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Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10021
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A large variety of octamer-binding transcription factors have been cloned, and these include the ubiquitous Oct-1 and B cell-restricted Oct-2, which are related through a conserved DNA-binding protein structure called the POU domain (6, 7). Initially, the cell-specific expression of Oct-2 and various in vitro and in vivo functional assays led to the idea that the B cell-enriched Oct-2 was responsible for octamer-dependent activation of Ig genes in B lymphocytes, but more detailed biochemical analyses clearly demonstrated that this was not the case. In fact, in vitro transcription data indicated that Ig promoters function equally well with either the B cell-restricted Oct-2 or the ubiquitous Oct-1 and that B cell-specificity is actually due to a novel Oct-interacting cofactor, designated OCA-B, found only in B lymphocytes (Ref. 8 ; reviewed in Ref. 9).
The biochemical identification of OCA-B was followed by cognate cDNA cloning using both biochemical purification and protein-protein interaction screening methods (9, 10, 11, 12). In agreement with previous biochemical data, recombinant OCA-B was shown to interact physically with either Oct-1 or Oct-2 through their highly related POU domains and to activate transcription from Ig promoters in combination with either Oct-1 or Oct-2. Although the isolated POU domains of Oct-1 or Oct-2 are sufficient to recruit OCA-B to the octamer site, the POU-OCA-B complex is unable to stimulate transcription significantly in the absence of Oct-1 or Oct-2 activation domains (9). Similarly, structure/function analyses have demonstrated that OCA-B possesses an intrinsic activation domain that is distinct from the POU interaction domain and functions synergistically with the Oct activation domain (9, 13, 14, 15).
The role of OCA-B in vivo has been examined using OCA-B-deficient mice created by gene targeting (9, 16, 17, 18). These studies show 1) that OCA-B is dispensable for Ag-independent B cell differentiation, with no observable defects in early stage B cell-specific gene expression; 2) that OCA-B is crucial for Ag-dependent B lymphoid development; although class switching itself appears unaffected, OCA-B-/- mice exhibit a severe deficiency in production of secondary Ig isotypes; 3) that OCA-B-deficient B cells show a reduced rate of proliferation in response to surface IgM cross-linking; and 4) that germinal center formation in the secondary lymphoid organs is dependent upon OCA-B. In more detailed analyses (19), histochemical studies have shown that OCA-B protein is expressed specifically in germinal center B cells, suggesting a more direct role for OCA-B in germinal center formation. Furthermore, OCA-B protein levels are nearly undetectable by Western blot in resting primary B splenocytes, but can be strongly increased by treatment with a combination of IL-4 and anti-CD40 (9, 19). Taken together, these data indicate that OCA-B is more important for later stages of B cell differentiation in vivo and that OCA-B is a target of crucial cell signaling pathways.
Although there is a significant amount of data regarding the molecular function of OCA-B, relatively little is known about the factors that regulate its B cell-specific expression. This information is especially important in light of the apparent function of OCA-B in late B cell differentiation and its induction by important lymphoid signals. In this study, we have cloned the OCA-B promoter and characterized its function throughout B cell development to better assess the regulation of OCA-B in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine genomic DNA was digested, circularized by ligation, and used in PCR with tail-to-tail primers designed against known OCA-B sequences (20). PCR fragments were cloned and sequenced at the Rockefeller University Protein DNA Technology Center. Resulting sequences were searched for transcription factor binding sites using TFSEARCH ver. 1.3 (Yutaka Akiyama, Kyoto University, Kyoto, Japan). Site-directed mutagenesis of the OCA-B promoter was carried using the "overlap PCR" method (21). Oligonucleotides used for mutagenesis are as follows: NF-AT mutant top, 5'-CAGGCAGAGCTGTACTTTTTTTTATTTAGCAACACCGCCT-3'; NF-AT mutant bottom, 5'-GTTGACAGGCGGTGTTGCTAAATAAAAAAAAGTACAGCTC-3'; cAMP response element (CRE)4 mutant top, 5'-CTGAGAGGGGCCCACGGCATCTCATCCTCCAAGCCATG-3'; CRE mutant bottom, 5'-GAAAAAAAAGTACAGCTCTGCA TGGCTTGGAGGATGAGA-3'.
