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INTRODUCTION |
During a primary immune response to foreign antigens, mature B
cells become activated and differentiate into pentamer IgM-secreting plasma cells. One of the critical events in this process is the synthesis of the immunoglobulin J chain protein required for the assembly and secretion of pentamer IgM antibody (1). Studies of both
normal B cells and model B cell lines have shown that J chain synthesis
is tightly regulated at the transcriptional level. For efficient
J chain transcription to occur, activation signals from both
the B cell receptor and the interleukin-2
(IL-2)1 or IL-5 receptors are
needed. Correlating with IL-2/IL-5 induced gene transcription is the
appearance of a DNase I-hypersensitive site (bp
170 to +88) on the
J chain promoter of activated B cells (2, 3). The full
activity of the J chain promoter is contained within this
hypersensitive region, as has been shown using 5' deletion mutants in a
CAT reporter system (4).
Three regulatory elements have been found to be present within this
region. These include a T-rich positive regulatory element denoted JA
(
74 to
60), a purine-rich sequence (
58 to
47) denoted JB, and a
repressive motif, JC (
127 to
110) (5-7). The factor interacting
with the JA sequence has recently been identified to be a member of the
myocyte enhancer factor-2 (MEF-2) family that is expressed in the B
cell lineage, denoted B-MEF2 (5). The JB element has previously been
shown to interact with transcription factor PU.1, a member of the Ets
family of transcription factors expressed in hematopoietic lineages
including monocytes, macrophages, and B lymphoid cells (6). Mutational
analyses in J chain positive cell lines have indicated positive
regulatory roles for both PU.1 and B-MEF2; base changes that prevent
either PU.1 or B-MEF2 from binding result in a 95% loss of promoter
activity (5, 6). The activity mediated by the JC motif has been shown
to be due to the binding of the transcription factor B cell
specific activator protein (BSAP),
or Pax-5 (7).
BSAP/Pax-5 is a member of the Pax family of transcription factors,
which are important regulators of embryonic cell development and
differentiation. Targeted gene disruption experiments have shown that
BSAP expression is essential both for B cell development as well as
development of the nervous system (8, 9). BSAP is highly expressed
during the early stages of B cell development, but expression ceases
during the antigen-driven stages of B cell development. BSAP is
considered a "master regulator" of B cell development, and at least
eight B cell-specific putative target genes have been identified so far
(10).
Depending on the target gene, BSAP can act either as an activator, a
repressor, or a docking protein (9, 11, 12). In the case of the J
chain promoter, BSAP acts as a repressor: base changes in the
BSAP-binding site result in a relief of repression in J chain negative
cell lines (7). An IL-2 or IL-5 signal delivered to mature B cells has
been shown to cause a progressive decrease in BSAP transcripts that
extends from the presecretor immunoblast through the plasma cell stages
(7). This pattern of expression inversely correlates with J
chain expression.
In addition to a role in J chain repression, a BSAP
repression motif has also been identified in the 3'
enhancer of the immunoglobulin heavy chain genes. Although the process of BSAP repression is not well understood, a possible mechanism has been suggested by in vivo footprinting studies by Neurath and
colleagues (13). This work showed that although two BSAP sites exist in the immunoglobulin 3'
enhancer, only the most 5' site is occupied by BSAP in mature B cells. As expected, no BSAP footprint was detected
in plasma cells, due to low levels of BSAP at this cell stage.
Importantly, the authors identified a second factor, NF-
P, which
bound to a position 50 bp downstream of the 5' BSAP-binding site in
plasma cells, but not in mature B cells. NF-
P, a member of the Ets
family of transcription factors, is expressed both in mature B and
plasma cells, and is necessary for maximal activity of the 3'
enhancer. Selective blocking of BSAP binding by triplex-forming oligonucleotides resulted in an NF-
P footprint in mature B cells and
an increased level of immunoglobulin gene transcription. Thus, it
appears that BSAP prevents NF-
P from activating the 3'
enhancer until the plasma cell stage.
