Developmental Regulation of the
Locus Involves Both Positive
and Negative Sequence Elements in the 3' Enhancer That Affect Synergy
with the Intron Enhancer*
Xiangdong
Liu,
Anila
Prabhu, and
Brian
Van Ness
From the Department of Biochemistry, Institute of Human Genetics
and the Cancer Center, University of Minnesota,
Minneapolis, Minnesota 55455
 |
ABSTRACT |
Expression of the mouse immunoglobulin
locus
is regulated by the intron and 3' enhancers. Previously, we have
reported that these enhancers can synergize at mature B cell stages.
Here we present our recent studies on the identification and
characterization of the 3' enhancer sequences that play important roles
in this synergy. By performing mutational analyses with novel reporter constructs, we find that the 5' region of the cAMP response element (CRE), the PU.1/PIP, and the E2A motifs of the 3' enhancer are critical
for the synergy. These motifs are known to contribute to the enhancer
activity. However, we also show that mutating other functionally
important sequences has no significant effect on the synergy. Those
sequences include the 3' region of the CRE motif, the BSAP motif, and
the region 3' of the E2A motif. We have further demonstrated that
either the 5'-CRE, the PU.1/PIP, or the E2A motif alone is sufficient
to synergize with the intron enhancer. Moreover, the PU.1 motif appears
to act as a negative element at pre-B cell stages but as a positive
element at mature B cell stages. We have also identified a novel
negative regulatory sequence within the 3' enhancer that contributes to
the regulation of synergy, as well as developmental stage and tissue
specificity of expression. While the levels of many of the 3' enhancer
binding factors change very little in cell lines representing different B cell stages, the intron enhancer binding factors significantly increase at more mature B cell stages.
 |
INTRODUCTION |
Expression of the immunoglobulin
(Ig
) gene is
tissue-specific and is developmentally regulated. In addition to
tissue-specific variable (V
) region promoters, at least two
enhancers also contribute to this tissue-specific and developmental
control (1-5). The intron enhancer (
Ei) is located between the
joining (J
) segments and the constant (C
) region, and the 3'
enhancer (
E3') lies approximately 9 kb1 downstream of the C
region. Both enhancers show tissue specificity and developmental
regulation. It has been shown that the transcription activities of
these enhancers are modulated through specific sequence motifs (6). The
activity of the
Ei is contributed by several motifs, including
A,
B, E1, E2 and E3; and the activity of the
E3' is mediated in part
through CRE, BSAP, PU.1, PIP, and E2A motifs (7-12). Specific DNA
binding proteins for many these motifs have been identified and
characterized (6, 13-16). Although no direct interactions among
binding factors of the
Ei motifs have formally been established,
interactions among binding factors of the
E3' motifs have been
described (10-12, 17).
Expression of a functionally rearranged Ig
gene is known to be
up-regulated during B cell development, and it reaches maximal activity
at mature B and plasma cell stages. Despite numerous reports on these
enhancers, it is still not entirely clear how both the enhancers
coordinately participate in this developmental up-regulation. While
some studies show that the
Ei is active at a low level in pre-B
cells, other studies suggest that it is completely silent in pre-B
cells (9, 10, 18). A more recent study, however, suggests that the
Ei is probably always active but at a relatively low level at early
B cell stages, and it is thus considered to play no significant role in
the activation of
locus during pro-B to pre-B transition (19). The
approximately 1-kilobase region of the
E3' is inactive, or active at
a low level, at early B cell stages, and its activity increases during B cell maturation and reaches full levels at mature B and plasma cell
stages (9, 10, 12, 20). However, a 132-base pair (bp) core within the
larger
E3' has been reported to be active in pre-B cells, and its
activity is suppressed by negative sequence elements in its flanking
regions (21, 22). Interestingly, like the
Ei, the
E3' can also be
activated by bacterial lipopolysaccharide (LPS) treatment, and this
inducibility is thought to be mediated by some sequences flanking the
core as well (22).
While the two enhancers individually contribute to the developmental
regulation of the
locus, we and others have reported that they can
synergistically activate
transcription at mature B cell stages and
the combined strength of the two is roughly equivalent to that of a
heavy chain µ core enhancer (18, 23, 24). Thus, it is important to
consider the interactions of the two enhancers in B cell development,
and the roles of individual sequence motifs should be examined not only
in the context of the individual enhancer, but within the natural
context of both enhancers. We previously determined some of the
sequence requirements of the
Ei for synergizing with the
E3'
(24). However, little has been done to characterize the
E3' with
respect to its involvement in the synergistic activation with the
Ei. Thus, in this current study we sought to identify and
characterize the
E3' sequence elements and the binding factors that
are important for the developmentally regulated synergistic
expression. Using novel reporter constructs, we demonstrate that both
positive and negative elements within the
E3' contribute to the
developmental regulation and the tissue specificity of
expression.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Construction and Mutagenesis--
Standard recombinant
DNA procedures were performed as described by Sambrook et
al. (25). A functionally rearranged
gene of mouse myeloma
MOPC41 was excised as an approximately 7-kilobase EcoRI
fragment from a vector
(pEotk.neo.short-MAR+ENH+) provided
by Dr. William Garrard (University of Texas Southwestern Medical
Center) (26). The fragment was then inserted into an EcoRI
restriction site in a multiple cloning region of pGEM1 (Promega) containing a previously inserted neomycin resistance
gene.2 The C
region of the
gene in this construct was replaced as a
SacII-BamHI fragment with a different C
region, a SacII-BamHI fragment from
pSPIg.neo-V
21C2 in order to generate a unique
BamHI restriction site and avoid ambiguity of sequence
information at the 3' end of the
gene. A luciferase gene was
amplified by polymerase chain reaction and inserted in-frame into a
HpaI site in the C
region. Constructs containing the
wild-type larger
E3' and the
E3' core were subsequently generated
by inserting these enhancer fragments into the BamHI site.
