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*
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10021; and
Department of Biological Sciences, Hunter College and The Graduate Center of the City University of New York, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). Immediately
upstream of each variable region lies a promoter containing the
conserved octamer sequence (5'-ATGCAAAT-3') that is important for B
cell-specific activity of IgH promoters in vitro and in vivo (1, 2). Between the rearranged VDJ segments and the Cµ constant
region gene lies the well-characterized intronic enhancer
(Eµ)3 (1, 2). The intronic enhancer contributes significantly to B
cell-specific activity throughout B cell differentiation and can
function in the absence of other more distal enhancers. The murine
intronic enhancer also possesses a functional octamer element that can
contribute to B cell-specific activity in vitro and in vivo
(3, 4, 5). Finally, downstream of the C
constant region
gene and spread over a distance of >30 kb lie the 3'-IgH enhancer
elements4: HS3A (3A, C3'E)
(6), HS1,2 (1,2, 3'E) (7, 8), HS3B (3B,
HS3), and HS4(4) (9) (reviewed in Ref. 10).
Although early studies suggested that these elements could function
individually as enhancers, few of the elements actually exhibit
significant levels of stimulatory activity when present alone and at a
more distal position from the promoter. Our previous data demonstrate,
in fact, that the 3'-IgH elements must be grouped together to exhibit
significant activity from a distance, indicating that the entire group,
rather than individual elements, contributes normal enhancer function
(11). All of the 3'-IgH enhancer elements possess matches
to the octamer consensus site, but there are currently no data to
indicate whether these putative octamer sites are important for the
activity of the 3'-IgH enhancer elements when they are grouped
together.
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Whereas the factors that act through the IgH enhancers comprise a large
group of ubiquitous and B cell-specific factors, the factors that act
via the IgH promoters are much better characterized. As mentioned
above, the octamer sequence (5'-ATGCAAAT-3') has been shown to be
crucial for B cell-specific activity of the IgH promoter in vitro and
in vivo. The octamer sequence is recognized by the Oct family of
transcription factors, including the ubiquitous Oct-1 and B
cell-restricted Oct-2, both of which interact functionally with the
cofactor OCA-B to generate full IgH promoter function
(14). OCA-B is a B cell-specific transcription coactivator
that was originally identified in B cell nuclear extracts
(15) and subsequently cloned by several laboratories
(16, 17, 18). Although OCA-B has been shown to function from a
promoter position, data regarding the possible function of OCA-B at
more distal enhancer positions are less clear. Thus, although OCA-B is
insufficient, even with Oct-2, to generate strong B cell-specific
transcription activation from an enhancer position in non-B cells
(19, 20), this does not preclude the possibility of a
crucial role for OCA-B in a higher order complex involving Oct-1 or
Oct-2 and some as yet uncharacterized cofactor in enhancer function.
Gene disruption studies in mice have shown that whereas OCA-B is not
necessary for early B cell differentiation, including IgM, IgD, and
light chain synthesis, it is essential for germinal center formation,
for normal rates of B cell proliferation in response to IgM
cross-linking, and for normal expression of secondary heavy chain
isotypes. The 
10-fold decrease in serum isotype levels does not
appear to be due to any defect in class switch recombination, because
switching occurs normally in vitro with B cells derived from knockout
mice (21, 22, 23). Therefore, as suggested by mRNA analysis in
OCA-B-/- B cells (21), the observed reductions in the
switched heavy chain isotype levels in serum could reflect reductions
in the transcription rates at the IgH locus. Given the previously
demonstrated role of OCA-B in IgH promoter function in vitro (15, 16), we sought to determine whether the observed immune defects
in the OCA-B knockout mice involved deficiencies in IgH promoter and/or
enhancer function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells were isolated for transient transfection essentially as described (24). Briefly, freshly isolated murine spleens were isolated and crushed to release cells in serum-free medium. RBC were depleted with lysis buffer (0.16 M NH4Cl, 10 mM potassium bicarbonate, 0.1 mM EDTA). T cells were removed by incubation with anti-Thy1.2 antisera (Sigma, St. Louis, MO) and subsequent treatment with guinea pig complement (Sigma). Macrophages and other adherent cell types were removed by overnight incubation in tissue culture flasks and subsequent decanting of suspension cells. The cells were preactivated by 50 µg/ml LPS (Sigma) for 48 h, and activated B cells were isolated via Percoll gradient centrifugation (25) and transfected using the DEAE-dextran method (Promega, Madison, WI).