In the work described here, a new DNA binding motif JE was identified
at positions
140 to
132 of the J chain promoter. This sequence resembles the µE3 element of the immunoglobulin heavy chain
enhancer and the related
E3 element of the
light chain enhancer
(14). We show that the helix loop helix protein upstream stimulatory factor (USF) binds to the J
chain promoter at the JE motif and positively regulates J
chain transcription. Recently, it was shown that USF increases the
activity of the immunoglobulin heavy chain gene intron enhancer, in
combination with two other factors, PU.1 and Ets-1 (15). In addition,
USF factors have been shown to interact with members of the basal
initiation complex (16, 17). We show here that USF may, at least in
part, be mediating its positive effect on J chain
transcription through a mechanism which necessitates interaction with
B-MEF2. Although these positive-acting factors are both expressed
throughout B cell development, it appears that they are only able to
bind weakly to the J chain promoter in the presence of BSAP.
This may provide a mechanism which ensures repression of J
chain transcription until the activated B cell stages. As BSAP
levels decrease during that time, USF and B-MEF2 replace BSAP on the
J chain promoter and this results in activation of transcription.
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MATERIALS AND METHODS |
Cell Culture--
PD31, 38C13, K46R, BCL1, and L cells were
maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 µM
2-mercaptoethanol, 100 units of penicillin/ml, and 100 µg of
streptomycin/ml. MOPC315 and S194 cells were cultured in Dulbecco's modified Eagle's medium supplemented as described above.
Preparation and Assay of Nuclear Proteins--
Large-scale
extracts were prepared from 109 cells by the detergent
lysis method of Peterson et al. (18) as modified by Lansford et al. (4). All buffers contained the following mixture of protease inhibitors: 0.5 nM phenylmethylsulfonyl fluoride,
0.5 mM dithiothreitol, 0.5 mM
Na2S205, aprotinin (10 units/ml),
leupeptin (5 mg/ml), and pepstatin A (5 mg/ml). Mini-extracts were
prepared from 107 cells as described (19).
Probes and DNA competitors used: J1, nt
83 to
9; J2, nt
168 to
84; JA:,
74 to
60; JB, nt
58 to
47; JC, nt
127 to
110;
JE, nt
140 to
132 of the J chain promoter. For gel
mobility shift assays, the specific oligonucleotides were end-labeled
with [
-32P]dCTP and Klenow enzyme (20). The binding
reactions with crude nuclear extracts were performed as described
previously (19) using 8-10 µg of extract, 4-6 µg of poly(dI-dC)
nonspecific competitor, and 104 cpm (0.1-1.0 ng) of probe.
For the antibody gel shift assays, crude nuclear extracts were
preincubated with 2 µl of a 1:10 dilution of either rabbit preimmune
sera or polyclonal rabbit anti-mouse USF-2 antiserum which is specific
for one of the two subunits of USF (p44) (Santa Cruz Biotechnology). In
each case the protein-DNA complexes formed were resolved from free
probe by electrophoresis through glycerol-containing 5% polyacrylamide
gels (29:1) containing 0.25 × TBE buffer.
Footprinting Assays--
Methylation protection footprinting was
performed with crude nuclear extracts from the mature B cell line,
K46R; 50 µg of nuclear protein was incubated 15 min at 0 °C with 2 µg of poly(dI-dC) and 106 cpm of DNA probe. One-half
microliter of dimethylsulfate was added for 45 s and quenched by
the addition of dithiothreitol to a final concentration of 23 mM. The remaining steps in the assay, isolation and
sequencing of free and protein-bound DNA, were performed according to
the standard protocol for methylation interference footprinting (21).
The probe J2 was an 84-bp XbaI/HindIII fragment
containing the J chain sequence
168 to
84 that was end-labeled on
the top or bottom strand.
Plasmid Constructions for Linked Promoter Analyses--
In the
p
42CassI vector (24) the CAT gene is under control of a truncated
-fibrinogen promoter (
54 to +36) that includes a TATA box and a
single Sp1-binding site. Fragments from the J chain promoter
(J1, bps
83 to
9; J2, bps
168 to
84; J1-J2, bps
168 to
9)
and oligonucleotides representing the JE element were synthesized with
XbaI linkers and inserted either singly or in multiple
copies into the polylinker upstream of the
-fibrinogen promoter. All
constructs were sequenced to determine oligomer copy number and orientation.
