From the Department of Biology, The College of
William and Mary, Williamsburg, Virginia 23187 and the
¶ Department of Molecular and Cellular Biology, the University of
California, Berkeley, California 94720
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ABSTRACT |
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The B cell-specific transcription factor Pax-5
has been shown previously to interact with the promoter of the
blk gene in vitro. blk encodes a tyrosine
kinase associated with the B cell receptor, which is expressed during
the early but not the final stages of B cell development. To
investigate whether Pax-5 regulates expression of the blk
gene in vivo during B cell development and/or activation,
Pax-5a was overexpressed in B cell lines. Increases in
blk promoter activity using a chloramphenicol
acetyltransferase reporter gene system suggested a role for Pax-5a as a
transcriptional activator. Subsequent site-specific mutagenesis studies
showed that mutations of the Pax-5 binding site on blk
significantly alter promoter activity, although results suggested that
other factors could bind to this region as well. Using mobility shift assays, we detected an inducible transcription factor that interacts strongly with a sequence overlapping the Pax-5 site on the
blk promoter and identified this as a homodimer of
NF-B/p50, a member of the NF-
B/Rel family of transcription
factors. This factor was present at high levels in
lipopolysaccharide-activated normal B cells and in plasma cell lines
but either at low levels or undetectable levels in resting normal B
cells or pre-B or mature B cell lines. In contrast, lipopolysaccharide
induction of a pre-B cell line (703/Z) induced a complex that contained
both NF-
B/p50 and p65. These studies suggest that different NF-
B
complexes are able to interact with a sequence overlapping the Pax-5
site on the blk promoter and that the relative levels of
"bound" factor influence levels of blk expression.
Since p50 homodimers and p50/p65 heterodimers of the NF-
B complex
should have opposing effects on blk transcription, this
could provide a mechanism to differentially regulate blk expression during B cell development and activation.
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INTRODUCTION |
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The activation and subsequent proliferation and differentiation of mature B cells into plasma cells is perhaps the most critical event within the B cell differentiation pathway, because it leads to the production of antibodies necessary for the elimination of foreign antigens. B cell activation is typically initiated by cross-linking of the B cell antigen receptor complex, but activation signals can also be generated through binding of certain cytokines or the B cell mitogen lipopolysaccharide (LPS).1 Activation involves a cascade of events associated with proliferation and differentiation, eventually resulting in the presence of plasma cells, which secrete large amounts of antibodies and memory B cells. In order to elucidate the molecular pathways involved in B cell development and activation, we have taken the approach of investigating specific transcription factors and the roles they play in the modulation of gene activation and/or repression during these processes.
A number of Src family kinases have been reported to be associated with the B cell receptor complex, including the B cell-specific Blk kinase, encoded by the blk gene (1). In this study, we use blk as a B cell-specific target gene whose expression is regulated, at least in part, by transcription initiation (1). Blk transcripts are present in all pro-B, pre-B, and mature B cell line studied but are undetectable at the plasma cell stage (1). As has been observed in many developmentally regulated genes, the 5'-flank of the blk gene contains at least four transcription start sites but no functional TATA box (1). Earlier work indicated that a 320-nucleotide (nt) promoter region of blk can promote transcription of a heterologous gene after transfection into B cell lines (2), although its activity is low. At least one B cell-specific transcription factor, the B cell-specific activator protein (BSAP) has been shown to interact specifically with a 5' blk sequence in vitro (2, 3).
BSAP is encoded by the Pax-5 gene and is a member of the paired domain family of transcription factors. BSAP (also named Pax-5) protein has been detected in B cells, adult testis, and the developing midbrain (3-5). Within the B cell lineage, Pax-5 transcripts and its encoded products have been detected in pro-B, pre-B, and mature B cell lines, whereas either very low or undetectable levels were found in the various plasma cell lines studied (3-5). Pax-5 is alternatively spliced, and at least four isoforms have been detected in nuclear extracts of B cell lines, although the expression levels of the full-length form, Pax-5a, are higher then any of the alternatively spliced forms Pax-5b, -5d, or -5e (3). The amino acid sequence of isoform Pax-5a is identical to that of BSAP (3).2
The in vivo function of Pax-5 has been
investigated through targeted gene disruption in mice (6). Homozygous
mutant mice display a complete developmental block at the late pro-B
cell stage, suggesting essential regulatory functions in early B cell development. Many putative target sequences for Pax-5 have
been reported, including the promoters of CD19, CD20,
5, Vpre-B, J-chain, blk, and mb-1 (2, 4, 7-11) as well as the
3'-
enhancers and switch regions of immunoglobulin (Ig) heavy
chain genes (12, 13). Based on the expression pattern of
Pax-5 target genes, it is likely that Pax-5 is required
during later B cell stages as well. Some evidence to support this comes
from a recent report showing that overexpression of isoform
Pax-5a in plasma cell lines can repress expression of two of
its target genes, the J-chain and the Ig heavy chain gene (10, 14,
15).