Plasmids constructs
VH LUC3. The IgH promoter, VH, was removed from pGL2 BV (described in Ref. 22) using KpnI and BglII and inserted into the identical sites in pGL3 Basic (Promega, Madison, WI).
Minimal promoter constructs.
The LBK minimal promoter (23, 24) from the LBK
44 CAT
construct was removed with BamHI and HindIII and
inserted into the BglII and HindIII sites
upstream of the luciferase gene in pGL3 Basic (Promega).
Oligonucleotides bearing a single copy of either an intact or mutated
octamer sequence were subsequently cloned between the SacI
and XhoI sites upstream of the minimal promoter. The
oligonucleotide sequences are as follows: octamer top,
5'-CACTAGTAATATGCAAATCAGTCTAGAC-3'; octamer bottom,
5'-TCGAGTCTAGACTGATTTGCATATTACTAGTGAGCT-3'; octamer mutant top,
5'-CACTAGTAATCTGAACATCAGTCTAGAC-3'; octamer mutant bottom,
5'-TC GAGTCTAGACTGATGTTCAGATTACTAGTGAGCT-3'.
Cell lines
The murine pre-B cell lines 18-8 and 18-81 and murine B cell line M12.4.1 were supplied by Dr. L. A. Eckhardt (Hunter College, New York, NY). The murine B cell line A20, human B cell lines Namalwa and Raji, the murine plasmacytoma cell line P3X63Ag8, and the murine fibroblastic cell line NIH3T3 were all obtained from American Type Cell Culture (Manassas, VA). The pre-B and B cell lines were grown in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% FBS (JRH Biosciences, Lenexa, KS), 1% penicillin-streptomycin (Life Technologies), and 2 mM L-glutamine (Life Technologies). Plasma and fibroblast cells were grown in DMEM (Life Technologies) supplemented with 10% FBS, 1% penicillin-streptomycin, and 2 mM L-glutamine. Primary B cells were cultured in RPMI 1640 (Sigma) containing 10% FBS (JRH Biosciences), 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate (Sigma), 1% nonessential amino acids (Sigma), 5 x 10-5 M 2-ME (Sigma), and 1% penicillin-streptomycin (Life Technologies). Cells were maintained in 8% CO2 at 37°C.
Primary B cell isolation
Spleens were freshly isolated from 4- to 6-wk-old C57BL/6 wild-type and OCA-B-/- mice. For each experiment, splenocytes from each genotype were isolated concurrently. After washing in serum-free media, the spleens were crushed between the frosted ends of microscope slides, releasing the cells. RBCs were depleted by lysis in buffer containing 0.16 M NH4Cl, 10 mM potassium bicarbonate, and 1 mM EDTA. The remaining cells were pelleted, washed, and resuspended in serum-free media and incubated at room temperature for 20 min with anti-mouse Thy1.2 monoclonal antisera (Sigma) diluted 1:1000. The T cells were then depleted by incubation with guinea pig complement (Sigma), diluted 1:400, for 45 min at 37°C. The cells were washed several times and resuspended at 1–2 x 106/ml in complete media containing 50 µg/ml LPS (Sigma) for 48 h. Macrophages and other adherent cell types were depleted by subsequent decanting of suspension cells. The B cells isolated via this method were shown by FACS to be >90% pure (data not shown). The activated B cells were pelleted, resuspended in PBS, and layered on a pad of 60% Percoll (Sigma). The cells remaining at the PBS/Percoll boundary after centrifugation were isolated and washed several times with PBS and counted. A total of 1–2 x 107 cells were used for each transfection sample, and cells were transfected using the DEAE-dextran method (Promega).
Transient transfections
Primary B, pre-B, and B cells were transfected by the
DEAE-dextran method (25), plasmacytoma cells were
transfected with Lipofectin (Life Technologies) (26), and
fibroblasts were transfected with Superfect (Qiagen, Chatsworth, CA).