Constructs that contain
E3' mutations were generated by inserting
different forms of the
E3' mutations into the BamHI site.
A set of linker scan mutations of the
E3' core, provided by Dr.
Michael Atchison (University of Pennsylvania) (12), was cloned into the
BamHI site. Constructs containing individual sequence motifs
were generated by inserting double-stranded oligonucleotides of the
individual motifs into the BamHI site. Additional mutations of specific sites within the
E3' core were generated by an
overlapping polymerase chain reaction method (27). The construct with
the deletion of the
Ei was made by replacing a part of the
gene with the one that had a 1042-bp deletion of both the matrix association region and
Ei from a vector
(pEotk.neo-MAR
ENH
) provided by
Dr. William Garrard (26). All mutations were confirmed by DNA
sequencing with SequiTherm EXCEL DNA Sequencing Kit (Epicentre Technologies).
Cell Culture--
The mouse pre-B cell lines 3-1 and 1-8, the
mouse nonsecreting mature B cell line A-20, and mouse mature B
plasmacytoma cell line S194 have been characterized and referenced
previously (18). The cell lines A-20 and S194 were obtained from the
American Type Culture Collection. The mouse pre-B cell line 38B9 was
obtained from Dr. Eugene Oltz (Vanderbilt University). The mouse
erythroid leukemia (Mel) cell line and the human Jurkat T lymphoma cell lines were provided, respectively, by Drs. Jane Little and Lizhen Gui
(University of Minnesota). Cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 25 units/ml penicillin, and 25 µg/ml streptomycin (all from Life Technologies, Inc.). 50 µM
-mercaptoethanol was added only to pre-B cell
cultures. For LPS induction, cells were treated with LPS (Difco) at 1 µg/ml. Transfection analyses were normally performed after 20-24 h
LPS induction.
Transient Transfection--
Mouse B cell lines were transfected
by a modified DEAE-dextran method (18). Briefly, 5 × 106 cells per transfection were harvested and washed in 25 ml of 1 × sterile TS solution warmed to 37 °C (137 mM NaCl, 5 mM KCl, 0.3 mM
Na2HPO4·7H2O, 25 mM
Tris, 1 mM MgCl2, 1 mM
CaCl2, pH 7.4]. The cells were then resuspended in 1.5 ml
of warm TS containing 500 µg/ml DEAE-dextran (Amersham Pharmacia
Biotech) with 1 µg of test plasmid and 1 µg of a control plasmid,
SV40-
-gal, a
-galactosidase expression vector, and the mixture
was incubated for 20 min at 37 °C. Then the cells were washed with
10 ml of RPMI 1640, pelleted, and resuspended into 10 ml of RPMI 1640 (10% fetal calf serum, 2 mM L-glutamine, 25 units/ml penicillin, 25 µg/ml streptomycin, and 50 µM
-mercaptoethanol (±1 µg/ml LPS if required)), and placed in a
37 °C, 7% CO2 incubator. Transfected cells were
harvested after 20-24 h and used in luciferase and
-galactosidase
assays. Transfection efficiency was normalized by cotransfecting with the
-galactosidase expression vector, dividing luciferase activity by
-galactosidase activity. The luminescence ratio was plotted in
arbitrary units. Typically, transfection assays for each construct were
performed in duplicate with a minimum of three transfections for each
construct, and average values were plotted with standard error of the
mean indicated.
Luciferase and
-Galactosidase Assays--
Luciferase assays
were performed as follows: at harvest, 2 × 106 cells
were transferred into 1.5-ml microcentrifuge tubes, washed once with
1 × phosphate-buffered saline, and pelleted. The cells were then
lysed for 10 min at room temperature in 50 µl of 1 × Reporter
Lysis Buffer (Promega). Luciferase Assay Reagent (Promega) (100 µl)
was added to 20 µl of the cell lysate and counted for 15 s in a
Lumat 9501 luminometer (EG & G Berthold) according to the
manufacturer's instructions. Relative light units are reported.
-Galactosidase activity was measured using a Galacto-Light kit
(Tropix, Bedford, MA). Ten µl of the above cell lysate were incubate
with 67 µl of 1 × Galacton substrate (diluted with 1 × Galacto-Light Reaction Buffer Diluent) for 45 min at room temperature in the dark. Light Emission Accelerator reagent (100 µl) was injected immediately prior to measurement in the luminometer.