Plasmid constructs
VH luc. The IgH promoter, VH, was removed from pGL2 BV (described in 11) using KpnI and BglII, and inserted into the identical sites in pGL3 Basic, a luciferase reporter construct obtained from Promega.
VH luc 3A.
The 1-kb HS3A (C3'E) element was removed from pGL2 BV HS3A
(described in Ref. 11) using BamHI and
SalI and inserted into the same sites in
VH luc.
VH luc 1,2.
The 4-kb HS1,2 (3'E) element was removed from pGL2 BV HS1,2
(described in Ref. 11) with BamHI and
SalI and inserted into the same sites in
VH luc.
VH luc 3B. The 1-kb HS3B element was isolated by high fidelity PCR (26) from pGL2 BV HS3B (described in Ref. 11) and inserted into pCR 2.1 via TOPO-TA cloning according to the manufacturer’s instructions (Invitrogen, San Diego, CA). HS3B was then removed with BglII and SalI and inserted into the identical sites of VH luc.
VH luc 4. The 1.4-kb HS4 element was isolated by high fidelity PCR (26) from pGL2 BV HS4 (described in Ref. 11) and inserted into pCR 2.1 via TOPO-TA cloning according to the manufacturer’s instructions (Invitrogen). HS4 was then removed with BglII and SalI and inserted into the identical sites of VH luc.
VH luc 3B 4. HS3B and HS4 were isolated by high fidelity PCR (26) from pGL2 BV HS34 (described in Ref. 11) and inserted into pCR 2.1 via TOPO-TA cloning according to the manufacturer’s instructions (Invitrogen). HS3B and HS4 were then removed with BglII and SalI and inserted into the identical sites of VH luc.
3A 1,2 3B 4. HS3A was cloned by high fidelity PCR (26) and inserted into pCR 2.1 (Invitrogen) according to the manufacturer’s instructions. pGL2 BV HS1,2 (described in Ref. 11) was digested with BamHI and SalI and inserted into the identical sites in pCR 2.1 3A. The paired elements HS3A and HS1,2 were removed by BglII and SalI digestion and inserted into VH luc 3B 4 between the BglII and XhoI sites, creating an intermediate vector possessing the group of 3'-IgH enhancer elements in proper relative order and orientation with respect to the IgH genomic locus.
VH luc 3A 1,2 3B 4. The unit of all the 3'-IgH enhancer elements was removed as a group from the intermediate '3A 1,2 3B 4' construct using BglII and SalI and inserted into VH luc digested with BamHI and SalI.
VH luc Eµ. The 1-kb Eµ enhancer was removed from pGL2 BV Eµ (described in Ref. 11) with BamHI and SalI and inserted into the same sites in VH luc.
LBK luc.
The LBK minimal promoter was removed from LBK
44CAT (described in
Refs. 27 and 28), using BamHI and
HindIII, and inserted into the identical sites in pGL3
Basic. This construct was used as the base for the following
constructs.
LBK luc Eµ. The Eµ enhancer was removed from pGL2 BV Eµ (described in Ref. 11) with BamHI and SalI and inserted into the same sites in LBK luc.
LBK luc 3A 1,2 3B 4. The entire unit was removed with BglII and SalI digestion from the intermediate '3A 1,2 3B 4' construct and inserted into the LBK luc construct digested with BamHI and SalI.