In experiments in which samples were further treated with both cell
lines and primary B cells, the inducer(s) was added to the media
immediately after transfection and left until harvesting of the samples
48 h later. For cell lines, 
2–4 x
106 cells were used per transfected sample, while
for primary B cells, 1–2 x 107 cells
were used per transfected sample. The amounts of inducers added to the
media are as follows: dibutyryl cAMP (Sigma) 1 mM, IL-4 (Sigma) 5
ng/ml, anti-CD40 (PharMingen, San Diego, CA) 10 µg/ml.
Transfection harvesting and reporter gene assays
Transfections of cell lines and primary B cells were harvested using 1x reporter lysis buffer after 48 h (Promega). Luciferase assays were conducted using Promega’s luciferase assay system. ß-galactosidase control vector assays were conducted (27), and resulting values were used to normalize the luciferase data for differences in transfection efficiency.
Western blotting
Cells were washed once in serum free media, then resuspended in cold buffer (20 mM HEPES, 400 mM KCl, 0.5 mM EDTA, 0.5 mM DTT, 0.02% Nonidet P-40, 20% glycerol, pH 7.5) at 5 x 106 cells/50 µl and subjected to three rounds of freezing and thawing on dry ice before use. Then, 10 µl (1 x 106 cell equivalents) of whole-cell extract were electrophoresed and transferred to Immobilon P (Millipore, Bedford, MA). The filter was blocked in TBST containing 5% nonfat dry milk, incubated with rabbit anti-OCA-B antisera (supplied by Dr. Y. Luo, The Rockefeller University), washed with TBST, then incubated with HRP-conjugated goat anti-rabbit antisera (Promega) and visualized using chemiluminescence (NEN, Boston, MA).
EMSA
The DNA probe for EMSA was made by digestion of plasmid DNA and labeling with Klenow (28). Binding reactions (15 µl) contained 10 mM Tris, 40 mM NaCl, 1 mM EDTA, 1 mM 2-ME, 4% glycerol (final concentrations), 1000 ng nonspecific competitior (poly dIdC or salmon sperm DNA), 2 x 104 cpm probe, and either 4 µg Namalwa or 5 µg HeLa nuclear extract prepared as described (28). (Nuclear extracts were previously titrated for optimal binding; data not shown.) For the antisera supershift experiments, 2 µl of NF-AT-, BCL6-, or activating transcription factor (ATF)/CRE binding protein (CREB)-specific antisera (Santa Cruz Biotechnology, Santa Cruz, CA) were added 5 min after initiating the binding reaction and further incubated for 10 min on ice before electrophoresis. Then, 0.5x TGE (12.5 mM Tris, 95 mM glycine, 0.5 mM EDTA, pH 8.5 final concentration) was used as both gel and chamber buffer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine genomic DNA was partially digested with the restriction
enzyme EcoRI, and the resulting fragments were circularized
with T4 DNA ligase. Based upon the genomic sequences generated during
the previous cloning of the OCA-B locus for gene targeting, we designed
tail-to-tail oligonucleotides for use as PCR primers. Employing the
circularized genomic DNA as a template, we amplified a series of DNA
fragments that were subcloned and then sequenced. These fragments were
inserted upstream of the luciferase reporter gene in the pGL3 Basic
vector and tested for their ability to express luciferase in plasma
cells. One of these clones, which contained 1.5 kb of DNA upstream of
the start codon of the OCA-B gene (see Fig. 1
), was the smallest construct that
retained all of the promoter activity and thus was used for further
analyses (GenBank accession no. AF241257).
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Comparison of the OCA-B promoter sequence to databases of known
transcription factor binding sites indicated a large number of matches,
some of which are shown in Fig. 1
. There is a TATA sequence 30 bp
upstream of the major start site (see Fig. 1). Relatively close to the
TATA sequence are near-perfect matches to the CREB/ATF (-64 to -57
bp) and NF-AT (-105 to -98 bp) consensus binding sites. Further
upstream, there are strong matches to binding sites for YY-1, Myb, and
Sox (or SRY-related) factors.