Recent studies suggest that Pax-5a can act as an activator, a docking protein, or a repressor, depending on the target gene and possibly the developmental state of the B cell. For example, the expression patterns of the CD19 and Pax-5 genes in B cells are identical, which suggests that Pax-5 is a positive regulator of the CD19 gene (4). This possibility is further supported by the observation that CD19 expression has ceased completely in homozygous Pax-5 mutant mice (6). In the regulation of the B cell-specific mb-1 gene, Pax-5a functions as a docking protein to recruit a group of more widely expressed transcriptional activators, the Ets proteins (16). In contrast, a suppressor function has been suggested by studies of both J-chain and Ig heavy chain genes, although the exact mechanisms by which this is accomplished by Pax-5a are not well understood (10, 12, 14, 17, 18). In regard to the regulation of blk gene expression, it has not yet been established what in vivo role, if any, Pax-5a plays. Notably, the levels of blk RNA in the pro-B cells from homozygous Pax-5 knockout mice were reportedly identical to those of their wild type (WT) littermates, which either implies that Pax-5a does not regulate blk levels during the earliest stages of B cell development or that the absence of Pax-5a in knockout mice leads to an artificial situation of redundancy in which other factors can take over Pax-5a functions.
In this study, we wished to investigate whether Pax-5a influences blk expression during B cell development and/or activation. We show here that overexpression of Pax-5a in B cell lines leads to an increase of blk promoter activity as measured using a reporter gene system, which suggests that it acts as a transcriptional activator. Subsequent site-specific mutagenesis studies of the blk promoter showed that mutations of the Pax-5 binding site can significantly alter blk promoter activity in vivo, although the results suggest that other factors with binding sites close to or even overlapping with the Pax-5 site may influence blk transcription as well. Using normal B cells activated with LPS, we subsequently identified an inducible transcription factor that binds strongly to a sequence overlapping the Pax-5 site in activated B cells.
This inducible factor was identified as a homodimer of NF-B/p50, a
member of the NF-
B/Rel family of transcription factors that in
mammals contains the protein members p50, p65, p52, c-Rel, and RelB;
these subunits can form either homodimers or heterodimers (19-22).
NF-
B is present in the cytoplasm during periods when it is not
needed, forming an inactive ternary complex with the so-called
inhibitory (I
B) factor, which prevents NF-
B from moving into the
nucleus. The NF-
B pathway can be activated in B cells by IgM
cross-linking or by the addition of LPS (19-22). In such situations,
the I
B protein is degraded, and the remaining (active) NF-
B dimer
is translocated to the nucleus, providing a system that is able to
respond very rapidly to cell activation events. Interestingly, p50
homodimers lack a transactivating domain, and it has been implied that
such dimers are involved in repression of transcription initiation
(22-25). In contrast, p50 heterodimers are thought to be involved in
transcriptional activation (22-25).
Unexpectedly, LPS induction of a pre-B cell line 703/Z did not induce formation of the p50 homodimer complex but instead induced a complex containing both p50 and p65. Further analysis in B cell lines representing different stages of B cell differentiation indicated that the relative levels of p50 homodimers, p50 heterodimers, and Pax-5a bound to the blk promoter strongly influence the levels of blk transcripts in these lines.
These studies suggest that different NF-B complexes are able to
interact with the blk promoter and that these complexes
compete for binding with Pax-5a. Because NF-
B/p50 homodimers and
heterodimers have opposite effects on transcription initiation, this
may provide a mechanism to differentially regulate blk
expression after cell activation, depending on the developmental state
of the B cell.