Mutagenesis of the p
42CassI plasmid containing the J1-J2 sequence
and the XBµ plasmid was performed with the TransformerTM
Site Directed Mutagenesis Kit (CLONTECH
Laboratories). The sequence 5'-CGTAAGTATGAACAATCTTCGTCTTTCCAGTGTAGC-3' (mJE) was used
to introduce a 3-bp change in the JE element (the underlined region
replaces the wt sequence CATGTG, see "Results"). The selected
plasmid was sequenced to verify the base substitutions. Mutations
introduced into the JC motif have been described previously (7).
Transfections--
Transient transfections of PD31 pre-B cells
were performed by the DEAE-dextran technique (22) and transfections of
MOPC315 myeloma cells by electroporation (23). In each case,
107 cells in logarithmic growth phase were transfected with
9 µg of supercoiled test plasmid or a combination of plasmids. Cell extracts were prepared 44-48 h after transfection and assayed for CAT
activity (4).
Immunoprecipitations and Western Blots--
Immunoprecipitation
reactions using antibody-Sepharose, were performed according to the
standard protocol (20). For Western blot analyses, nuclear extract
samples were boiled for 5 min, size fractionated by SDS-polyacrylamide
gel electrophoresis (10%), and transferred to a nitrocellulose filter.
After pretreatment with 5% dry milk in 1 × phosphate-buffered
saline, the filters were incubated for 3 h with a 1:10,000
dilution of antibody specific for BSAP, PU.1, MEF-2 (Santa Cruz
Biotechnology Inc.), or USF-2 (Santa Cruz Biotechnology Inc.). The
filters were then washed 3 times with 1 × phosphate-buffered
saline and incubated for 1 h with 1:5,000 dilution of horseradish
peroxidase-conjugated goat anti-rabbit IgG. After three 1 × phosphate-buffered saline washes, the filters were developed using an
enhanced chemiluminescence kit (Amersham).
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RESULTS |
Identification of the JE Element by Deletion
Analyses--
J chain transcription has been
shown to be regulated by at least two positive elements and one
repressor element on its promoter (4-7). To investigate whether
additional inducible elements are present which are necessary for
regulation of J chain transcription, deletion analyses of
the 5' region of the minimal J chain promoter was performed.
The constructs used for the deletion analyses have been described
previously and were generated through progressive 5' deletions of
upstream J chain sequences from the hypersensitive region (4, 7). These
fragments were tested for their ability to drive expression of the CAT
gene, as shown in Fig. 1 (4). In addition
to J chain promoter sequences and the CAT gene, each of the
constructs also contained the intronic immunoglobulin µE, inserted
downstream of the CAT gene in opposite transcriptional orientation
(24). The µE sequences were included because levels of CAT expression
obtained with constructs lacking a heterologous enhancer were too low
to yield reliable values. The constructs were transiently transfected
into the J chain-expressing and IgA-secreting myeloma MOPC315, and
assayed for CAT activity (Fig. 1). Data obtained from transfection of
these constructs into the J chain-silent pre-B cell line PD31 has been
shown previously (7) and is shown here for the purpose of comparison.

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Fig. 1.
Identification of a positive regulatory
element in the 5'-flanking region of the J chain
promoter. Schematic diagram of the 5'-flanking region of the
J chain gene showing regulatory motifs. Deletion constructs
were transfected into J chain-negative PD31 pre-B cells or J
chain-expressing MOPC315 plasmacytoma cells and assayed for CAT
activity. Data is shown as relative CAT activity.