Primer extension analysis shows transcription initiation from one major
site (indicated as +1 in Fig. 1) and several minor sites within the
promoter in B cells (data not shown). These latter sites are consistent
with previously published data (13, 14) and therefore were
not further investigated.
Developmental regulation of the OCA-B promoter
To test whether the putative promoter region of OCA-B has B
cell-specific activity, we conducted a series of transfections with
cell lines representative of the pre-B, B cell, and plasma cell stages
of B cell differentiation and with fibroblasts as a negative control.
Each cell line was transfected with a luciferase reporter containing
either the full-length -1.5 kb OCA-B promoter or a truncated OCA-B
promoter extending from -40 bp through the TATA homology and
transcription start site. This minimal construct shows a low
constitutive activity in all the cell lines tested (data not shown) and
therefore was used to establish a baseline value; the values for the
full-length promoter are expressed as fold activity relative to the
value for the minimal promoter. The data are shown in Fig. 2A. In NIH3T3 fibroblasts,
which were used as representative non-B cells, there is little activity
of the full-length OCA-B promoter above basal promoter levels. In
contrast, the full-length OCA-B promoter activity is 17-fold above the
basal promoter level in 18-81 pre-B cells, 44-fold above the basal
level in M12.4.1 B cells, and 240-fold above the basal level in
P3X63Ag8 plasma cells. Similar values were obtained in transfections of
other representative non-B, pre- B, B, and plasma cell lines (data not
shown).
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To ensure that the progressive increase in promoter activity
observed throughout B cell development reflects an actual increase in
endogenous OCA-B expression in vivo, whole-cell extracts were made from
the cell lines representative of each stage in B cell differentiation
and checked for OCA-B protein levels via Western blotting. Equal cell
equivalents were loaded onto each lane, and transfer efficiency was
checked using Ponceau S staining (data not shown). OCA-B is normally
detectable in Western blots as a doublet of 34 and 35 kDa (10, 19). As shown in Fig. 2B, the OCA-B doublet is not at
all detectable in the NIH3T3 fibroblasts and only weakly visible in the
pre-B cells, 18-81. The OCA-B protein levels are further increased in B
cells and highest in the plasmacytoma cells. Because each lane contains
equal cell equivalents of whole-cell extract, the amounts of protein
detected represent the relative levels of OCA-B expression in each of
the cell lines.
Functional analysis of the OCA-B promoter
To delineate the functional regions of the promoter sequence, a
series of deletion mutants of the OCA-B promoter were generated via PCR
and cloned into the pGL3 Basic vector. Deletion endpoints were
selected, in part, on the basis of the positions of various
transcription factor binding site matches, as indicated in Fig. 1. To
observe both positive and negative activities, we cotransfected plasma
cells and non-B cells. The results from three to five separate
transfections of both plasma cells and fibroblasts are shown in Fig. 3. The values for each of the deletion
mutants are expressed as fold activity relative to the values of the
minimal promoter construct. Endpoints of the deletions are indicated at
the bottom of figure.
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, the non-B cell activities of all the deletion
mutants are low and relatively similar (signified by the open bars),
indicating that repression may not be a significant mechanism for
control of the OCA-B promoter. The results observed with plasma cells
(the crosshatched bars) are more complex. First, the intact promoter is
over 200-fold more active in plasma cells than in fibroblasts. Second,
there appear to be three regions that contribute positively to OCA-B
promoter function, as their deletion reduces overall activity in plasma
cells. The first region, between -0.56 kb and -0.55 kb, contains no
obvious transcription factor binding site matches, as determined by
computer analysis; the second region, between -0.27 kb and -0.09 kb,
contains a site related to the NF-AT binding sequence; and the third
region, between -0.07 kb and -0.04 kb, contains the CREB/ATF binding
site. These regions appear to be the most significant for promoter
activity in plasma cells because deletions of intervening sequences
have little effect on activity. Similar results, albeit at lower
overall levels of activation, were observed in B cells and pre-B cells
(data not shown). Analysis of function of CREB/ATF and NF-AT binding site mutants
The data derived from the promoter deletion studies indicated that
the CREB/ATF and NF-AT sites may be important for OCA-B promoter
function. Therefore, we used PCR-based mutagenesis to generate
site-specific mutants in the CREB/ATF and NF-AT binding site matches.