Nuclear Extract Preparation--
Nuclear extracts were prepared
according to the method of Dignam et al. with modifications
(28). Briefly, cells were washed in cold 1 × phosphate-buffered
saline and resuspended in 5 packed cell volumes of cold buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.1%
Nonidet P-40) and incubated for 2 min on ice to lyse the cells. The
nuclei were pelleted by centrifugation at 9,000 × g
for 30 s at 4 °C. The supernatant was removed, and the pellet
was then resuspended in 0.5 packed cell volume of protein extraction
buffer (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride,
0.5 mM DTT). Nuclei were rocked for 30 min at 4 °C and
then centrifuged at 12,000 × g for 20 min to remove
insoluble debris. The supernatant (nuclear extract) was then dialyzed
against 20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M NaCl, 0.2 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, 1.0 mM DTT for over 4 h
and stored at
70 °C. Protein concentrations were determined by the
Bradford colorimetric assay with Protein Assay Dye Reagent (Bio-Rad)
according to the manufacturer's recommendations.
Electrophoresis Mobility Shift Assays--
Electrophoresis
mobility shift assays were carried out with 20,000 cpm of the
32P-end-labeled oligonucleotide probes, which were
incubated for 30 min at room temperature with 10 µg of nuclear
extracts in a final volume of 20-µl binding reactions (50 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA,
1 mM DTT, and 5% glycerol, pH 7.9), including 0.5-2 µg
of poly(dI-dC) (Sigma). Samples were separated on 5% nondenaturing polyacrylamide gels. The specificity of protein-DNA complexes was
confirmed in competition experiments with unlabeled specific competitors.2 The images were acquired with a
PhosphorImager (Molecular Dynamics). The sequences of the
oligonucleotide probes (upper strand shown) are as follows.
Oligonucleotides--
All oligonucleotides were synthesized by
the MicroChemical Facility at the University of Minnesota. Sequences of
the oligonucleotides used for generating constructs containing the
E3' individual motifs are as follows.
Sequences of the oligonucleotides used for replacing the
negative regulator region are as follows.
Sequences of the oligonucleotides used for polymerase chain
reaction amplification of the linker scan mutations are as follows.
 |
RESULTS |
Previous reporter constructs that have been developed introduced
enhancer elements and heterologous promoters into artificially arranged
vectors (9, 10, 12, 18). In this study we designed reporter constructs
that preserve the natural context of enhancer elements. The base
construct (Fig. 1) contains a
functionally rearranged V
J
1 with the
Ei and
E3' in a more
natural context than previous constructs (18, 24, 26), and a luciferase gene fused in-frame within the C
region. To identify sequence elements important for the developmental regulation, a series of
modifications, including small deletions, linker scanner mutations, and
substitution mutations, were designed and are presented in each
subsequent figure. In this study our particular focus is on
modifications to the
E3'. We used these constructs to transiently transfect a number of cell lines representing different stages of B
cell development. The advantage of this approach is the ability to
examine clonal responsiveness of early and late B cells without the
selective expansions of heterogeneous B cell populations in transgenic
or embryonic stem cell-generated mice.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 1.
A schematic diagram of the rearranged gene and construct design. At the top, the rearranged
gene with an integrated luciferase gene used as a backbone in the
generation of all constructs is shown. The E3' core and its sequence
motifs are also indicated below. P, M,
Ei, E3', V , J ,
C , and LUC represent variable region promoter,
nuclear matrix association region, intron enhancer, 3' enhancer,
variable region, joining segments, constant region, and a luciferase
gene, respectively.
|
|
The Intron and 3' Enhancers Synergistically Activate
Transcription at Mature B Cell Stages but Not at Pre-B Cell Stages, and
the Activities of These Enhancers Are Up-regulated from Pre-B to Mature
B Cell Stages--
In previous studies we designed constructs in which
the
Ei and
E3' were placed adjacent to each other upstream of a
V
region promoter, driving a luciferase reporter (18, 24). We showed that in this context the intron and 3' enhancers together
synergistically activate transcription at mature B and plasma cell
stages, but not at pre-B cell stages (24). To be sure that the newly
generated constructs possess the same synergistic property and to
retest the previous observation with the
regulatory elements in a
more natural context, we performed transient transfection analysis with
new constructs that had different combinations of a V
region promoter with the two enhancers (Fig.