Cell lines
The pre-B cell lines 18-8 and 18-81, and the B cell line M12.4.1 have been described previously (11). The B cell lines A20, Namalwa, and Raji; the plasma cell line P3X63Ag8; and the fibroblastic cell line NIH/3T3 were obtained from American Type Cell Culture Collection (Manassas, VA). The pre-B and B cell lines were grown in RPMI 1640 (Life Technologies, Gaithersburg, MD) 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.
Transient transfections
Cells were transiently transfected as follows: primary B, pre-B and B cells with the DEAE-dextran method (24, 29), plasma cells with Lipofectin (Life Technologies) (30), and fibroblasts with Superfect (Qiagen, Chatsworth, CA). In experiments in which samples were further treated, the inducer(s) was added to the medium immediately after transfection and left until harvesting of the samples. The amounts added to the medium are as follows: IL-4 (Sigma), 5 ng/ml; Ab to CD40 (anti-CD40) (PharMingen), 10 µg/ml; goat anti-mouse IgM (F(ab')2) (Sigma), 15 µg/ml.
Transfection harvesting and reporter gene assays
Cells were harvested using 1x passive lysis buffer after 48 h (Promega). Luciferase assays were conducted using the Promega dual-luciferase assay system.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We cotransfected cell lines representative of various stages in B
cell differentiation with newly constructed reporter vectors,
diagrammed schematically in Fig. 1
B, with a modified luciferase
reporter gene (Promega) and bearing either the VH
promoter alone, the VH promoter in combination
with the intronic enhancer Eµ, or the VH
promoter combined with the entire group of 3'-IgH enhancer elements. In
these constructs, the enhancers are positioned relative to one another
such that they more accurately reflect their organization in the
genome. The results from several independent transfections of each cell
line are presented in Fig. 2. Data are
presented as fold activation of the enhancer-containing constructs
relative to the enhancerless construct.
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The 3'-IgH elements exhibit synergistic activity in primary B cells
Another important feature of the 3'-IgH enhancer elements is
their synergistic activity when combined with one another
(11). We tested whether this synergy is also observed in
primary B cells using newly constructed reporter vectors with a
modified luciferase reporter gene (Promega) and bearing the
VH promoter alone, the VH
promoter in combination with the intronic enhancer Eµ, the
VH promoter combined with one or two of the
3'-IgH enhancer elements, or the VH promoter
combined with the entire group of 3'-IgH enhancer elements (Fig. 1B). The results of several transfections, presented as fold
activation relative to the enhancerless construct, are shown in Fig. 3. Consistent with our previous results
in transformed cell lines, the 3'-IgH enhancer elements, with the
exception of HS4, show little function in primary B cells when assayed
alone. However, when assayed together, the 3'-IgH enhancer elements
show significant synergistic activity. Thus, pairing either HS3A
(C3'E) with HS1,2 (3'E) or HS3B (HS3) with HS4 generates levels
of activity equal to or greater than the activity of the construct
bearing the intronic enhancer. A construct containing all of the 3'-IgH
enhancer elements together exhibits activities much greater than the
activities observed with Eµ alone or with the pairwise combinations
of 3'-IgH enhancer elements. Along with our previous data, these
observations indicate that the IgH enhancer elements function similarly
in both primary and transformed cells, indicating the potential
physiological significance of the synergistic activity of the combined
3'-IgH enhancer elements.
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The OCA-B knockout mice are defective in both germinal center formation and expression of class switched isotypes, despite the fact that class switch recombination itself appears to be normal (21, 22, 23). OCA-B levels can be increased in primary B cells by treatment with IL-4 and anti-CD40 (31). There is also evidence that anti-CD40 stimulation can activate HS1,2 when assayed independently of the other elements and from a proximal position (32). We therefore examined the effects of IL-4 and anti-CD40 treatment on IgH enhancer function in wild-type and OCA-B-/- primary B cells.