The sequences of the mutations were designed on the basis of previously
published reports of mutations that eliminate NF-AT or CREB/ATF DNA
binding. The mutations were made in the full-length (-1.5 kb) OCA-B
promoter, subcloned into the luciferase reporter pGL3 Basic, and
assayed by transient transfection. Interestingly, as shown in Fig. 4, mutation of the NF-AT binding
site match has absolutely no effect upon OCA-B promoter function in
plasma cells. More significantly, disruption of the CREB/ATF binding
site reduces overall promoter activity >90% in plasma cells. It is
important to note that the mutation of the CREB/ATF site is in the
context of the entire -1.5 kb OCA-B promoter, indicating a crucial
role for this sequence in overall promoter function and no significant
redundancy with other elements.
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To test whether any factors bind to the region of the OCA-B
promoter bearing both the NF-AT and CREB/ATF binding site homologies,
we conducted a series of EMSAs with a probe comprising the -270 to
-40 region of either the wild-type OCA-B promoter or an OCA-B promoter
bearing a mutation in the CRE region, with nuclear extracts from non-B
cells (HeLa) and B cells (Namalwa). As shown in Fig. 5A, multiple complexes are
generated with wild-type probe in both HeLa extract (lane
1) and Namalwa extract (lane 3). Nearly all of
the complexes are eliminated with mutated probe in HeLa extract
(lane 2) and Namalwa extract (lane
4), indicating that they depened upon the presence of the CRE for
binding.
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B, the
HeLa extract yields three major complexes of intermediate mobility
(lane 2), and the Namalwa extract yields two major
complexes of intermediate mobility (lane 6). However,
there are qualitative differences in the mobilities of the intermediate
complexes produced with each extract, perhaps suggesting that different
protein complexes are binding to the promoter in B cell vs non-B cell
extracts. We found that additions of an antiserum that specifically
recognizes a region common to most NF-AT family members
(lanes 3 and 7) and an antiserum to BCL6,
which was used as a negative control (lanes 5 and
9), have no effect upon the observed complexes. In contrast,
an antiserum that recognizes a region common to many of the CREB/ATF
family members almost quantitatively eliminates and/or supershifts the
major intermediate complexes in both HeLa (lane 4)
and Namalwa (lane 8) extracts. These data indicate
that all the major intermediate complexes (three in the case of HeLa
extracts, two in the case of Namalwa extracts) must contain at least
one member of the CREB/ATF family, whether derived from B cell or non-B
cell extracts, and provide evidence for the specificity of the
complexes. However, the data do not exclude the possibility that the
complexes also contain other factors bound to other DNA elements or to
ATF/CREB factors. Dibutyryl cAMP stimulates the OCA-B promoter via the CREB/ATF site
Another hallmark of functional CREB/ATF sites and cognate factors is their ability to be stimulated with dibutyryl cAMP, which activates the cAMP-dependent protein kinase signaling pathway and results in activation of CREB/ATF family members (29). We tested the OCA-B promoter for dibutyryl cAMP responsiveness by transiently transfecting B cells in duplicate with either the full-length -1.5 kb OCA-B promoter, the minimal -0.04 kb OCA-B promoter, or the full-length -1.5 kb OCA-B promoter with a mutated CREB/ATF binding site and then stimulating one set with 1 mM dibutyryl cAMP. We chose to use B cells because the promoter is less active in these cells than in plasma cells and thus can be further stimulated.