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MATERIALS AND METHODS |
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Cell Lines--
All cell lines were grown in RPMI 1640 medium
containing 10% fetal calf serum (BioWhittaker, Inc.), 2 mM
glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, and 50 µM -mercaptoethanol.
DNA Constructs--
The construction of the
p(191)blkCAT2µE reporter construct has been described
elsewhere (2). A promoterless chloramphenicol acetyltransferase (CAT)
construct,
CAT2µE, was derived from p(
191)blkCAT2 µE by restriction digestion with the endonucleases StuI
and SalI, resulting in the specific deletion of
blk sequence only. The effector construct pcDNA3.Pax-5a
was made by digesting both the expression vector pcDNA3
(Invitrogen) and the pBS-1.2 plasmid (2), containing the full-length
cDNA sequence of Pax-5a, with the restriction endonuclease
NotI, followed by gel purification of the 1.2-kilobase pair
Pax-5a insert. The NotI insert was then cloned directly into calf intestine phosphatase-treated, NotI-digested
pcDNA3, and the orientations of the resulting clones were
determined using dideoxy sequencing. This resulted in clone
pcDNA3.Pax-5a, which expressed Pax-5a protein as determined by
in vitro transcription and translation in the presence of
[35S]methionine followed by SDS-polyacrylamide gel
electrophoresis and radiography. The pcDNA3 construct was used as a
negative control effector construct, and the HBIICAT construct (2) was
used as a control for transfection efficiency.
Transfections and CAT Assays--
The B cell lines A20/2J, M12,
and S194 were transiently transfected using DEAE-dextran, and
chloramphenicol acetyl transferase was assayed as described elsewhere
(2). The generation of clones S194-Tf1 and S194-Tf4, which are stable
transfectants containing the full-length isoform Pax-5a in S194 (clone
Tf1 expresses low levels of Pax-5a; clone Tf4 expresses high levels)
has been described elsewhere (10, 14). The relative CAT conversion was
determined by calculating the ratio of the acetylated to the acetylated
plus unacetylated counts per minute, subtracting the activity of the mock transfection (transfection without DNA), followed by subtraction of the promoterless construct (CAT2µE) and normalization for protein content as determined by the Bradford assay. To enable comparison of different cell lines, values were normalized for transfection efficiency by dividing each activity by the relative CAT
conversion of the HBIICAT control construct (see above). Relative CAT
activity values for each transfection were calculated as -fold change
compared with the activity of the wild type
(
191)blkCAT2µE construct.
In Vitro Transcription and Translation of Pax-5a-- The plasmid (pBluescript) pBS-1.2 (containing full-length Pax-5a cDNA; see Ref. 2) was transcribed in the sense direction with T3 RNA polymerase. Translation was carried out using a rabbit reticulocyte lysate (Promega) according to the supplier's directions. Between 1 and 2 µl of extract was used for mobility shift assays.
Antibodies-- To generate an antibody against Pax-5a isoforms, the paired domain sequence of the Pax-5 gene was cloned downstream from the glutathione S-transferase sequence in the vector pGEX2T (Amersham Pharmacia Biotech). The fusion protein containing 148 amino acids of the paired domain sequence of Pax-5 was purified using glutathione-agarose beads. New Zealand White rabbits were immunized intradermally with 500 µg of purified protein, followed by five booster immunizations of 100 µg each that were administered every other week beginning 21 days after the initial immunization. Antiserum, named Ed-1, was obtained at 12 weeks, analyzed by enzyme-linked immunosorbent assay, and used at a 1:2000 dilution in Western blot analysis or at 1:5 or 1:10 dilution for the mobility shift assays. Antibodies to p65, p50, and Sp1 were purchased from Santa Cruz Biotechnology, Inc., and used at a 1:5000 dilution in Western blot analysis or 1:5 in mobility shift assays. The anti-p65, anti-Pax5, and anti-Sp1 antibodies were detected using a horseradish peroxidase-conjugated donkey-anti-rabbit secondary antibody (Amersham Pharmacia Biotech). The p50 antiserum was directed against the nuclear localization domain of p50, and a rabbit anti-goat, horseradish peroxidase-conjugated antibody was used as its secondary antibody in Western blot analysis (Amersham Pharmacia Biotech).