To examine whether the effects of IL-4 and anti-CD40 stimulation
are promoter dependent, we constructed a separate set of reporters
using a minimal promoter (designated LBK) derived from the
liver/bone/kidney alkaline phosphatase gene, and previously shown to
exhibit low level constitutive function in a variety of cell types
(Refs. 27 and 28 ; Fig. 1B). The
minimal LBK promoter does not itself possess an octamer element and, as
expected, does not respond to OCA-B in cotransfection experiments (data
not shown).
Primary B cells of each genotype were isolated and transiently
transfected in duplicate with constructs bearing either the minimal LBK
or the VH promoter alone or the minimal LBK or
VH promoter in combination either with Eµ or
with the entire group of 3'-IgH enhancer elements. One set of the
samples was then treated with IL-4 and anti-CD40. The results from
several independent experiments are found in Fig. 4, expressed as relative light units.
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CD40 treatment (Fig. 4A). The
activity of the VH promoter construct is
up-regulated by IL-4 and anti-CD40 stimulation in wild-type primary
B cells, whereas the activity of the minimal LBK promoter construct
appears to be unaffected (Fig. 4A). In
OCA-B-/- primary B cells, the minimal LBK
promoter activity remains unaffected by IL-4 and anti-CD40
treatment, whereas the function of the VH
promoter is again induced by IL-4 and anti-CD40 treatment, albeit
at lower overall levels (Fig. 4A).
As shown in Fig. 4B, the intronic enhancer significantly
stimulates activity of both the VH and the
minimal LBK promoter constructs in both uninduced wild-type and
OCA-B-/- primary B cells, indicating that
intronic enhancer function itself is relatively unaffected in the
absence of OCA-B (Note the change in scale from the promoter only
assays in Fig. 4A.). Furthermore, in both wild-type and
OCA-B-/- primary B cells, treatment with IL-4
and anti-CD40 moderately stimulates both the Eµ-containing
VH and the Eµ-containing minimal promoter
constructs.
As shown in Fig. 4C, the VH and the
minimal LBK promoter constructs bearing the combined 3'-IgH enhancer
elements exhibit significantly higher activity than the reporters
containing the promoters alone in uninduced primary B cells of either
genotype (Fig. 4A), indicating that the function of the
combined 3'-IgH enhancer elements is not impaired by the lack of OCA-B.
However, although the 3'-IgH enhancer elements are significantly
stimulated by IL-4 and anti-CD40 treatment in wild-type primary B
cells, this same treatment has no effect on function of the combined
3'-IgH enhancer elements in OCA-B-/- cells
(Fig. 4C). This latter result contrasts with the results
obtained with the intronic enhancer-containing constructs (Fig. 4B). The lack of stimulation of the combined 3'-IgH enhancer
elements in OCA-B-/- primary B cells is
independent of the nature of the promoter and thus appears to be
intrinsic to the 3'-IgH enhancer elements themselves.
Surface IgM cross-linking fails to repress intronic or 3'-IgH elements in OCA-B-/- primary B cells
Another defect observed in the OCA-B-/- B
cells is a reduced ability to proliferate in vitro in response to
surface Ig cross-linking (21). Previous studies have
indicated that surface Ig cross-linking can repress intronic enhancer
function, but there are conflicting data regarding the effects on the
3'-IgH enhancer elements (33, 34, 35, 36). We examined the effects
of surface Ig cross-linking on IgH enhancer activity by transiently
transfecting both wild-type and OCA-B-/-
primary B cells with the above-described constructs. For each group of
experiments, two sets of constructs were transfected; and one set was
induced by addition of anti-IgM (µ chain) to the medium. The
results of several independent experiments are presented in Fig. 5. In wild-type cells, the promoter-only
constructs are unaffected by surface Ig cross-linking (Fig. 5A), whereas both the Eµ and 3'-IgH enhancer
element-containing constructs are significantly repressed by this
treatment (Fig. 5, B and C). In
OCA-B-/- cells, the promoters are again
unaffected by surface Ig cross-linking; however, unlike the wild-type
primary B cells, neither Eµ nor the combined 3'-IgH enhancer elements
are repressed. These data show that both Eµ and the group of 3'-IgH
enhancer elements are strongly repressed by surface Ig cross-linking,
in agreement with previous reports (34, 35, 36). More
importantly, in these studies, the observed repression of both Eµ and
the 3'-IgH enhancer elements is totally absent in the
OCA-B-/- primary B cells, regardless of the
