein kinase II. A, bacterially
expressed C/EBP
was incubated without casein kinase II (CKII) (lane 1), with casein kinase II (lane
2), and with casein kinase II and bacterial alkaline phosphatase (BAP) (lane 3) and then analyzed by
SDS-polyacrylamide gel electrophoresis. Proteins were detected by
silver staining. The arrows designate phosphorylated (upper) and nonphosphorylated (lower) C/EBP
. B, shown is the gel shift analysis of phosphorylated
C/EBP
. Increasing amounts (0.11, 0.33, and 1 ng) of C/EBP
(nontreatment (lanes 2-4), treatment with casein kinase
II (lanes 5-7), and treatment with casein kinase II and
bacterial alkaline phosphatase (lanes 8-10)) were
incubated with the probe (pal) and separated by native polyacrylamide
gel electrophoresis.
Figure 4:
DNA binding specificity of phosphorylated
C/EBP. Probes containing palindrome consensus sequence were
incubated with C/EBP
phosphorylated by casein kinase II in the
absence (lane 1) or presence (A, lanes
2-13; B, lanes 2-9) of increasing
amounts of the competitor (50- and 250-fold molar excesses) for gel
shift analysis. Competitor sequences (A, double point mutants; B, position +3 mutants) are indicated in Fig. 2.
Figure 5:
Relative affinities of bacterially
expressed C/EBP isoforms and phosphorylated C/EBP for the native
binding sequences found in the genes regulated by the C/EBP family. A, shown are the nucleotide sequences of binding sites in the
promoter of C/EBP regulated genes. The bases distinct from those of the
consensus binding site are underlined. B, the probe
(pal) containing palindrome consensus sequence was incubated with
bacterially expressed proteins or phosphorylated C/EBP
in the
absence (lane 1) or presence (lanes 2-10) of
increasing amounts of the competitor (10-, 50-, and 250-fold molar
excesses) for gel shift analysis.
A gel shift selection and PCR amplification using randomized
oligonucleotides and bacterially expressed proteins allowed us to
identify the preferred DNA-binding sites for C/EBP, C/EBP
,
and C/EBP
. Most of the selected clones, 76.6% (C/EBP
), 79.8%
(C/EBP
), and 45.8% (C/EBP
), contained 5`-RTTGC-3` (R = A or G) sequence (Fig. 1A). The
consensus binding site for each member of the C/EBP family was
determined by an alignment of the binding site selection data, which
were summarized by the frequency of nucleotide selection at each
position of a composite pentanucleotide as well as flanking
nucleotides. The consensus binding site,
5`-RTTGCGYAAY-3` (R = A or
G, and Y = C or T), for all three proteins is similar,
except for small differences. Many sequences of the binding sites
selected by C/EBP
are not identical to the consensus binding
sites. The hierarchy of DNA binding affinities for the consensus
sequence of C/EBP isoforms is C/EBP
> C/EBP
>
C/EBP
(data not shown) (7) . In the case of C/EBP
and
C/EBP
, there is a difference between the high and low affinity
binding sites, with the former being mainly selected. However, in the
case of C/EBP
, because of small differences in affinity, many of
the low affinity binding sites were also selected. It was reported that
C/EBP
and C/EBP
could recognize a consensus sequence derived
from regulatory elements in virus and acute-phase response genes,
TKNNGYAAK or TKNNGNAAK (K = T or G, and Y = C or T),
respectively(2, 13) . These consensus sequences are
not palindromic, although they resemble our results. As our system
included all possible combinations of the 10-nucleotide randomized
sequence, the selected binding sites were unbiased. Our data revealed
that C/EBP isoforms could recognize the palindrome sequence. The
precise nucleotide identities of positions +/-3 and
+/-4 were the most critical determinants of recognition
specificity and affinity. Position +/-2 bases were the third
most important. Bases at positions +/-1 and +/-5
were also selected weakly, indicating that C/EBP isoforms interact at
these positions with a moderate degree of sequence preference. The
crystal structure of the GCN4 and AP-1 sites revealed that one of the
bases near the center of the AP-1 site is not contacted by the basic
leucine zipper structure(31) . Because position +/-1
bases are located in the center, these bases are not in contact with
the basic leucine zipper structure and show higher flexibility compared
with positions +/-2, +/-3, and +/-4.