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First we compared the relative CAT activity of MOPC315 cells with the
J chain negative and BSAP positive pre-B cell line PD31. Transfection of the
192 construct containing the entire
hypersensitive region resulted in approximately 50-fold higher CAT
activity in MOPC315 cells compared with PD31 cells, in agreement with
activity of the J chain promoter in plasma cell lines, but
not in early B cell lines (data not shown). Deletion of base pairs from
192 to
136 led to an 85% decrease in J chain promoter
activity in MOPC315 cells, as shown in Fig. 1. This suggested the
presence of a positive regulatory element, denoted JE, located at the
5' distal portion of the hypersensitive site, upstream from the
previously identified JC motif (Fig. 1). Finally, we found that further
deletion of the promoter element from
135 to
76 did not cause any
additional change in CAT activity (Fig. 1). This is not surprising
since the repressive factor BSAP that binds to the JC motif (base pairs
127 to
110), is undetectable in MOPC315 cells (13). However, as had
been identified previously, evidence for BSAP repression is observed in
the BSAP-containing and J chain negative PD31 cells, where removal of
the JC binding motif (nt
136 to
76) resulted in an increased CAT
activity (7).
Characterization of a Nuclear Factor Interacting with the JE
Element--
Next we examined the presence of a putative factor NF-JE
that could interact with the JE element on the J chain
promoter. Electrophoretic mobility shift assays (EMSAs) were performed
using a DNA fragment covering bps
168 to
84 of the J
chain promoter (J2) as a probe. This probe contains the JC and JE
elements, but not the JA or JB elements (see Fig. 1). Distinct binding
patterns were observed using nuclear extracts from the immature B cell line 38C.13, the mature B cell line K46R, the B presecretor BCL1, the
plasmacytoma lines S194 and MOPC315, the fibroblast line L (Fig.
2A), and the pre-B cell line
PD31 (not shown). Only S194 and MOPC315 express the J chain.

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Fig. 2.
EMSA analyses of NF-JE using the J2 probe
which covers base pairs 168 to 84 of the J chain
promoter. Arrow 1 indicates the complex containing
BSAP, arrow 2 the complex containing NF-JE. NS
indicates nonspecific probe binding. A, nuclear extracts
from five B cell lines representing different stages of development:
38C.13 (J ), immature B; K46R (J ), mature B; BCL1 (J ), presecretor
B; S194 (J+) and MOPC315 (J+), plasmacytoma; and one fibroblast cell
line, L (J ). B, competition EMSA using 100 × excess
unlabeled double-stranded oligonucleotides JB, JC, and JE and nuclear
extracts from the pre-B cell line PD31.
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The EMSA pattern showed three complexes on the gels. The fastest
migrating band (complex 1) was detected only in extracts from 38C.13,
K46R, BCL1 (Fig. 2A, arrow 1), and PD31 (not shown). This
suggested that this complex contained BSAP, based on the presence of a
BSAP-binding site on this probe (JC) and its pattern of expression in
the B cell lineage (see below). An intermediate migrating complex
(complex 2) was detected in all cell lines of the B cell lineage (Fig.
2A, arrow 2), but not in L cells. The relative amounts of
the two complexes varied between early and late B cell lines: early B
cell lines had high levels of complex 1, but low levels of complex 2. In contrast, the two plasmacytoma lines had very low or undetectable
levels of complex 1, but high levels of complex 2. All cell lines
tested showed a slow migrating complex which could not be competed with
excess unlabeled J2 DNA and thus represents nonspecific (NS)
protein-probe complexes (Fig. 2A, arrow NS).
The complexes 1 and 2 (Fig. 2A, arrows 1 and 2)
were analyzed further using competition EMSAs and nuclear extracts from
the pre-B cell line PD31. The unlabeled double-stranded (ds)
oligonucleotides JB (nt
58 to
47) and JC (nt
127 to
110), were
used as competitors and contain the PU.1 and BSAP-binding sites,
respectively. As expected, the JB competitor was unable to compete for
binding with either complex. The JC oligonucleotide was able to prevent binding of BSAP to the J2 probe (Fig. 2B, arrow 1), but
could not compete with the second, slower migrating complex (Fig.
2B, arrow 2). In contrast, this second complex was
specifically competed by a second double-stranded oligonucleotide JE,
which covers
140 to
132 of the J chain promoter.
To analyze the JE region further, we performed copper phenanthroline
and methylation protection footprinting using the J2 probe (Fig.
3). The retained band corresponding to
the JE region gave an extended footprint on the noncoding strand (Fig.