promoter used.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Until recently, studies of the 3'-IgH enhancer elements have focused on their individual roles in activating transcription of reporter genes from promoter-proximal positions, in transfection and transgenic studies (reviewed in Ref. 10). Our previously published data, as well as the results described here, have demonstrated that these types of studies may give an incomplete view of 3'-IgH enhancer element function (11). Thus, when assayed independently in single copies positioned at a distance greater than 1 kb from a promoter, the 3'-IgH enhancer elements function weakly. In contrast, grouping the 3'-IgH enhancer elements together creates a significant synergistic activity that is both distance and orientation independent, more consistent with the definition of enhancer activity. This may be a more accurate reflection of 3'-IgH enhancer element function in vivo, because the 3'-IgH enhancer elements are all present in the IgH locus at a distance of >200 kb from the promoter after VDJ recombination. The activity of the combined 3'-IgH enhancer elements is shown here, and in previously published studies (11), to vary during B cell development; the entire group of 3'-IgH enhancer elements are inactive in pre B cells but strongly active at the B cell and plasma cell stages. Consistent with these earlier data from studies with transfected cell lines, the current studies show that all of the 3'-IgH enhancer elements other than HS4 are weak in primary B cells when assayed independently (in single copies) and at a distance from the promoter. When grouped together, the 3'-IgH enhancer elements show significant synergistic activity, such that the entire group of 3'-IgH enhancer elements stimulate promoter activity to a much greater level than the intronic enhancer, Eµ. These results demonstrate that the strong synergistic activity of the 3'-IgH enhancer elements is generated in primary B cells as well as in transformed cell lines and indicate the potential physiological relevance of the synergistic activation by the 3'-IgH enhancer elements.
IL-4 and anti-CD40 stimulation of the 3'-IgH enhancer elements is affected in OCA-B-/- cells
B cell-Th cell interactions play an important role in germinal center formation and in expression of class-switched heavy chain isotypes in vivo, and both processes are greatly affected in OCA-B-/- mice (21, 22, 23). A number of studies have suggested a preferential role for the 3'-IgH enhancer elements in the expression of IgH genes of the class-switched isotypes (37, 38, 39, 40, 41, 42). These observations led us to examine whether there is a link between OCA-B and 3'-IgH enhancer element function. IL-4 and anti-CD40 stimulation of primary B cells, a treatment that mimics B cell-Th cell interactions, has previously been shown to up-regulate OCA-B expression (31).
In agreement with previous studies, treatment with IL-4 and
anti-CD40 was found to enhance the function of all the IgH
transcription control elements in wild-type primary B cells transfected
with corresponding reporters: the VH promoter
alone; the VH promoter in combination with the
intronic enhancer; and the VH promoter combined
with the entire group of 3'-IgH enhancer elements (Fig. 4). The
stimulation of enhancer function is not promoter specific because
similar results were observed when these elements were assayed in
combination with a minimal LBK promoter that is unresponsive to IL-4
and anti-CD40 treatment in wild-type B cells. Therefore, all the
IgH transcription control elements appear to be responsive to IL-4 and
anti-CD40. In contrast with the results obtained with wild-type
cells, in primary OCA-B-/- B cells, IL-4 and
anti-CD40 failed to enhance the function of the combined 3'-IgH
enhancer elements when linked either to the minimal LBK promoter or the
VH promoter (Fig. 4). These data indicate (1)
that the inability of IL-4 and anti-CD40 treatment to stimulate
3'-IgH enhancer element function is not simply due to reduction of
VH promoter function in the absence of OCA-B and
(2) that the factor(s) responsible for the stimulation of 3'-IgH
enhancer element function are missing or inactive in
OCA-B-/- B cells. Although previous data have
suggested that OCA-B, either with or without Oct-2, is insufficient to
activate transcription from an enhancer position (19, 20),
it is important to note that these experiments involved cotransfections
of non-B cells and thus do not eliminate the possibility of an OCA-B
requirement for another B cell-specific component(s) to activate
transcription from a distal position. Therefore, the observed defects
in 3'-IgH enhancer element stimulation by IL-4 and anti-CD40 in
OCA-B-/- primary B cells could reflect the lack
of an OCA-B-dependent activation mechanism that directly involves one
or more of the 3'-IgH enhancer elements. Alternately, these data are
consistent with the possibility that OCA-B plays a role in the
expression and/or activation of another factor(s) necessary for IL-4
and anti-CD40 induction of enhancer activity. This could involve
either increased expression of a responsive transcription factor(s),
decreased expression of a repressor(s), or even an effect on
intermediate factors in cell signaling. These possibilities are
currently under investigation.