Many binding sites of C/EBP family members have been previously
identified. Most of them, however, are not completely coincident with
the consensus sequence reported here. When significant base(s) for the
DNA binding are not identical to the consensus sequence and other bases
are conserved, C/EBP members could weakly bind to the DNA. Binding site
sequence that is more similar to the consensus sequence, especially at
positions +/-3 and +/-4, showed a much higher
affinity. We used bacterially expressed DNA-binding domain fusion
proteins, such as glutathione S-transferase,
-galactosidase, and histidine tag. Gel shift analysis using mutant
binding sites revealed that the same binding specificities were shown
regardless of the kind of fusion protein (data not shown).
Sequence-specific transcription factors are composed of structural
domains for DNA binding and transcriptional regulatory functions, and
DNA binding specificities are usually determined by the DNA-binding
domain. However, it seems possible that the transcriptional regulatory
domain affects the DNA binding specificities. These possibilities
remain to be resolve.
The binding activity of C/EBP isoforms was
modified by phosphorylation. It is known that phosphorylation of the
DNA-binding domain of C/EBP, containing Ser
in the
basic region of the basic leucine zipper structure, which is the
phosphorylation site by protein kinase C, results in attenuation of DNA
binding(20) . Phosphorylation of C/EBP
at Ser
within the DNA-binding domain by protein kinase A results in a
decrease in its binding to DNA, while phosphorylation of Ser
within the transcriptional activation domain has no effect on DNA
binding(21) . In the case of C/EBP
, dephosphorylation of
the nuclear extracts reduces the DNA binding ability of endogenous
C/EBP
in vitro, although the phosphorylation site and the
kinase and/or phosphatase for this protein have not been identified yet in vivo(22) . When C/EBP
was phosphorylated by
incubation with casein kinase II, DNA binding activity was promoted (Fig. 3). Casein kinase II phosphorylates serine residues
located in acidic regions of proteins(32) . Five serine
residues exist in the C/EBP
DNA-binding domain, and one of them,
Ser
, exists in acidic regions
(Glu-Leu-Ser
-Ala-Glu-Asn-Glu), although it is unclear
whether Ser
is actually phosphorylated by casein kinase
II in vitro and in vivo. We could not rule out the
possibility that other sites of phosphorylation on C/EBP
may also
affect the structure and therefore the specificity and affinity of the
protein for its DNA targets in vivo.
We found that the
consensus binding sites for the C/EBP family members are similar to
each other, suggesting that same target genes might be regulated in the
cells. The function of the C/EBP-binding site is classified in three
ways. AlbD and CRP act as a constitutive and an inducible enhancer,
respectively(3, 4) . On the other hand, GPS1 functions
as a silencer(29) . The order of the affinity of the three
binding sites depends on the similarity to the consensus sequences.
These sites show different functions, but they behave similarly when
binding. Considering the gene expression regulated by the C/EBP family,
the amount of expression and the affinity for DNA of C/EBP proteins are
important factors. In the cell, many parameters must collaborate to
define the function of a given C/EBP-binding site located in chromatin
structure, and it is possible that these interact synergistically with
other transcription factors. As C/EBP interacts with NF-
B-p50
polypeptide and glucocorticoid receptor and C/EBP
associates with
TFIIB and TATA-binding protein, the differential activity of C/EBP
isoforms in vivo may be caused by distinct interactions with
other proteins(33, 34, 35) . In vivo experiments will be required to clarify the functional specificity
of the C/EBP transcription factor family.