Isolation of Normal B Cells-- B cells were isolated from spleens from 6-8-week-old BALB/c mice. After removal of the spleens, cells were resuspended and washed in Hanks' balanced salt solution. Small resting B cells were isolated from a Percoll gradient (Amersham Pharmacia Biotech) according to the supplier's instructions. This population of cells, containing 75% Ig-positive cells, 24% Thy-1-positive cells, and 1% non-lymphocytes, was either processed immediately to obtain nuclear extracts or used for activation experiments by growing in complete RPMI medium (as above) including 20 µg/ml LPS.
Western Blot Analysis-- Nuclear and cytoplasmic extracts from B cell lines or normal B cells were prepared as described elsewhere (10). Extracts were separated on 12% SDS-polyacrylamide gels and electrophoretically transferred onto nitrocellulose filters (Schleicher and Schuell). Membranes were first incubated for 2 h in blocking solution of phosphate-buffered saline (154 mM NaCl, 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, pH 7.3) containing 5% nonfat milk, followed by a 1-h incubation with the primary antiserum in blocking solution. Next, membranes were washed tree times for 10 min in phosphate-buffered saline, followed by a 1-h incubation in the presence of a secondary, horseradish peroxidase-conjugated antibody (1:10,000; Amersham Pharmacia Biotech). Membranes were then washed three times for 10 min in phosphate-buffered saline, the blots were developed with an enhanced chemiluminescence kit (ECL; Amersham Pharmacia Biotech), and bands were visualized on Kodak XAR5 film for 1 or 10 min.
Electrophoretic Mobility Shift Assays--
Binding assays were
carried out for 20 min at 30 °C in 10-µl reactions containing 60 mM KCl, 12 mM HEPES, pH 7.9, 4 mM
Tris-Cl, pH 7.9, 1 mM EDTA, 1 mM
dithiothreitol, 30 ng of BSA, 12% glycerol, 1-5 µg of nuclear
extract, 0.1 ng of 32P-labeled DNA probe, and 1-5 µg of
poly(dI·dC). The F3 DNA probe was identical to the one used in
earlier studies (2, 3) and spans nt 70 to +136 of the murine
blk promoter. The double-stranded blk.BSAP oligonucleotide
probes (wild type and mutants) were made by annealing sense and
antisense oligonucleotides spanning nt
70 to
39 of the murine
blk promoter and labeled using [32P]
-dCTP
as described previously (2, 3). For shift assays involving antibodies
or antisera, reactions were incubated for 10 min at 30 °C in the
presence of nuclear extract and antibody prior to the addition of the
radioactive probe. Products were separated by gel electrophoresis
through a 5% polyacrylamide gel in a buffer containing 30 mM Tris-Cl, pH 7.5, 29.3 mM boric acid, and
0.66 mM EDTA. Gels were dried and exposed to Kodak XAR5
film.
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RESULTS |
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The murine blk promoter contains a Pax-5 binding site
in the region covering nt 68 to
37, as determined previously
in vitro. We wished to determine whether this sequence
represents a functional site on the blk promoter. Initially,
we tested whether overexpression of Pax-5a could increase
transcription from a blk minimal promoter fragment driving
expression of the CAT reporter gene. Transient co-transfections of the
Pax-5a expression construct (pcDNA3.Pax-5a) and the
blk reporter construct ((
191)blkCAT2µE)) into
the mature B cell lines M12 and A20/2J were performed using
DEAE-dextran. The expression vector without insert (pcDNA3) was
used in parallel in co-transfections as a negative control.
Overexpressed Pax-5a was able to increase the activity of the
blk promoter construct 4.6-fold (±0.55) and 2.8-fold
(±0.41) in cell lines M12 and A20/2J, respectively, as shown in Fig.
1A.
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As B cell lines generally have a low transfection efficiency, we performed these experiments in stable cell lines that expressed Pax-5a protein. A previous study had shown that clones from the plasmacytoma line S194 that had been stably transfected with Pax-5a were able to repress endogenous J-chain expression (10, 14). These S194 clones were used to transiently transfect the blkCAT reporter construct using DEAE-dextran. CAT conversion of nontransfected S194 cells was compared with that of a stable clone that expressed low levels of transfected Pax-5a protein (S194-Tf1) and one clone that expressed high levels of Pax-5a (S194-Tf4), which resulted in a 6.6-fold (±0.5) and 44-fold increase (±3.9) of CAT activity, respectively. Results of the stable transfections are shown in Fig. 1B. Together, the transfection studies indicated that Pax-5a functions as an activator of transcription on the murine blk gene.