2A). In S194 mature B cells, the addition of the
Ei alone increased transcription level by 25-35-fold above the promoter alone, and the addition of the
E3' increased transcription level by 5-7-fold. The two enhancers together increased transcription level by more than 100-fold compared with the
promoter alone (Fig. 2B). In 3-1 pre-B cells, however,
either enhancer alone showed minimal activity, and the two enhancers together only slightly increased transcription level compared with the
promoter alone (Fig. 2C). Because the activities of these enhancers reportedly can be induced by LPS stimulation at pre-B cell
stages, we then treated transfected 3-1 cells with LPS and found that
even in LPS-stimulated 3-1 cells, the up-regulation of transcription
was rather modest by these constructs (Fig. 2C). Similar
effects were also observed in other mature and pre-B cell lines (data
not shown). These results suggest that the activities of both enhancers
are significantly up-regulated from early to late B cell stages, and
with these constructs the synergistic activation is still a
developmentally regulated phenomenon that is evident only at mature B
cell stages but not at pre-B cell stages.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
Synergistic activation of expression by the
intron and 3' enhancers in mature B cells but not in pre-B cells and
developmental up-regulation of the enhancer activities.
A, constructs containing different combinations of a V
region promoter with the two enhancers. IM represents matrix
association region and intron enhancer. 3'E(800) represents
the 800-bp 3' enhancer. B, transient transfection and
synergistic activation in S194 plasmacytoma cells. C,
transient transfection and transcription activities in untreated and
LPS-treated 3-1 pre-B cells.
|
|
The 132-bp 3' Enhancer Core Alone Is Sufficient to Synergize with
the Intron Enhancer--
The synergistic activation of
expression
is conferred by the intron and 3' enhancers together. Previously, we
have carried out detailed studies on the sequence requirements of the
Ei for the synergy (24). In this current study, we sought to
determine specific
E3' sequences that are functionally important for
the synergy and therefore contribute to the developmental regulation of
expression. In all previous studies, including experiments shown in
Fig. 2, we used the 800 bp
E3' (Ref. 24, and see "Experimental Procedures" for details). However, several reports have suggested that the 132-bp
E3' core accounts for most of the activity that the
800-bp enhancer has in regulating
transcription (9, 10). To test
whether the core is sufficient to confer synergy we carried out
transfection analysis with constructs in which the 800-bp enhancer was
replaced by the 132-bp core (Fig.
3A). We find that the core by
itself is sufficient to synergize with the
Ei to achieve high level
transcription in S194 cells (Fig. 3B).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
The 132-bp 3' enhancer core is sufficient to
synergize with the intron enhancer in S194 cells. A, an
illustration of constructs containing either the 800-bp 3' enhancer or
its 132-bp active core. 3'Ecore represents the 132-bp 3'
enhancer core. B, transient transfection shows that the
132-bp core alone is capable of synergizing with the intron enhancer.
Constructs number 6 and number 11 are two constructs that were
generated and confirmed independently.
|
|
Linker Scanner Analysis of Enhancer Synergy--
Because the
E3' core is sufficient for the synergistic activation, we then
investigated what specific core sequences are required for the synergy
by incorporating linker scanner mutations of the
E3' into the
reporter vector (Fig. 4A) Each
linker scan mutation has a 10-bp sequence substitution across the
E3' core sequence (12). As shown in Fig. 4B, three
regions were identified that, when mutated, significantly decreased the
enhancer synergy. These three regions overlap with the 5' region of the
CRE, the PU.1/PIP, and the E2A motifs. All of these motifs have been
shown previously to be important for independent enhancer activity in the context of heterologous promoters (9, 10, 12). However, other
motifs that contribute to the independent enhancer activity had no
significant effects on the synergistic activity, including the 3'
region of the CRE motif, the BSAP motif, and the region 3' of the E2A
motif (12). Our results suggest that while some motifs are dispensable
for the synergy, the 5'-CRE, PU.1/PIP, and E2A motifs are required for
the full level synergistic activity.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 4.
Distinct involvement of different 3' enhancer
core sequences in the synergy in S194 cells. A, a
diagram of the constructs containing a set of linker scan mutations of
the core. LSM stands for linker scan mutation. 13 linker
scan mutations are designated as A-M.
B, effects of the linker scan mutations on the synergy in
S194 cells. p values are indicated only for those that have
shown significant changes in transcription activities.
|
|
As is evident in Fig. 4B, we also identified a region that
when mutated significantly increased the overall transcription activity
by 2-3-fold compared with the wild-type
E3' or the core (LSM(H) in Fig. 4B). This region covers a 10-bp
sequence (ACAGAACCTT) located between the PU.1/PIP and the E2A motifs,
and it defines a novel negative activity for reporter expression. We
therefore refer to this region as the
E3' negative regulator
(
E3'NR). To role out the possibility that the linker scanner
mutation H artifactually caused the increase in reporter activity, we
independently introduced a completely different 10-bp sequence at
position H, and observed the same 2-3-fold increase in activity (data
not shown).
The Individual 5'-CRE, PU.1/PIP, and E2A Motifs Are Sufficient to
Synergize with the Intron Enhancer, and the Activities of These Motifs
Are Developmentally Regulated--
To further examine the importance
of the 5'-CRE, PU.1/PIP, and E2A motifs in the developmentally
regulated synergistic activation, we tested whether each of these
motifs alone was sufficient to synergize with the
Ei. We performed
transfection analysis with constructs in which the wild-type
E3' was
replaced by either the 5'-CRE, PU.1/PIP, or E2A motif alone (Fig.