Data from several transfection assays are summarized in Fig. 6A. While the full-length
OCA-B promoter is significantly stimulated by dibutyryl cAMP treatment,
both the minimal OCA-B promoter and the full-length OCA-B promoter with
a mutated CREB/ATF site are unaffected by dibutyryl cAMP addition.
These data indicate that the CREB/ATF site in the OCA-B promoter is
indeed functional.
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Because the OCA-B promoter is stimulated by dibutyryl cAMP, and because OCA-B is important for octamer-dependent promoter function in B cells, we examined whether dibutyryl cAMP has any effect upon octamer-dependent promoters. We transiently transfected B cells with a minimal promoter alone or with a minimal promoter linked either to an intact octamer sequence or to a mutant octamer sequence; consistent with previous reports (30) the minimal construct bearing a single octamer exhibits B cell-specific activity (data not shown). We also analyzed an intact VH promoter containing functional octamer and TATA elements. The cells were transfected in duplicate with these constructs in the absence or presence of 1 mM dibutyryl cAMP.
The data from several independent experiments indicate that dibutyryl
cAMP treatment can stimulate octamer-dependent promoter function in B
cells (Fig. 6B). Thus, a single copy of the wild-type
octamer sequence linked to the minimal promoter, as well as the natural
octamer-dependent VH promoter, are significantly
stimulated by the addition of dibutyryl cAMP. In contrast, the minimal
TATA-containing promoter, as well as the minimal promoter linked to a
single copy of a mutated octamer site, are unaffected by dibutyryl cAMP
treatment. The fact that the mutant and minimal promoters are not
stimulated by dibutyryl cAMP shows that the observed activities are due
to the octamer sequences themselves and not the minimal promoter
sequences.
Stimulation of the OCA-B promoter by IL-4 and anti-CD40 treatment
OCA-B protein levels in resting primary B splenocytes are greatly increased by treatment with IL-4 and anti-CD40 (19). CREB/ATF sites are known to be targets of cell signaling pathways in a variety of systems, including B cells (31). Therefore, we examined whether the OCA-B promoter, and, more specifically, the CREB/ATF site within the promoter, was a target of IL-4 and anti-CD40 stimulation. We transiently transfected either the full-length -1.5 kb OCA-B promoter, the minimal -0.04 kb OCA-B promoter, or the full-length -1.5 kb OCA-B promoter with a mutated CREB/ATF binding site into primary B splenocytes in duplicate then added IL-4 and anti-CD40 to one set immediately after transfection.
The results in Fig. 7A
indicate that while IL-4 and anti-CD40 treatment has little or no
effect upon the minimal promoter construct, it strongly activates the
intact OCA-B promoter. This stimulation of the OCA-B promoter is
largely abrogated by mutation of the CREB/ATF site, suggesting that
this site is a target of the IL-4 and anti-CD40 signaling pathways in
primary B cells.
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Because treatment of primary B splenocytes with IL-4 and anti-CD40 clearly elevates both OCA-B promoter activity and protein levels (19), we tested whether these increases resulted in changes in the activity of octamer-dependent promoters. We transiently transfected primary B cells in duplicate with the intact VH promoter, a minimal TATA-containing promoter alone, and a minimal promoter linked either to an intact octamer site or to a mutant octamer site (see above). One set of samples was treated with IL-4 and anti-CD40 immediately after transfection.
As shown in Fig. 7B, IL-4 and anti-CD40 treatment activates
both the VH promoter and the synthetic promoter
bearing a single copy of the intact octamer site. In contrast, there is
no significant stimulation of either the minimal TATA-containing
promoter or the minimal promoter linked to the mutant octamer sequence,
clearly showing that the stimulation is specific to the octamer
site.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The OCA-B promoter is B cell-specific and developmentally regulated
The OCA-B promoter is clearly more active in B cells than non-B cells, as indicated by transient transfection analysis. More importantly, the OCA-B promoter shows increasing activity throughout B cell development, consistent with data from gene targeting studies indicating that OCA-B is more crucial at later stages of B cell development (16, 17, 18). While the OCA-B promoter is relatively weak in pre-B cells, it shows an increased activity in B cells and the highest activity in plasma cells. The increase in OCA-B promoter activity throughout B cell differentiation is not due to variations of individual cell lines, because transfection of other cell lines representative of similar stages in B cell development exhibited equivalent levels of OCA-B promoter function (data not shown).