Next, we tested whether mutations within or in the vicinity of the
Pax-5 binding site would affect the activity of the blk promoter in vivo. Earlier work (2) using mobility shift
assays had already established that mutations at the 5'-end and center of the Pax-5 binding site (mutA and mutB), but not at the 3'-region (mutC), prevented Pax-5a from binding to the blk promoter
in vitro. The location of the mutated blk
sequences relative to the Pax-5 binding site is shown in Fig.
2A. The WT
(191)blkCAT2µE or one of three mutated constructs (mutA,
mutB, and mutC; see Fig. 2A), were transiently transfected
into the highly differentiated, mature B cell line A20/2J (which
expresses moderate levels of Pax-5a). Unexpectedly, CAT assays showed
that mutations at the 5'-end of the binding site (mutA) led to a
significant increase in promoter activity. As expected, mutation of the
central region of the Pax-5 site (mutB) significantly reduced promoter
activity, whereas mutation mutC had no significant effect on promoter
activity. Results from the CAT assays are shown in Fig. 2B.
These data at least partially agreed with the mobility shift assays
mentioned above, because both in vivo and in
vitro studies pointed toward the importance of the central region
(mutB) but not the 3'-region (mutC) of the Pax-5 binding site for
interaction with Pax-5a. The observation that mutA causes an increase
in blk activity will be discussed in more detail below.
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Blk levels drop when mature B cells differentiate into
plasma cells, and plasma cells have undetectable levels of
blk transcripts (1). If Pax-5a is an activator of
blk transcription, a decrease of promoter-bound Pax-5a would
be expected when mature B cells are stimulated by LPS or anti-IgM. To
obtain data on a range of B cell activation stages, we decided to use
small resting B cells isolated from mice spleens using Percoll
gradients. These cells were activated with LPS and collected at various
time points after the LPS addition, and nuclear extracts were
prepared. To investigate the levels of Pax-5 proteins in these samples,
mobility shift assays were used with a labeled blk DNA
fragment F3, which covers nt 70 through +136 of 5'-blk.
Results of the mobility shift assays are shown in Fig.
3A.
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The data showed that the amount of Pax-5a interacting with the blk promoter decreased after 48 h of activation by LPS to very low levels at 73 h (Fig. 3A, arrow 2). To distinguish between a true decrease in total Pax-5a levels in the nucleus and a change in the amount of Pax-5a bound to the blk promoter, Western blot analyses were performed on the nuclear extracts. An anti-Pax-5 serum, Ed-1, which recognizes all four isoforms of the Pax-5 gene, was generated for this purpose (see "Materials and Methods"). Results of the Western blots are shown in Fig. 3B. Surprisingly, these data indicated that Pax-5a levels in the nucleus did not change significantly during this 73-h period.
This observation could suggest that blk-bound Pax-5a is replaced by a second transcriptional regulator, which is expressed after LPS induction. In agreement with this hypothesis, we noticed the presence of a new, slower migrating complex in activated cells (Fig. 3A, arrow 3). This complex was present at almost undetectable levels in resting B cell nuclei but became a major complex present on the blk probe after 24-48 h of LPS stimulation. At the 73-h time point, this higher complex was still very prominent, in contrast to the Pax-5a complex. A second induced complex was found to bind very weakly to the blk promoter as well (Fig. 3A; indicated by arrow 4).
A somewhat unexpected finding was that in nuclear extracts prepared from resting B cells, a protein that is likely to represent the alternatively spliced isoform Pax-5d bound to the blk promoter at levels almost identical to Pax-5a (see Fig. 3A, arrow 1). Interestingly, after 24 h of LPS stimulation, amounts of this protein had already dropped to a base-line level. Because no antisera specific to Pax-5d are yet available, we could not determine the exact nature of this protein. Although it was recognized by the anti-Pax-5 antiserum Ed-1, it could also be a proteolytic fragment derived from isoform Pax-5a. This possibility is still under investigation.
The LPS-inducible complex was studied in more detail using mobility
shift assays with the F3 probe. For practical reasons, we used the
presecretor B cell line BCL1 instead of normal activated B
cell extracts. BCL1 has high levels of both Pax-5a and the
unidentified complex and has a mobility shift pattern identical to that
of normal B cells after 24-48 h of LPS stimulation. Fig.
