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*
Immunobiology and Cancer Research Program, Oklahoma Medical Research Foundation, and
Department of Microbiology and Immunology, Oklahoma University Health Sciences Center, Oklahoma City, OK 73104
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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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Several transcription factors have been implicated as playing important roles in late-stage B cell differentiation. Knockout mice containing disruptions of Oct-2 suggested that Oct-2 expression is not only required for postnatal survival but also for B cell maturation (1). Likewise, mice that do not express the coactivator protein OCA-B/Bob-1/OBF-1 (2, 3) also show defects in late stage B cell differentiation. However, the precise stages of differentiation that are affected in these mice as well as the functional importance of these proteins in Ig expression during those stages is not clear. These proteins are thought to exert their effects, at least in part, through binding to the intronic heavy chain enhancer sequences.
The B cell regulator of Ig heavy chain transcription (Bright)3 is another DNA-binding protein that interacts with the murine Ig heavy chain enhancer and may play an important role in Ig expression at late stages of differentiation. It was first identified as an inducible protein complex in a mature, transfected B cell line after stimulation through the IgR and IL-5R (4). This 70-kDa protein, which is thought to bind to A+T-rich sequences as a tetramer, represents a new family of DNA-binding proteins (5). The yeast SWI1 and Drosophila dri proteins share regions of high homology with Bright (5, 6). Although the function of D. dri is unknown (6), SWI1 is part of a larger protein complex that affects the transcription of some genes by altering chromatin structure (7, 8, 9). The first Bright binding sites observed were found 5' of the S107 family V1 heavy chain promoter, where Bright binding correlated with two- to sixfold increases in µ heavy chain mRNA levels (4). Transfectants in which the Bright binding sites had been deleted from the V1 gene did not exhibit increased V1-specific heavy chain mRNA levels in response to IL-5 and Ag (10), implying a role for Bright in heavy chain expression. Subsequently, cotransfection studies also suggested that Bright may function as a transcription factor when bound to intronic enhancer sequences (5).
Preliminary analyses of murine tissues suggested that Bright might be expressed in a B lymphocyte-specific fashion, while analyses of transformed B cell lines representative of varying stages of differentiation suggested that Bright expression was limited to the mature B cell stage (Ref. 5 and our unpublished observations). Bright activity was expressed in some immature and mature B cell lines and in a few plasma cells at very low levels, but it was not expressed in pre-B, T, or nonlymphoid cell lines. However, little is known about the expression of Bright in normal B cell counterparts. In fact, Bright expression was not detected in the RNA from normal spleen, a B lymphocyte-rich tissue (5). Determination of the expression pattern of Bright during normal B lymphocyte differentiation could lead to important new insights into the function(s) of this protein and its role in Ig gene expression. Therefore, we have analyzed normal murine tissues for the presence of Bright protein. In addition, we have used RT-PCR analyses to determine the mRNA expression pattern of Bright in isolated normal B lymphocytes from varying stages of differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both male and female BALB/c mice that were 2- to 6-mo-old were used for the adult spleen and bone marrow (BM) analyses. For B cell activation, adult spleen cells were teased into single-cell suspensions, washed with PBS, and resuspended at 106 cells/ml in RPMI 1640 with 20% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 x 10-5 M 2-ME. LPS from Escherichia coli 0111:1B4 (Sigma, St. Louis, MO) was used at a concentration of 10 µg/ml, and both LPS-stimulated and unstimulated controls were cultured for 3 days as previously described (11) before preparation of nuclear extracts. Uncultured fresh tissues were used for cell-sorting experiments. As a source of enriched thymic progenitors, 16 male recombination-activating gene (RAG)-2-/- mice (12) were used that had been bred and maintained in our laboratory animal resource center. Fetal tissues were pooled from 15 to 40 fetuses that were obtained at days 11, 12, 14, 15, 17, and 19 of gestation from either BALB/c or C57BL/6 mice. EL-4 is a murine T cell line that does not express Bright but does express the NFµNR complex that binds to Bright-binding sites in non-B cells (5). BCg3R-1d is a transfectant of the BCL1B1 murine lymphoma line and is the first cell line in which Bright activity was demonstrated (4, 10).