The observed defects in IL-4- and anti-CD40-induced enhancement of 3'-IgH enhancer element function may help explain the reduced expression of class-switched isotypes in OCA-B-/- mice. If indeed the 3'-IgH enhancer elements are more critical for expression of the class-switched isotypes, whereas Eµ is more important for IgM and IgD expression, as previously suggested (10), then defects in 3'-IgH enhancer element stimulation would preferentially affect expression of the class-switched isotypes.
Surface Ig cross-linking represses Eµ and 3'-IgH elements in wild-type but not OCA-B-/- primary B cells
Surface Ig cross-linking induces a variety of effects on B cells, one of which is an enhanced B cell proliferation that is reduced in OCA-B-/- mice (21). Several studies have demonstrated that surface Ig cross-linking reduces IgH transcription and intronic enhancer activity (34, 35, 36). We chose to examine the effects of surface Ig cross-linking on the activity of the combined IgH transcription control elements in transiently transfected primary B cells of both wild-type and OCA-B knockout mice.
Both the VH and minimal LBK promoters were unaffected by surface Ig cross-linking in wild-type B cells. Thus, all the observed effects are intrinsic to the enhancers themselves. The intronic enhancer and combined 3'-IgH enhancer elements were significantly repressed in wild-type primary B cells on surface Ig cross-linking, whether linked to the VH promoter or to the minimal LBK promoter. These results support previously published data indicating that IgH transcription is reduced by surface Ig cross-linking (34, 35, 36). Other previous data suggested that surface Ig cross-linking could induce activity of a subregion of the HS1,2 element (32, 33). The differences between these earlier observations and our present studies may be due to differences in the types of constructs used, or, more likely, the use of an isolated piece of the HS1,2 element instead of the entire group of 3'-IgH enhancer elements.
As expected, surface Ig cross-linking has no effect on either the VH promoter or the minimal LBK promoter in OCA-B-/- primary B cells. In strong contrast to the wild-type cells, neither the intronic enhancer nor the combined 3'-IgH enhancer elements are repressed in OCA-B knockout B cells. Unlike IL-4 and anti-CD40-induced activity, the defect in surface Ig-mediated repression is not specific to the 3'-IgH enhancer elements. One possibility is that OCA-B is normally a target for repression by surface Ig cross-linking, such that the signaling pathways are no longer able to reduce IgH enhancer function in cells that lack OCA-B. Another possibility is that OCA-B controls one or more of the intermediates in the signaling pathway(s) utilized by surface Ig cross-linking, and thus the signals from the B cell receptor fail to be propagated within the cell.
These results demonstrate an important role for the B cell-specific cofactor OCA-B in activation and repression of IgH enhancer activity and, therefore, IgH expression. Subsequent to the submission of this article, data published by Tang and Sharp (43) support the notion of OCA-B involvement in 3'-IgH enhancer function. Taken together, all of the data help to explain some of the immune defects observed in OCA-B knockout mice, indicating the importance of OCA-B in lymphocyte function in vivo.