4A shows a sequence comparison
of the various WT and mutant double-stranded oligonucleotides used in
this and following competition shift assays. Using a double-stranded
oligonucleotide covering nt 37 to
70 of blk as unlabeled
competitor, we showed that this inducible complex (as well as Pax-5a)
binds to this region of the blk gene (Fig. 4B,
arrow 2, lane 3). To narrow
down the exact position of binding, additional competition assays with
mutant double-stranded oligonucleotides were performed. From these
experiments, we conclude that nt
57 to
50 (mutB) as well as nt
45
(mutH) were necessary for the binding of this unidentified factor,
whereas mutations of nt
66 to
60 (mutA) had no effect on binding
(shown in Fig. 4B (arrow 2) and/or
summarized in Fig. 4A). This suggested that the new complex
bound most strongly to a region overlapping with the central region of
the Pax-5 site on the blk promoter (Fig. 4A). In
addition, using oligonucleotides containing Pax-5 binding sites from
the CD19 and J-chain promoters showed that only
oligonucleotides containing the blk sequence can compete for
binding with the induced complex (Fig. 4, A and
B).
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To identify the new complex, we tested antibodies specific for various
putative factors and competitor oligonucleotides containing binding
sites for such factors. Results from competition shifts with a
double-stranded oligonucleotide containing the class I major
histocompatibility complex NF-B site (20) as well as antisera
against NF-
B/p50 or NF-
B/p65 suggested that the inducible complex in nuclei from activated normal B cells as well as from presecretor cell line BCL1 contained the
transcription factor NF-
B/p50, but not p65, as shown in Fig.
5 (arrow 2).
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NF-B proteins bind their targets either as homodimers or
heterodimers. Based on the position of the inducible complex on shift
gels (discussed with Fig. 6), as well as
its inability to be recognized by p65 (see arrow
2 in Fig. 5), we hypothesize that this complex represents a
homodimer of p50. One study showed that the optimal DNA binding site
for p50/p50 homodimers is different from that of p50/p65 heterodimers
(26). Preferential binding of p50 homodimers on the blk
promoter is in agreement with the NF-
B site on blk: 9 of
10 nt correspond to a p50/p50 homodimer consensus sequence, whereas
only 8 of 10 nt correspond to a p50/p65 consensus site (see Fig.
4A). Because the p50 subunit lacks a transactivating domain,
it has been implicated in repressor functions. The increased levels of
p50 homodimers accessible to the blk promoter may provide a
mechanism to down-regulate blk expression after B cells
become activated.
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In addition to this p50 homodimer, a faint, slower migrating complex
was detected after cell induction (arrow 3). This
complex was found to contain p50 but not p65 and could be competed by an oligonucleotide containing the NF-B binding site. The fact that
both inducible complexes represent members of the NF-
B family is
perhaps not surprising, considering that such factors are normally induced in B cells by either IgM cross-linking or LPS.
The DNA binding sites for Pax-5 and NF-B/p50 are very close together
and may in fact overlap (Fig. 4A). We wished to investigate whether the two factors bind to the blk promoter together,
which would be indicative of a "docking" function for Pax-5a,
similar to the situation on the mb-1 promoter (16). To
address this possibility, co-immunoprecipitation experiments were
performed using antibodies specific to both Pax-5a and NF-
B/p50.
Nuclear extracts from B cell presecretor line BCL1 and
pre-B cell line PD36 were incubated with antibody-coated beads
overnight, the immunoprecipitated proteins were separated on
SDS-polyacrylamide gels, and Western blot analysis was performed. We
were unable to demonstrate any association between NF-
B/p50 and
Pax-5a in these experiments (data not shown). Some further evidence for a "replacement" but not a "cooperation" mechanism was supported by mobility shift assays in which the addition of excess in
vitro translated Pax-5a to nuclear extracts from BCL1
(containing both p50 and Pax-5a protein) resulted in increased levels
of bound Pax-5a and a simultaneous decrease of bound p50 (data not
shown). Together, these data suggested that at least in
vitro, Pax-5a and NF-
B/p50 compete for binding to the
65 to
45 region of blk rather then binding to the sites
together.
To investigate whether the induction of an early B cell line would also
result in binding of p50 homodimers to the blk promoter, we
used the pre-B cell line 703/Z, which has been shown by others to be
inducible by LPS (21). Cells were activated with LPS as described
(above), and time points were collected at 0, 4, 48, and 120 h.