5A). We found that in S194
mature B cells each motif alone is sufficient to synergize with the
Ei (Fig. 5B). In fact, each motif conferred an activity even higher than the wild-type
E3' or the 132-bp core. Notably, the
individual
E3' motifs did not contain the complete
E3'NR sequence; thus, the result further supports the apparent negative effect of the
E3'NR. In contrast, the
E3' core, or the individual motifs that showed synergy in S194 cells, showed minimal increases in
induced pre-B cells (Fig. 5C), again indicating the
synergistic activity is developmentally regulated.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 5.
The individual 5'-CRE, PU.1/PIP, and E2A
motifs are sufficient to synergize with the intron enhancer in S194
cells, and their activities are developmentally up-regulated from pre-B
to mature B cell stages. A, a diagram of the constructs
containing either the 5'-CRE, PU.1/PIP, or E2A motif of the 3' enhancer
core. B, transfection results in S194 cells. C,
transfection results in LPS-stimulated 3-1 cells.
|
|
The role of individual linker scanner mutations of the
E3' was also
assessed in induced pre-B cells. Synergy is not observed by the
inclusion of both enhancers in pre-B cells (shown in Fig. 2). Indeed,
when the intron enhancer was paired with LSMs B (5'-CRE motif), G (PIP
motif), and I (E2A motif), there was very little effect on total
reporter activity (Fig. 6). However, we
noted that in contrast to the negative effect mutation of PU.1 motif had in mature B cells (Fig. 4, LSM(F)), this same mutation
served to increase activity in 3-1 pre-B cells (Fig. 6). Similar
effects were seen in 38B9 and 1-8 pre-B cells (data not shown). The
results we obtained with the PU.1 mutation are also consistent with
previous reports, suggesting that the PU.1 motif may serve as a
negative regulator in early B cell development (29, 30).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Linker scanner mutations of the 3' enhancer
have different effects in pre-B cells. Transient transfections
were done for linker scanner mutations B, F, G, and I (see legend to
Fig. 4A).
|
|
The Negative Regulator Participates in the Developmental and
Tissue-specific Regulation of
Expression--
While the positive
regulators defined by the 5'-CRE, PU.1/PIP, and E2A motifs play
significant roles in the developmental synergistic regulation of
expression, we also identified a novel negative transcription regulator
in the
E3' core (Fig. 4). Considering that
expression is
up-regulated during B cell maturation, we examined whether this region
might participate in the developmental regulation. We conducted
transfection analysis with the wild-type
E3' core and the
E3'NR
mutation (LSM H) in the B cell lines representing different
developmental stages. The cell lines included the 3-1 and 1-8 pre-B
cell lines, the A-20 B cell line, and the S194 plasma cell line. We
then compared transcription activities conferred by the two constructs
in the same cell lines. We found that the
E3'NR mutation caused a
significantly more dramatic increase (>12-fold) in transcription
activity in 3-1 pre-B cells, and the magnitudes decreased progressively
from 3-1 cells to S194 cells where the increase of transcription
activity was around 2-3-fold (Figs. 4B and
7A). These results suggest
that the negative regulator plays a more prominent role in suppressing
transcription at early B cell stages, thus participating in the
developmental regulation of the locus.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7.
The negative regulator participates in
developmental regulation of expression. Constructs containing
either a negative regulator-mutated 3' enhancer core or a wild-type
core were transfected into model B cell lines presenting different
developmental stages: the 3-1 and 1-8 pre-B cell lines, the A-20 B
lymphoma cell line, and the S194 plasmacytoma cell line. Transcription
activities conferred by the two constructs were compared (the mutant
versus the wild-type) in the same cell lines. The average
luciferase/ -galactosidase values for each wild-type construct
expressed in each cell line are: 0.698 ± 0.071 (3-1), 0.652 ± 0.120 (1-8), 44.984 ± 9.960 (A20), and 44.601 ± 8.100 (S194). The 3-1 and 1-8 cells were treated with LPS for 24 h
before luciferase and -galactosidase assays.
|
|
It is known that
expression is restricted to B cells. Some
cis-acting elements including
E3' sequences have previously been
implicated in this cell type-specific control (29, 31, 32). To
determine whether the identified negative regulator also participates
in the determination of the tissue specificity, we conducted similar
transfection studies in several non-B cell lines. The results from the
Mel cell line and human Jurkat T lymphoma cell line are presented in
Fig. 8, A and B.
They show that mutating the negative regulator significantly activates
reporter expression in both cell lines, suggesting that the negative
regulator may also be involved in determining the tissue-specificity of
expression.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 8.
The negative regulator is involved in the
determination of the tissue specificity of expression.