3A) from bp
138 through the BSAP-binding site to bp
124
(which also contained a footprint). The coding strand showed protection
in the 5' region of the noncoding strand footprint (Fig. 3B)
in the region between bp
138 through
134. These results are in
agreement with a putative factor NF-JE binding to this region of the
J chain promoter.

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Fig. 3.
Copper phenanthroline and methylation
protection footprints of NF-JE using nuclear extract from the J chain
negative B cell lymphoma K46R. Arrows indicate regions
of footprints. B, bound probe; F, free probe.
A, copper phenanthroline cleavage reaction footprint of
NF-JE with the anti-coding (MnlI to PstI)
restriction fragment of the J chain promoter as a probe. G + A, Maxam-Gilbert chemical cleavage at guanines and adenines on the
anticoding strand. B, methylation protection footprint of
NF-JE with the coding strand of the MnlI to PstI
restriction fragment as the probe. The G + A reaction was performed
with the coding strand probe. C, schematic representation of
the NF-JE footprint on the sequence of the J chain promoter.
Underlined residues indicate regions of protection.
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Identification of NF-JE as the Helix Loop Helix Transcription
Factor USF--
To search for possible candidates for NF-JE, known
consensus DNA-binding motifs were compared with the sequence in the JE region. One motif that contained high homology to the sequence recognized by NF-JE (CATGTG) was one recognized by E-box family proteins, particularly the µE3 (CACATG) and
E3 (CATGTG) motifs present on the immunoglobulin µ heavy chain intron enhancer and
light chain enhancer, respectively (14, 25, 26).
To test whether NF-JE represents an E-box factor, EMSAs were performed
using a polyclonal rabbit antiserum against one of the two subunits of
USF, namely USF-2. When tested with PD31 nuclear extracts this
antiserum was able to block NF-JE binding to the JE oligonucleotide
probe (Fig. 4, lane 4). Next,
we tested whether mutation of the core binding sequence for E-box
proteins would result in prevention of binding of the factor NF-JE,
using competition EMSAs. A mutant JE oligonucleotide (mJE) was
synthesized containing a 3-base pair mutation in the region
corresponding to the consensus E motif CANNTG (changing CATGTG to
AATCTT). We found that the NF·JE complex could not be inhibited by
this mutant JE oligonucleotide (Fig. 4, lane 2). In Fig.
2B we already showed that a wt JE oligonucleotide could
compete with this complex. DNA-protein complexes were not observed when
the mJE oligonucleotide was used as a labeled probe (data not shown).
Together, these findings provide evidence that NF-JE represents the
helix loop helix transcription factor USF and will be referred to as
such hereafter.

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Fig. 4.
Identification of NF-JE as the helix loop
helix transcription factor USF. EMSA's using the JE motif ( 146
to 124) as a probe and nuclear extract from the J chain negative B
cell lymphoma K46R. Lane 1, extract alone; lane
2, 100 × excess mJE oligonucleotide competitor; lane
3, preimmune serum; lane 4, anti-USF-2 antibody.
Arrows indicate complexes as specified.
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USF Needs Downstream Regulatory Elements to Affect J Chain
Transcription--
To determine the contribution of the JE element to
J chain transcription in vivo, we performed
transient transfections in the J chain-expressing plasma cell line
MOPC315, using either a wild-type (wt) or mutated J chain
promoter (bp
1150 to +88) driving expression of a CAT reporter gene.
The mutated J chain promoter contained a 3-bp replacement
mutation of the JE sequence, mJE (see Fig. 4 and text). Comparison of
relative CAT activity between wt and mutated constructs revealed a 66%
reduction in CAT activity in the JE-mutated J chain
promoter, as shown in Fig. 5A.
This is in agreement with the 5' deletion studies (Fig. 1) and
indicates that JE represents a positive regulatory element on the
J chain promoter.

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Fig. 5.
Functional analysis of J chain
promoter elements in the J chain-expressing plasma cell line
MOPC315. A, relative CAT conversion of wild-type (wt)
or JE-mutated J chain promoter (base pairs 1150 to +88)
driving expression of a CAT reporter gene. The mutated J chain
promoter contains a 3-nt replacement mutation of the JE sequence
(indicated by an "X"). B, CAT reporter
constructs containing different combinations of wt or mutated fragments
of the J chain promoter, linked to a minimal fibrinogen
promoter. See text for details.