Relative levels of p50 and Pax-5a from uninduced or induced cells were
compared using mobility shift assays. As shown in Fig. 6A
(lane 2), reasonably high levels of bound Pax-5a
were present in unstimulated 703/Z cells, but no or very low levels of
p50 homodimers were detectable. After 4 h of LPS stimulation, a
higher molecular weight complex was detected, at a level equal to that
of bound Pax-5a (Fig. 6A, lanes 3-5).
The mobility shift pattern at 4 h of LPS stayed unchanged for the
remainder of the 120 h (not shown). Interestingly, the induced
complex appeared to run at a higher position in the gel compared with
the p50 homodimer complex that we had previously identified in
activated B cells and BCL-1 cells (Fig. 4B, arrow
2). Levels of p50 homodimers were not affected by LPS
stimulation. Further analysis showed that the induced complex
represents a heterodimer containing both p65 and p50 subunits of
NF-B, which are normally involved in activation of transcription
(Fig. 6A, lanes 6 and 7).
Thus, at least in vitro, LPS stimulation of an early B cell
line leads to interaction of the p50/p65 with the blk
promoter, but not p50/p50, whereas in activated mature B cells or
presecretor B cell lines, the p50/p50 homodimer binds
preferentially.
To see whether this distribution reflected patterns present during B
cell ontogeny, a number of B cell lines representing different stages
of B cell development were analyzed for the presence of p50/p50
homodimers versus p50-containing heterodimer complexes on
the blk promoter. Using mobility shift assays, we found that early B cell lines, including pro- and pre-B cell lines, have very low
levels of promoter-bound p50 homodimers. Mature B cells, presecretors,
and plasma cell lines have increasing levels of bound p50 homodimer.
Levels of p65/p50 or other heterodimeric NF-B complexes varied in
these lines. Results are shown in Fig. 6B.
Both p50/p65 and p50/Rel run at a similar position in the shifts, making it difficult to distinguish between the two unless anti-p65 antisera were used. To address this question, additional mobility shift assays as well as Western blot analyses were performed on a number of B cell lines including A20/2J, CH12, BCL1, SP2/0, and S194. Results from mobility shift assays (not shown) indicated that the slower migrating complex in these lines contained p50 but no, or only a relatively small amount of, p65. Interestingly, Western blot analysis showed that all tested lines had both p50 and p65 present in the nucleus, illustrating the difference between total levels of these factors present in the nucleus versus the amount bound to the blk promoter.
Interestingly, the experiments shown in Fig. 6B suggest that the ratio of bound p50 homodimers to bound Pax-5a strongly increases when B cells progress through their various developmental stages, from very low in pro- and pre-B cells to very high in plasma cell lines. These data agree with the patterns seen in LPS-activated normal B cells and may be indicative of a mechanism that regulates the levels of blk transcripts during B cell development.
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DISCUSSION |
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In the studies described here, we sought to explore the
functionality of the Pax-5 binding site on the blk promoter,
which was shown in earlier studies to interact with Pax-5a and its
alternatively spliced isoform Pax-5d in vitro. Our results
indicate that the activity of the blk promoter is regulated
by the relative level of at least three factors, including Pax-5a, the
NF-B/p50/p50 homodimer, and the NF-
Bp50/p65 heterodimer. Whether
a fourth factor, Pax-5d, plays a functional role in transcription
regulation of blk or other Pax-5 target genes is still under
investigation.
Pax-5a Is a Transcriptional Activator of the blk
Gene--
Evidence for a role for Pax-5a as an activator was shown
when Pax-5a protein was overexpressed in various B cell lines. In all
transfected cell lines tested, activity of the blk promoter had increased significantly. This result is important, because it rules
out the possibility that Pax-5a acts upon the blk promoter as a repressor. How exactly Pax-5a exerts its functions on the blk promoter is still unclear. From the experiments
described here, we cannot distinguish between Pax-5a functioning as an
activator of transcription through (indirect) interactions with the
initiation complex, or alternatively, its functioning as a
"gatekeeper" by keeping the blk promoter region
accessible to other factors. Also, a role for Pax-5a as a docking
protein cannot be ruled out, although it is unlikely that it would
interact with NF-B proteins in this function (see below).