Transfection analysis with the indicated constructs in Mel and human
Jurkat T lymphoma cell lines.
|
|
Correlation of Transcription Factor Binding Activities with
Transcription Activity during B Cell Development--
We demonstrated
that the activities of both enhancers and the individual
E3' motifs
are up-regulated from early to late B cell stages. This result is
consistent with previous reports on
expression pattern during B
cell development (6). To further investigate whether there is any
correlation between our reporter expression and transcription factor
binding activities of the enhancer sequence motifs, we performed
electrophoresis mobility shift assays to examine the protein complexes
formed at individual enhancer sequence motifs at different B cell
stages. These sequence motifs included the
A,
B, E2, and E3
motifs of the
Ei and the 5'-CRE, PU.1/PIP, and E2A motifs of the
E3' (Fig. 9). Radiolabeled double-stranded oligonucleotide probes
containing individual motifs and nuclear extracts from untreated and
LPS-treated 3-1 pre-B cells, and S194 mature B cells were used. To
quantitatively measure the differences of the complex formation between
different B cell stages, we quantified the shifts of protein-DNA
complexes (normalized to Oct-1 binding) (plots shown in Fig.
9). Interestingly, when comparing factor
binding activities of uninduced pre-B, induced pre-B, and mature B cell
extracts, we consistently found that there was a modest increase in
factor binding to the individual motifs of the
E3' (less than
2-fold) and a significant increase in most of the factors binding to
the
Ei (up to 20-fold). We have made repeated attempts to identify
binding to the
E3'NR and have been unable to identify specific
binding to the region encompassing the
E3'NR defined by the linker
scanner mutations.

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 9.
Transcription factor binding patterns of the
individual enhancer sequence motifs at different B cell stages.
Electrophoretic mobility shift assays were performed with radiolabeled
oligonucleotide probes of the indicated sequence motifs and nuclear
extracts of untreated and LPS-treated 3-1 cells and S194 cells. The
major protein-DNA complexes were indicated by arrowheads for
all tested motifs. The OCTA-1 shifts (upper band) were used
as a loading control. The intensities of the major complexes were
quantified and normalized to the octamer shifts and plotted for each
cell line. When more than one band is indicated, the graphs represent
the sum of the signals from all the bands. The value (1) of the shifts
at all tested motifs in untreated 3-1 cells is an arbitrary
value.
|
|
 |
DISCUSSION |
In most previous in vitro studies of
enhancer
function, the activities of each enhancer have been examined
independently. The linker scanner mutations used in this study were
originally used to identify sequence motifs that impact on the
independent activity of the
E3' driven by a heterologous herpesvirus
thymidine kinase promoter (12). There are a number of conflicting
reports on the role of each enhancer at different stages of B cell
development. We reported that the
Ei appeared to be solely
responsible for early expression, particularly germ line transcription
at the pre-B cell stage (18); however, other reports have indicated the
E3' may be active at very early stages of B cell development as well
(19, 20). In addition, the roles of the enhancers in regulating tissue
specificity of transcription as well as the developmental regulation of
V
J
rearrangements have also been addressed (29, 33-36). In this
current study we attempted to re-address the roles of individual motifs
of the
E3' in coordinating expression with the
Ei and a
representative V
promoter. The use of heterologous promoters and
artificial spatial organizations was avoided, and relevant interactions
of the locus were preserved. The fusion of the luciferase gene in-frame
within the C
region provided a convenient reporter that reflected
reasonable tissue specificity and developmental regulation of the locus
in B cell lines. While the use of transformed lines has limitations,
the clonal responsiveness and enhancer regulation we observe certainly reflect elements that are important for coordinated enhancer regulation of expression. Indeed, transgenic studies of the endogenous
locus
confirm the developmental regulation and synergistic characteristics we
are observing (23). Although targeted knockout of either enhancer in
mice has only a modest effect on B cell development and overall
expression, direct comparison of cellular expression levels from an
intact and mutated allele has not been done. Moreover, selective
pressures in the developing immune response of the mouse can obscure
even major effects on expression levels (34, 35).
It is not surprising that sequence motifs that are important for the
independent activity of the
E3' are important in synergistic activation with the intron enhancer. However, based on the linker scanner mutation analysis, not every sequence motif previously identified to contribute to the enhancer activity is required for the
synergistic activity. In addition, we identified a sequence that
appears to serve as a negative regulator of
expression. The
increase in activity we observed when using LSM H is a little surprising, since this same linker scanner mutation caused a
significant decrease in activity of the enhancer alone when paired with
a thymidine kinase promoter (12). In addition, we see no effect of BSAP
motif (LSMs D and E) on the synergistic activity; whereas mutating this
site had a significant negative effect with the previously reported
TK-
E3' construct (12). In comparing these results it is notable that
our approach differs in that it is examining the mutations in the
context of intron-3' enhancer synergistic activation of the
locus.
We observed that increases in enhancer activity associated with stages
of B cell maturation correlate to increases in relative binding
activity of key transcription factors, particularly those within the
intron enhancer. Changes in factor binding to the
E3' motifs have
been previously noted during the pro-B to pre-B transition (19).
Consistent with our previous results with artificially assembled
constructs, synergy between the enhancers was only observed in mature B
cells. Somewhat surprisingly, we found that even individual sequence
motifs within the
E3' (5'-CRE, PU.1/PIP, E2A) are capable of
synergizing with the
Ei. Because a number of factor interactions have been reported within complexes forming at the
E3' (6, 9, 10,
12), it is certainly possible that factor interactions can occur
between the enhancer elements that result in different effects of
sequence mutations.