Nuclear extract preparation and electrophoretic mobility shift assays (EMSAs)
Tissues and cell lines were homogenized in hypotonic lysis
buffer with the protease inhibitors PMSF and leupeptin as previously
described (13) by the method of Dignam et al. (14). Protein
concentrations were quantitated using Bradford Reagents (Bio-Rad
Laboratories, Richmond, CA) and ranged in concentration from 2 to 10
µg/µl. EMSAs were performed in 4% nondenaturing gels as described
previously (13), using a [
-32P]bf150 DNA fragment
from the V1 heavy chain locus that contains the prototype
Bright-binding site (10). Competition assays were performed by
preincubating extracts for 5 min at room temperature with 100, 10, or 1
molar excesses of unlabeled ds oligonucleotides before the addition of
labeled bf150 fragment. The 46-base pair (bp) sequence containing the
Bright-binding site from -515 to -476 of the V1 promoter was used as
a specific competitor, while a ds oligonucleotide containing the
octamer and heptamer sequences of the native BCL1 promoter was used as
an unrelated competitor (10). Ab supershifting experiments were
performed by preincubating extracts for 5 min at room temperature with
1 to 2 µl of goat preimmune or anti-p29 mitogen-activated protein
peptide antisera that were prepared by Ferrell Farms (Oklahoma City,
OK). The mitogen-activated protein peptide containing the topoisomerase
II-conserved peptide P29 (SNYDDDEKKVTGGRN), which we had previously
shown elicited Abs that were cross-reactive with topoisomerase II and
Bright (15), was produced by Dr. K. Jackson (Oklahoma Center for
Molecular Medicine, Oklahoma City, OK). The Ab supershifted both in
vitro-translated Bright and the native Bright complex from BCg3R-1d but
did not react with octamer proteins or the NFµNR non-B cell complex
that also binds to the bf150 Bright site. The plasmid FLBrCMV-pBK (5)
containing Bright cDNA and polyclonal rabbit anti-Bright sera were
the kind gifts of Dr. P. Tucker (University of Texas, Austin, TX). The
TNT Rabbit Reticulocyte Lysate System (Promega, Madison, WI) was used
according to the manufacturer’s directions to produce in
vitro-translated Bright.
FACS and Abs
BM cells from adult BALB/c mice were harvested and then enriched for lymphoid cells by incubating cell suspensions with Gr-1, Ter119 (PharMingen, San Diego, CA), and Mac-1 (10x concentrated culture supernatant, hybridoma purchased from American Type Culture Collection, Rockville, MD) mAbs and subsequently washing them with staining buffer (PBS without Ca2+/Mg2+ supplemented with 3% heat-inactivated FBS). BM cells were then incubated with goat anti-rat IgG-coated magnetic beads (PerSeptive Diagnostics, Framingham, MA) followed by magnetic separation to deplete myeloid and erythroid lineage cells. The lymphoid-enriched cells were incubated with the appropriate combinations of Abs that allowed the identification of specific populations of B lineage precursors as defined by Hardy et al. (16). Fraction A cells were sorted as CD45R+ (B220+), CD43+, CD24/HSA-, and surface IgM- (sIgM-). Fraction B cells were sorted as B220+, CD43+, CD24/HSA+, BP-1-, and sIgM-. Fraction C cells were sorted as B220+, CD43+, BP-1+, and sIgM-. Fraction D cells were sorted as B220+, CD43-, BP-1+/-, and sIgM-. Fractions E and F were B220+, CD43-, IgM+, IgD- and B220+, CD43-, IgM+, and IgD+ cells, respectively. Adult spleens were teased into single-cell suspensions and stained for 15 min on ice. In some cases, IgM- cells were enriched before sorting by depletion over anti-IgM-coated beads. T lymphocytes were identified with anti-CD3, monocytes were identified with anti-Mac-1, and B lymphocytes were identified with anti-IgM and anti-CD19. Anti-CD24/HSA, anti-IgM, and anti-IgD were used to identify immature B lymphocytes, while peanut agglutinin (PNA)-FITC, anti-IgD, and anti-CD19 were used to identify populations enriched for germinal center B lymphocytes. We passed 16 adult male RAG-2-/- thymuses through a wire mesh screen to produce single-cell suspensions. The thymuses were subsequently pooled, washed, and stained with anti-CD19/FITC, anti-CD8/APC, and anti-CD4/phycoerythrin (PE) (all from PharMingen, San Diego, CA). Two adult BALB/c thymuses were treated similarly.