NF-B/p50 Homodimers Bind to the blk Promoter during Activation
of Mature B Cells--
An NF-
B site was identified on the
blk promoter, which overlaps with the Pax-5 binding site.
The NF-
B family contains at least five members, including p50, p52,
p65, RelB, and c-Rel, which share a conserved Rel (DNA-binding) domain
that interacts with NF-
B sites on its target genes (20, 26).
Relatively high levels of p50 homodimers, but not p50 heterodimers,
were found to bind to the blk promoter during the mature B,
activated B, and plasma cell stages, although high levels of p50
heterodimers are reportedly present in the nucleus at these cell stages
as well (21). One explanation for this could be that the blk
sequence has a slightly higher homology with the NF-
B/p50 homodimer
consensus site as compared with the p50/p65 dimer consensus site (26), although the combination of other factors already binding to the blk promoter may influence this selective behavior as well.
No evidence was found for interaction of the p52 subunit with the blk promoter, in agreement with the relatively low levels of
this subunit during all B cell stages except the plasma cell stage (21).
Relative Levels of Bound Pax-5 and NF-B Dimers Regulate blk
Expression--
Although a second transcription factor, NF-
B/p50,
binds to a site overlapping the Pax-5 site, no evidence pointed toward collaboration or interaction between Pax-5a and p50, using mobility shift assays and co-immunoprecipitations. In contrast, our experiments suggested that either Pax-5a or p50, can bind to this region on the
blk promoter at a given time and that the relative
concentration of the two factors in the nucleus influences their
binding frequency.
Heterodimers of the NF-B Complex May Increase blk Promoter
Activity--
LPS activation of the pre-B cell line 703/Z induces the
formation of a p50/p65 heterodimer complex on the blk
promoter. This result is in agreement with studies by others who
activated 703/Z cells using LPS (20). They found that in pre-B cell
lines, both p50 and p65, but not p52, RelB, or Rel are induced after
short term (4-h) LPS exposure. In addition, these authors report that in mature B cell lines, p50, p65, and Rel are present constitutively, whereas plasmacytomas mostly express the p52 and RelB subunits. Since
the p50/p65 complex induced in 703/Z cells contains a functional activation domain, this would be expected to lead to increased levels
of blk transcripts.
Functional Significance of the p50 Homodimer-- We did not detect a decrease in Pax-5a levels in nuclei of activated B cells, even after 73 h of LPS stimulation, which may be explained by the fact that B cells do not differentiate into plasma cells until 3-5 days after antigen exposure. It may be important that blk expression is down-regulated early in the B cell response; therefore, it may need to be shut off by p50/p50 and not by Pax-5a. This mechanism may be necessary because only a subset of Pax-5 target genes is down-regulated at this time, and the exact time point of down-regulation may vary among its target genes. Down-regulation is likely to depend on the unique function of each target gene during activation processes. It will be interesting to investigate blk levels in B cells of p50 knockout mice, which reportedly do not respond to LPS stimulation (27). Such mutant mice may have a delayed decrease in blk expression, which may prevent the cells from efficiently proliferating and differentiating into plasma cells. One report in agreement with this hypothesis suggests that blk is a growth inhibitor signaling molecule; the authors show that antisense oligonucleotides to blk prevent growth inhibition and apoptosis of an anti-µ-chain activated B cell lymphoma (30).
Regulation of gene expression through differential usage of NF- ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. W. Sha for NF-B reagents, Dr.
Yi Zhang for mouse spleens, and Ilsa Kaattari and Joan Fujita for
excellent technical support.
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FOOTNOTES |
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* This work was supported by National Science Foundation Research in Undergraduate Institutions Grant MCB-9419067.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Biology, The College of William and Mary, Millington Hall, Williamsburg, VA 23187. Tel.: 757-221-1969; Fax: 757-221-6483; E-mail: Pxzwol{at}facstaff.wm.edu.
1 The abbreviations used are: LPS, lipopolysaccharide; nt, nucleotide(s); BSAP, B cell-specific activator protein; WT, wild type; CAT, chloramphenicol acetyltransferase; BSA, bovine serum albumin.
2 We refer to the full-length Pax-5 protein as Pax-5a. Italics are used to designate the Pax-5 gene.
3 J. Wallin, personal communication.
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REFERENCES |
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