The individual enhancer activities appear to be significantly low at
early B cell stages. As a result, the impact of the
E3'NR may be
more significant at these stages of development. Mutation of the
E3'NR has a much more significant effect in pre-B cells than mature
B cells (12-fold versus 2-3-fold). Thus, as shown in Fig.
10, the net expression of the locus is
developmentally regulated by both positive and negative elements, and
activity in mature B cells is less sensitive to the effect of the
negative regulator. With low enhancer activity in early B cells, the
net activity of the locus may be tempered by the NR sequence, whereas the NR sequence has less impact in mature B cells where enhancer activity has increased and synergistic effect is apparent. It appears
that the
E3'NR can also contribute to tissue specificity, as
mutation of this region resulted in reporter activation in non-B cells.
Significantly, the 10-bp sequence encompassed by the
E3'NR linker
scanner mutation is completely conserved between mouse and human (9,
37).

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 10.
Schematic representation of positive and
negative regulation conferred by enhancers at different stages of B
cell development. Magnitudes of plotted values are schematic
representations showing the relative activities of the enhancers and
negative regulator and net activity resulting from coordinated
activities of the enhancers (with synergy restricted to mature B cell
stage).
|
|
Previous reports suggest the PU.1 motif may act as a negative regulator
and may contribute to tissue specificity and developmental timing of
V
-J
rearrangements (29, 30). In our study, we find that mutation
of the PU.1 motif significantly reduces transcription conferred by both
enhancers in mature B cells (Fig. 2), suggesting its role as a positive
regulator at this stage of development. However, the same mutation
appears to increase expression in pre-B cells, suggesting its role as a
negative element early in development. Therefore, it appears that there
is a correlation between regulation of
rearrangement and
transcription by the PU.1 motif. With respect to the
E3'NR we have
not yet determined its impact on rearrangement events. In addition, we
have been unable to identify specific protein-DNA complexes at this
region. We also have compared DNA footprints of CRE, PU.1/PIP, and E2A
within the context of wild-type and mutant
E3'NR sequences and not
found any differences (data not shown). Thus, the mechanism for the
negative effect has not been resolved.
As demonstrated in this study, the developmental regulation of the
expression must be examined in the context of the
locus, because
apparently it is the combined effect of enhancer sequence motifs that
coordinately affect expression. Moreover, since there is strong
evidence that both enhancers affect V
J
rearrangement, the
coordinate regulation conferred by both enhancers is important to consider.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Michael Achitson for
providing us the linker scan mutations of the 3' enhancer core and to
Dr. William Garrard for constructs with the rearranged kappa locus. We
also like to thank members of the Van Ness laboratory for their useful comments.
 |
FOOTNOTES |
*
This work was supported in part by research funds from the
University of Minnesota.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: Cancer Center Research
Bldg., Box 806 UMHC, 425 East River Rd., SE, University of Minnesota,
Minneapolis, MN 55455. Tel.: 612-624-9944; Fax: 612-626-7031; E-mail:
vanne001{at}maroon.tc.umn.edu.
The abbreviations used are:
kb, kilobase(s); CRE, cAMP response element; bp, base pair(s); LPS, lipopolysaccharide; Mel, mouse erythroid leukemia; DTT, dithiothreitol;
E3'NR,
E3'
negative regulator; LSM, linker scan mutation.
2
X. Liu and B. Van Ness, unpublished data.
 |
REFERENCES |
-
Parslow, T.,
and Granner, D.
(1982)
Nature
299,
449-451[Medline]
[Order article via Infotrieve]
-
Queen, C.,
and Baltimore, D.
(1983)
Cell
33,
741-748[Medline]
[Order article via Infotrieve]
-
Emorine, L.,
Kuehl, M.,
Weir, L.,
Leder, P.,
and Max, E. E.
(1983)
Nature
304,
447-449[Medline]
[Order article via Infotrieve]
-
Meyer, K. B.,
and Neuberger, M. S.
(1989)
EMBO J.
8,
1959-1964[Abstract]
-
Meyer, K. B.,
Sharpe, M. J.,
Surani, M. A.,
and Neuberger, M. S.
(1990)
Nucleic Acids Res.
18,
5609-1615[Abstract]
-
Staudt, L. M.,
and Lenardo, M. J.
(1991)
Annu. Rev. Immunol.
9,
373-398[CrossRef][Medline]
[Order article via Infotrieve]
-
Lenardo, M.,
Pierce, J. W.,
and Baltimore, D.
(1987)
Science
236,
1573-1577[Medline]
[Order article via Infotrieve]
-
Nelms, K.,
Hromas, R.,
and Van Ness, B. G.
(1990)
Nucleic Acids Res.
18,
1037-1043[Abstract]
-
Pongubala, J. M. R.,
and Atchison, M. L.
(1991)
Mol. Cell. Biol.