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To determine whether the JE element can act as an independent activator
of transcription, the truncated rat
-fibrinogen promoter was used to
drive expression of a CAT reporter gene (p
42CassI). This vector has
basal promoter activity in J chain-expressing myeloma lines
(24). Two or three copies of the wild-type JE sequence were cloned into
the p
42CassI construct, and the JE constructs transfected into
MOPC315 cells. The presence of the JE sequences had no effect on the
activity of the truncated rat
-fibrinogen promoter, suggesting that
the NF-JE factor needs to be in the context of other, J chain-specific
regulatory factors in order to have transcriptional activity (Fig.
5B, bottom two constructs).
We next examined the effect of the JE element in the context of other
known J chain promoter regulatory motifs. For these analyses, the following sequences were inserted into the p
42CassI vector upstream of the minimal
-fibrinogen promoter: the J1-J2 fragment (bp
168 to
9, which contains all four regulatory motifs JA, JB, JC, and JE), the J1-J2 fragment with a mutant JE-binding site
(J1-J2mE), J1-J2 with a mutant JC site (J1-J2mC), the J2 fragment alone
(bp
168 to
84, which contains the JC and JE motif only), the J2
fragment with a mutant JE site (J2mE), and the J2 fragment with a
mutant JC site. A 4-bp mutation (underlined sequences) changes
CAGTGTAGCATGCAGT to CAGTGTAGGTCACAGT in the JC
motif and this results in the absence of BSAP binding to this region
(7). These constructs were then transfected into MOPC315 cells. The constructs containing J1 as well as both JC and JE (J1-J2 and the
J1-J2mC) induced a 5- and 5.5-fold increase in the basal level of CAT
expression, respectively (Fig. 5B). Alternatively, the J1-J2mE construct which contained a 3-bp mutation in the JE motif, exhibited a 62% loss of activity in comparison to the other J1-J2 constructs. In contrast to the results obtained with the J1-J2 constructs, the wild-type J2 fragments alone (which did not contain JB
or JA), did not show detectable changes in basal CAT activity (Fig.
5B). These results suggest that USF can act as an activator of J chain transcription only if in the context of other
promoter elements present on both the J1 and J2 regions.
Based on the above results, we hypothesized that USF may be mediating
its positive influence on J chain transcription by
interacting with downstream activating elements PU.1 and/or B-MEF2,
both of which bind to the J1 region of the J chain promoter
(to JB and JA, respectively). To test this possibility, we performed
co-immunoprecipitation reactions using antibodies to B-MEF2, PU.1, and
USF-2. We were able to co-precipitate B-MEF2 with USF-2 antibody (Fig.
6) and vice versa (data not shown), both
in the J chain negative pre-B cell line PD31 as well as in the J chain
positive line MOPC315. In contrast, no evidence was found for
interactions between either PU.1 and B-MEF2 or PU.1 and USF using this
approach (data not shown). These results are in agreement with the
above deletion analyses data of the J chain promoter (Fig.
5B) and suggest that promoter activity involves functional
interactions between B-MEF2 and USF. This interaction may be necessary
to assemble a functional transcription complex necessary for efficient
J chain transcription.

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Fig. 6.
Detection of protein interactions between USF
and B-MEF2. Nuclear extracts from PD31 or MOPC315, and antibodies
to USF were used in co-precipitation reactions, and resulting complexes
analyzed by Western blot analysis using an anti-MEF2 antibody
(right two lanes). As a negative control,
immunoprecipitations were performed with preimmune serum (left
two lanes). Molecular weight markers are indicated on the
right.
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BSAP Decreases USF and B-MEF2 Binding to Their Regulatory Motifs in
the J Chain Promoter--
Transcription of the J chain
gene is initiated when a mature immunocompetent B cell receives both an
antigen signal and a cytokine signal from IL-2 or IL-5. It has been
shown previously that the transcription factor BSAP is responsible for
repressing J chain transcription until this time (7, 10).