The observed increases in OCA-B promoter activity during B cell differentiation are paralleled by endogenous OCA-B protein levels, as shown by Western blot. This correlation suggests that OCA-B is regulated, at least in part, at the transcriptional level.
The role of the CREB/ATF site in OCA-B promoter function
Deletion and site-directed mutations of the CREB/ATF binding site
show that it is crucial for overall OCA-B promoter function, and
mobility shift analysis suggests that multiple complexes are able to
form at this site. These complexes contain genuine CREB/ATF family
members, because most are supershifted by antisera that recognize
CREB/ATF family members but not by control antisera. These data do not
eliminate the possibility that other factors are present, but simply
indicate that one component of any putative multiprotein complex is a
member of the CREB/ATF family. It is important to note that complexes
were observed with both B cell and non-B cell nuclear extracts. This is
to be expected, because some members of the CREB/ATF family are
expressed in all cells (32, 33, 34). More importantly,
however, published reports have already demonstrated a role for
CREB/ATF factors both in B cell-related signaling and in expression of
B cell-specific genes such as the Ig 
light chain (29, 31). There also is evidence for differential expression of
specific spliced forms of ATF-1 and CRE modulator over the course of B
cell development (29, 35). Furthermore, a B cell-enriched
ATF factor, designated B-ATF, has recently been cloned
(36). Therefore, there is a significant amount of data
implicating the CREB/ATF family of factors in B cell-specific signaling
and gene expression.
Induction of OCA-B promoter activity
An important functional aspect of CREB/ATF binding sites is promoter inducibility through these sites. Reagents such as dibutyryl cAMP stimulate the activity of factors binding to the CREB/ATF site by inducing cell signaling pathways leading to activation of CREB/ATF family members via phosphorylation (29, 31). We find that the OCA-B promoter is strongly activated by dibutyryl cAMP treatment and that this stimulation is dependent upon the CREB/ATF site. These data are in accord with previously published data suggesting that OCA-B is the target of multiple cell signaling pathways in vivo (19). Even more significantly, dibutyryl cAMP treatment is able to stimulate the activity of octamer-dependent promoters. Mutation of the octamer element abolishes the dibutyryl cAMP effect, demonstrating that factors binding directly (e.g., Oct-1) or indirectly (e.g., OCA-B) to the octamer site itself are being induced. In toto, these data strongly suggest that the endogenous OCA-B is being induced by dibutyryl cAMP, which in turn activates the octamer-dependent promoters.
In primary B cells, the OCA-B promoter is a target for stimulation by IL-4 and anti-CD40, a treatment that previously has been shown to increase OCA-B protein levels in primary B splenocytes (19). Activation of the OCA-B promoter by IL-4 and anti-CD40 appears to be mediated at least in part by the CREB/ATF site, because mutation of the site eliminates the stimulatory effect. These data suggest that CREB/ATF factors are at least one target of the IL-4 and anti-CD40 signaling pathways, further establishing the role of CREB/ATF factors in B cell-specific gene expression. More importantly, IL-4 and anti-CD40 treatment specifically activates octamer-dependent promoters in primary B cells, implicating the octamer site, and the interacting Oct-1 (or Oct-2) and OCA-B, in B cell-specific signaling events.
Taken together, the results presented here show that the OCA-B promoter is regulated throughout B cell development and is the target for cell signaling events via one or more members of the CREB/ATF family. OCA-B promoter activation appears to result in increased activity of octamer-dependent promoters, implicating OCA-B in B cell signaling events in the nucleus.
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Acknowledgments |
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Footnotes |
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2 Current address: Third Wave Technologies, 502 South Rosa Road, Madison, WI, 53719-1256.