11,
1040-1047[Medline]
[Order article via Infotrieve]
-
Pongubala, J. M. R.,
Nagulapalli, S.,
Klemsz, M. J.,
Mckercher, S. R.,
Maki, R. A.,
and Atchison, M. L.
(1992)
Mol. Cell. Biol.
12,
368-378[Abstract]
-
Pongubala, J. M. R.,
Beveren, C. V.,
Nagulapalli, S.,
Klemsz, M. J.,
Mckercher, S. R.,
Maki, R. A.,
and Atchison, M. L.
(1993)
Science
259,
1622-1625[Medline]
[Order article via Infotrieve]
-
Pongubala, J. M. R.,
and Atchison, M. L.
(1995)
J. Biol. Chem.
270,
10304-10313[Abstract/Free Full Text]
-
Sen, R.,
and Baltimore, D.
(1986)
Cell
46,
705-716[Medline]
[Order article via Infotrieve]
-
Murre, C.,
McCaw, P. S.,
and Baltimore, D.
(1989)
Cell
56,
777-783[Medline]
[Order article via Infotrieve]
-
Henthorn, P.,
Kiledjian, M.,
and Kadesch, T.
(1990)
Science
247,
467-470[Medline]
[Order article via Infotrieve]
-
Eisenbeis, C. F.,
Singh, H.,
and Storb, U.
(1995)
Genes Dev.
9,
1377-1387[Abstract]
-
Nagulapalli, S.,
Pongubala, J. M. R,
and Atchison, M. L.
(1995)
J. Immunol.
155,
4330-4338[Abstract]
-
Fulton, R.,
and Van Ness, B. G.
(1993)
Nucleic Acids Res.
21,
4941-4947[Abstract]
-
Shaffer, A. L.,
Peng, A.,
and Schlissel, M. S.
(1997)
Immunity
6,
131-143[Medline]
[Order article via Infotrieve]
-
Meyer, K. B.,
Teh, Y.-M.,
and Neuberger, M. S.
(1996)
Int. Immunol.
8,
1561-1568[Abstract]
-
Park, K.,
and Atchison, M. L.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
9804-9808[Abstract]
-
Meyer, K. B.,
and Ireland, J.
(1994)
Nucleic Acids Res.
22,
1576-1582[Abstract]
-
Blasquez, V. C.,
Hale, M. A.,
Trevorrow, K. W.,
and Garrard, W. T.
(1992)
J. Biol. Chem.
267,
23888-23893[Abstract/Free Full Text]
-
Fulton, R.,
and Van Ness, B. G.
(1994)
Nucleic Acids Res.
22,
4216-4223[Abstract]
-
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
-
Blasquez, V. C.,
Xu, M.,
Moses, S. C.,
and Garrard, W. T.
(1989)
J. Biol. Chem.
264,
21183-21189[Abstract/Free Full Text]
-
Ho, S. N.,
Hunt, H. D.,
Horton, R. M.,
Pullen, J. K.,
and Pease, L. R.
(1989)
Gene (Amst.)
77,
51-59[CrossRef][Medline]
[Order article via Infotrieve]
-
Dignam, J. D.,
Lebovitz, R. M.,
and Roeder, R. G.
(1983)
Nucleic Acids Res.
11,
1475-1489[Abstract]
-
Hiramatsu, R.,
Akagi, K.,
Matsuka, M.,
Nakamura, H.,
Kingsbury, L.,
David, C.,
Hardy, R. R.,
Yamamura, K.,
and Sakano, H.
(1995)
Cell
83,
1113-1123[CrossRef][Medline]
[Order article via Infotrieve]
-
Hayashi, R.,
Takemori, T.,
Kodama, M.,
Suzuki, M.,
Tsuboi, A.,
Nagawa, F.,
and Sakano, H.
(1997)
J. Immunol.
159,
4145-4149[Abstract]
-
Pierce, J. W.,
Gifford, A. M.,
and Baltimore, D.
(1991)
Mol. Cell. Biol.
11,
1431-1437[Medline]
[Order article via Infotrieve]
-
Saksela, K.,
and Baltimore, D.
(1993)
Mol. Cell. Biol.
13,
3698-3705[Abstract]
-
Takeda, S.,
Zou, Y.-T.,
Bluethman, H.,
Kitamura, D.,
Muller, U.,
and Rajewsky, K.
(1993)
EMBO J.
12,
2329-2336[Abstract]
-
Xu, Y.,
Davidson, L.,
Alt, F. W.,
and Baltimore, D.
(1996)
Immunity
4,
377-385[Medline]
[Order article via Infotrieve]
-
Gorman, J. R.,
van der Stoep, N.,
Monroe, R.,
Dividson, L.,
and Alt, F. W.
(1996)
Immunity
5,
241-252[Medline]
[Order article via Infotrieve]
-
Sleckman, B. P.,
Gorman, J. R.,
and Alt, F. W.
(1996)
Annu. Rev. Immunol.
14,
459-481[CrossRef][Medline]
[Order article via Infotrieve]
-
Judde, J.-G.,
and Max, E. E.
(1992)
Mol. Cell. Biol.
12,
5206-5216[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.