The concentration of BSAP present in activated B cells decreases over
the course of a primary immune response until it becomes almost
undetectable at the plasma cell stage (7). Overexpression of BSAP in
plasma cell lines, however, reverses this trend and results in
BSAP-mediated repression of J chain transcription (7,
10).
To further our understanding of the regulation of J chain
transcription, we analyzed the in vitro DNA binding
characteristics of all four factors that influence J chain
promoter activity, including BSAP, USF, B-MEF2, and PU.1. DNA binding
was analyzed by EMSA using nuclear extracts from the mature B cell line
K46R. Two different probes were used, either the wild-type J1-J2
segment (bp
168 to
9) which contains binding sites for all four
factors, or the same probe with a mutated BSAP-binding site (mJC).
In none of the EMSAs did we observe the binding of all four factors at
the same time, using either the wild-type or the BSAP mutant probes
(Fig. 7, A and B).
This was surprising, especially since we could show by Western blot
analysis that all four factors were present in the nuclear extracts of
the K46R cells (Fig. 7C). For example, B-MEF2 was unable to
bind to the J chain probe (Fig. 7A, left three
lanes) except when the BSAP motif was mutated (Fig. 7, A,
right three lanes, and B). In the presence of BSAP
binding, some USF was able to bind to the JE motif (using the wild-type probe), but USF binding increased greatly in the absence of BSAP (using
the BSAP mutant probe), as shown in Fig. 7, A and
B. Interestingly, the binding of USF to the JE motif was
reduced by competition with B-MEF2 oligos (Fig. 7B). In
contrast, PU.1 binding was unaffected by the presence of bound BSAP
(Fig. 7, A and B). Together, these results imply
that BSAP mediates its repression on the J chain gene by
preventing two activator factors, USF and BMEF-2, from binding to their
regulatory elements.

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Fig. 7.
Protein analyses of transcription factors
that influence activity of the J chain promoter.
A, EMSA using the J1-J2 probe ( 168 to 9) or the J1-J2
probe with the BSAP-binding site mutated, and extracts from the mature
B cell line K46R. 100-fold excess of unlabeled double-stranded
competitors representing the BSAP-binding site (JA, 127 to
110), or the PU.1-binding site (JB, 58 to 47) were
added as indicated on top. The various protein-probe
complexes are indicated on the left. B, EMSA using the J1-J2
probe with the BSAP-binding site mutated, and extracts from the mature
B cell line K46R. 100-fold excess of unlabeled double-stranded
competitors representing the PU.1-binding site (JB, 58 to
47) or the B-MEF2-binding site (JA, 74 to 60) were
added as indicated on top. The various protein-probe
complexes are indicated on the left. C, Western blot
analysis showing the relative amounts of B-MEF2, PU.1, USF, and BSAP in
nuclear extracts from the mature B cell line K46R (K) or the
plasmacytoma line MOPC315 (M), using antibodies as specified
in the text.
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DISCUSSION |
Regulation of gene expression during B cell development is
extremely complex and involves combinatorial binding activities of
multiple transcription factors at each target gene. In this study we
sought to further explore the transcriptional regulation of the
immunoglobulin J chain gene promoter. This promoter is relatively well defined, and the nuclease hypersensitivity region between base pairs
170 and +88 has been shown to be sufficient for
full activity in J chain positive cells (4). Earlier studies defined
three regulatory elements on the J chain promoter, JA, JB,
and JC, which were shown to associate with the transcription factors
B-MEF2, PU.1, and BSAP, respectively (5-7). This report defines a
fourth binding motif denoted JE, with a consensus E-box factor binding
sequence. The protein binding to this motif was identified as USF, a
member of the E box family of basic helix loop helix transcription
factors. Since we have not observed the binding of other nuclear
factors within the nuclease hypersensitivity region, we believe that
USF represents the fourth and final major inducible DNA-binding protein
necessary for activation of the J chain promoter.