3 Address correspondence and reprint requests to Dr. Robert G. Roeder, Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue Box 166, New York, NY 10021.
4 Abbreviations used in this paper: CRE, cAMP response element; ATF, activating transcription factor; CREB, CRE binding protein.
Received for publication December 6, 1999. Accepted for publication April 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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bacteriophage templates. Proc. Natl. Acad. Sci. USA 91:2216. 3' enhancer. J. Biol. Chem. 270:10304.This article has been cited by other articles:
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 J. T. Blois, J. M. Mataraza, I. Mecklenbrauker, A. Tarakhovsky, and T. C. Chiles B Cell Receptor-induced cAMP-response Element-binding Protein Activation in B Lymphocytes Requires Novel Protein Kinase C{delta} J. Biol. Chem., July 16, 2004; 279(29): 30123 - 30132. [Abstract] [Full Text] [PDF]
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J. R. Podojil, N. W. Kin, and V. M. Sanders CD86 and {beta}2-Adrenergic Receptor Signaling Pathways, Respectively, Increase Oct-2 and OCA-B Expression and Binding to the 3'-IgH Enhancer in B Cells J. Biol. Chem., May 28, 2004; 279(22): 23394 - 23404. [Abstract] [Full Text] [PDF]
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M. Salas and L. A. Eckhardt Critical Role for the Oct-2/OCA-B Partnership in Ig-Secreting Cells J. Immunol., December 15, 2003; 171(12): 6589 - 6598. [Abstract] [Full Text] [PDF]
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C. Brunner, H. Laumen, P. J. Nielsen, N. Kraut, and T. Wirth Expression of the Aldehyde Dehydrogenase 2-like Gene Is Controlled by BOB.1/OBF.1 in B Lymphocytes J. Biol. Chem., November 14, 2003; 278(46): 45231 - 45239. [Abstract] [Full Text] [PDF]
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 M. Jankovic and M. C. Nussenzweig OcaB regulates transitional B cell selection Int. Immunol., September 1, 2003; 15(9): 1099 - 1104. [Abstract] [Full Text] [PDF]
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 U. Kim, R. Siegel, X. Ren, C. S. Gunther, T. Gaasterland, and R. G. Roeder Identification of transcription coactivator OCA-B-dependent genes involved in antigen-dependent B cell differentiation by cDNA array analyses PNAS, July 22, 2003; 100(15): 8868 - 8873. [Abstract] [Full Text] [PDF]
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C. S. Malone and R. Wall Bob1 (OCA-B/OBF-1) Differential Transactivation of the B Cell-Specific B29 (Ig{beta}) and mb-1 (Ig{alpha}) Promoters J. Immunol., April 1, 2002; 168(7): 3369 - 3375. [Abstract] [Full Text] [PDF]
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L. A. Vereshchagina, M. Tolnay, and G. C. Tsokos Multiple Transcription Factors Regulate the Inducible Expression of the Human Complement Receptor 2 Promoter J. Immunol., May 15, 2001; 166(10): 6156 - 6163. [Abstract] [Full Text] [PDF]
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U. Kim, C. S. Gunther, and R. G. Roeder Genetic Analyses of NFKB1 and OCA-B Function: Defects in B Cells, Serum IgM Level, and Antibody Responses in Nfkb1-/-Oca-b-/- Mice J. Immunol., December 15, 2000; 165(12): 6825 - 6832. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
CD40) stimulate both the OCA-B promoter and
octamer-dependent promoters. A, Induction of OCA-B
promoter activity by IL-4 and anti-CD40. Results are presented as
relative light units. White bars indicate uninduced activity, and
crosshatched bars indicate activity induced by 5 ng/ml IL-4 and 10
µg/ml anti-CD40. B, Octamer-dependent promoter
induction by IL-4 and anti-CD40. Results are presented as relative
light units. White bars indicate uninduced activity, and crosshatched
bars indicate activity induced by 5 ng/ml IL-4 and 10 µg/ml
anti-CD40. Murine primary B cells were used for the transfections shown
in each panel.