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Acknowledgments |
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Footnotes |
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2 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.
3 Abbreviations used in this paper: Eµ, IgH intronic enhancer; 3A, HS3A (C3'E); 1,2, HS1,2 (3'E); 3B, HS3B (HS3); 4, HS4; luc, luciferase reporter gene; VH, IgH promoter, LBK, liver/bone/kidney alkaline phosphatase minimal promoter; anti-CD40, Ab to CD40.
4 For the sake of clarity and convenience, in this article we refer to the transcriptional control regions found 3' of the C constant region gene collectively as the 3'-IgH enhancer elements. Each of the 3'-IgH enhancer elements is a complex region of DNA and consists of many putative transcription factor binding sites.
Received for publication December 28, 1999. Accepted for publication March 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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constant region. Mol. Cell. Biol. 13:1547.
bacteriophage templates. Proc. Natl. Acad. Sci. USA 91:2216.This article has been cited by other articles:
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 A. I. Kuraishy, S. W. French, M. Sherman, M. Herling, D. Jones, R. Wall, and M. A. Teitell TORC2 regulates germinal center repression of the TCL1 oncoprotein to promote B cell development and inhibit transformation PNAS, June 12, 2007; 104(24): 10175 - 10180. [Abstract] [Full Text] [PDF]
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N. W. Kin and V. M. Sanders CD86 Stimulation on a B Cell Activates the Phosphatidylinositol 3-Kinase/Akt and Phospholipase C{gamma}2/Protein Kinase C{alpha}beta Signaling Pathways. J. Immunol., June 1, 2006; 176(11): 6727 - 6735. [Abstract] [Full Text] [PDF]
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M. A. Sepulveda, A. V. Emelyanov, and B. K. Birshtein NF-{kappa}B and Oct-2 Synergize to Activate the Human 3' Igh hs4 Enhancer in B Cells J. Immunol., January 15, 2004; 172(2): 1054 - 1064. [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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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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J. R. Podojil and V. M. Sanders Selective Regulation of Mature IgG1 Transcription by CD86 and {beta}2-Adrenergic Receptor Stimulation J. Immunol., May 15, 2003; 170(10): 5143 - 5151. [Abstract] [Full Text] [PDF]
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 K. Zhang Accessibility control and machinery of immunoglobulin class switch recombination J. Leukoc. Biol., March 1, 2003; 73(3): 323 - 332. [Abstract] [Full Text] [PDF]
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 D. Tantin and P. A. Sharp Mouse Lymphoid Cell Line Selected To Have High Immunoglobulin Promoter Activity Mol. Cell. Biol., March 1, 2002; 22(5): 1460 - 1473. [Abstract] [Full Text] [PDF]
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J. Laurencikiene, V. Deveikaite, and E. Severinson HS1,2 Enhancer Regulation of Germline {epsilon} and {gamma}2b Promoters in Murine B Lymphocytes: Evidence for Specific Promoter-Enhancer Interactions J. Immunol., September 15, 2001; 167(6): 3257 - 3265. [Abstract] [Full Text] [PDF]
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 X. Shi and L. A. Eckhardt Deletional analyses reveal an essential role for the hs3b/hs4 IgH 3' enhancer pair in an Ig-secreting but not an earlier-stage B cell line Int. Immunol., August 1, 2001; 13(8): 1003 - 1012. [Abstract] [Full Text] [PDF]
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J. Hess, P. J. Nielsen, K.-D. Fischer, H. Bujard, and T. Wirth The B Lymphocyte-Specific Coactivator BOB.1/OBF.1 Is Required at Multiple Stages of B-Cell Development Mol. Cell. Biol., March 1, 2001; 21(5): 1531 - 1539. [Abstract] [Full Text]
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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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,
uninduced activity; 
, samples treated with IL-4 (5 ng/ml media) and
anti-CD40 (10 µg/ml media).