USF Interacts with the MADS Box Family Member B-MEF2--
Results
from the 5' deletion analyses and co-immunoprecipitation assays suggest
that USF interacts with a second, downstream binding transcription
factor, B-MEF2. The ubiquitously expressed factor USF commonly consists
of two subunits, USF-1 and USF-2 (26), and has been shown to be a
transcriptional activator in combination with other factors (27-30).
For example, in the immunoglobulin µ heavy chain enhancer, activity
is detected only in the presence of a three protein-DNA complex
consisting of USF, PU.1, and Ets-1 (15). In this case, the restricted
expression pattern of PU.1 and Ets-1 ensures that its activity is
limited to lymphoid cells. The observed association between USF and
B-MEF2 is supported by studies on protein-protein interactions between
MEF2 family members and E box proteins, and it has been proposed that
such interactions may lead to increased assembly of the basal machinery
(29, 30).
BSAP Prevents B-MEF2 and USF from Binding to the J Chain
Promoter--
The expression pattern of BSAP throughout B cell
development has been well established and has been found to be
inversely correlated with expression of the J chain (7, 9, 12, 13). In
addition, it has been shown that BSAP is responsible for repression of
the J chain gene during the antigen-independent stages of B cell development (7). In the present study, the mechanism of BSAP
repression of the J chain gene was explored further. EMSAs were used to analyze the DNA binding characteristics of BSAP as well as
the three other factors that interact with the J chain promoter, using nuclear extracts from the mature B cell line K46R. The data suggest that the binding of BSAP to the J chain
promoter reduces binding of the ubiquitously expressed USF to the JE
motif and simultaneously prevents B-MEF2 from binding to its JA motif. In contrast, BSAP binding does not influence the binding of the fourth
factor, PU.1. Since both B-MEF2 and USF are activators of J
chain transcription, the presence of BSAP may provide an efficient
mechanism to prevent activation of the J chain gene during
the antigen-independent stages of B cell development. This would also
be in agreement with the observation (in Fig. 2A) that extracts from early B cell lines show relatively low amounts of USF
bound to the J2 probe, but plasmacytoma lines (which have no BSAP) show
significantly higher amounts of bound USF.
The exact mechanism by which binding of BSAP prevents B-MEF2 and USF
binding to their respective motifs on the J chain promoter is not clear. Perhaps the easiest explanation is that the failure of
USF to bind to its JE motif is a direct result of its close proximity
to the BSAP binding motif JC. In this scenario, BSAP would prevent the
access of USF to its binding motif by either occluding its binding
site, or changing the structure of the DNA within this region. A local
unwinding of the DNA helix has been proposed as a mode of repression in
the major histocompatibility complex class II promoter (31).
The mechanism by which B-MEF2 binding is inhibited in the presence of
BSAP, which binds 35 bp upstream of this activating factor, is also not
clear. The situation is, however, analogous to what has been observed
in the 3'
enhancer, where BSAP has been shown to prevent the
binding of an activator factor whose binding motif is 50 bp downstream
(13). One explanation would be that USF and B-MEF2 each need to
interact with their DNA-binding sites as well as with each other, to
enable formation of a stable complex on the J chain promoter.
The analyses of J chain gene regulation presented here
supports a model where the key regulatory switch for J chain
expression is determined entirely by nuclear levels of BSAP protein.
The three other regulatory factors that can bind to the J
chain promoter are unable to influence J chain
transcription despite their expression throughout B cell development.
When BSAP concentrations decrease as a result of B cell activation, all
three activator factors are now able to rapidly bind to the J
chain promoter and presumably interact with each other to promote
efficient transcription of the J chain gene.
Regulation of J chain expression provides a good example of
combinatorial regulation: although all four factors are expressed during B cell development, a unique combination of three is necessary to activate J chain transcription. The fourth factor, BSAP,
not only prevents J chain activation until the final stages
of B cell development, but plays other, essential roles on distinct
target genes during earlier stages of development as well. USF, unable to activate transcription of the J chain gene until the
final stages of B cell development, plays essential roles in the
expression of the immunoglobulin µ heavy chain gene during early B
cell development (15). Thus, although many different transcription
factors are expressed throughout B cell development, the interactions
of unique combinations of factors with their target
promoters will largely determine the level of specific gene expression
during each developmental stage.