The following Abs were used: CD45R/APC (6B2) or CD45R/PE (14.8 mAb), biotin-labeled CD43 (S7), biotin-labeled or CD24 (M1/69) PE, biotin-labeled or BP-1 FITC, and biotin-labeled anti-CD19 (all from PharMingen). Anti-Mac-1 FITC (Boehringer Mannheim, Indianapolis, IN), anti-CD3 biotin (Life Technologies, Grand Island, NY), and PNA-FITC (Vector Labs, Burlingame, CA) were also used. IgM FITC polyclonal antiserum and anti-IgD-PE were purchased from Southern Biotechnology (Birmingham, AL). Biotinylated reagents were revealed by a second incubation with Ultra-avidin Texas Red (Leinco, Mallwin, MO). The isotype-matched controls used were: rat IgG2a-biotin, goat Ig dichlorotriazinyl aminofluorescein, goat IgG-PE, rat IgG2b-PE, and rat IgG2b-biotin. Four-color sorting was performed using a FACStarPlus (Becton Dickinson) with the assistance of the Flow Cytometry and Cell Sorting Laboratory, Oklahoma Center for Molecular Medicine, University of Oklahoma Health Sciences Center. Postsort analyses typically yielded >97% purity.
RT-PCR analyses
Total RNA from tissues and sorted cell populations was prepared using TriReagent (MRC, Cincinnati, OH) according to the manufacturer’s instructions. Fetal liver cells from each stage of gestation were depleted of TER119+ erythroid cells (17) using magnetic anti-Ig-coated beads (PerSeptive Diagnostics) to enrich for lymphocyte precursors before RNA extraction. The remaining fetal tissues were used without depletion. Synthesis of cDNA was performed at 42°C for 1.5 h with avian myeloblastosis virus reverse transcriptase in a 20-µl volume in the presence of 15 U of RNAsin. After heat inactivation at 68°C for 15 min, 3 µl of cDNA was PCR amplified by annealing at 61°C for 45 s, extending at 72°C for 1 min, and denaturing at 93°C for 45 s for 40 cycles for nonabundant Bright transcripts. ß-actin primers were used as a measure of the relative RNA quantities; 25 cycles of amplification were determined to be within the linear range of amplification of the abundant actin mRNA for samples containing >10,000 cell equivalents of RNA. Bright primer pairs spanning at least one intron were used to assess Bright expression. The forward primers used were BR669 5'-GGAAGAGCAAGAGCTGGAAG-3' and BR998 5'-GGAAAGAGTTCCTGGATGAC-3'. The reverse primers used were BR1781R 5'-CTCTCAGAGGCTTGGCTGTT-3' and BR1830R 5'-CTGTTGCTCCGGTTGGCAC-3'. The oligonucleotides are named according to the nucleotide numbering of the cDNA from Herrscher et al. (5). Because our data suggested that alternative splice variants of Bright might exist, each sample was subjected to PCR analysis with both primer pairs (BR669 and BR1781R, 1.1-kilobase (kb) product; BR998 and BR1830R, 850-bp product). In most instances, data are shown for only one primer pair.
Samples were electrophoresed through 1.2% agarose gels and transferred to nitrocellulose for Southern blotting. A 2.2-kb XhoI fragment from the FLBrCMV-pBK plasmid containing a full-length coding sequence for Bright (5) was labeled with 32P by random priming and used as a probe. PCR products from fetal tissues and the BCg3R-1d cell line were isolated from ethidium bromide-stained gels, reamplified for an additional 15 to 20 cycles, excised from agarose gels, and then subcloned into the pGEM-T vector (Promega) to confirm the identity of the putative Bright products by sequence analyses. Plasmid minipreps were prepared using Wizard kits (Promega), and dideoxy sequencing was performed with a Sequenase Version 2.0 kit (USB, Cleveland, OH).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a means of learning more about possible functions of
Bright in Ig expression in nontransformed cells, we asked whether
spleen cells could be induced to express Bright using the polyclonal
mitogen LPS, as we had shown for the BCg3R-1d-transfected cell line
(10). We also wanted to determine whether Bright activity might be
present in other tissues that contained large numbers of B lineage
cells, such as BM and fetal liver. Nuclear extracts from these tissues
were used in EMSAs and compared with the Bright-binding activity
observed in the BCg3R-1d B cell line (Fig. 1
). Unstimulated spleen cells did not
contain detectable Bright-binding activity, although they did contain a
mobility-shifted species that migrated faster than Bright. On the other
hand, spleen cells stimulated with LPS for 1 to 3 days contained a
mobility-shifted species that migrated identically to Bright. In
addition, unstimulated adult BM and fetal liver from day 16 of
gestation also contained mobility-shifted species that migrated
similarly to Bright.
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), neither stimulated nor
unstimulated spleen cells contained a species that migrated with this
mobility, despite the presence of large numbers of normal T cells in
this tissue. Nor did whole fetal liver or adult BM exhibit large
quantities of NFµNR activity, even though both contained large
numbers of nonlymphoid cells. A faint higher mobility band was detected
in fetal liver extracts that might be attributed to NFµNR binding,
although its mobility is slightly higher than NFµNR.
To determine whether the protein complexes formed from normal tissues
contained Bright, we used polyclonal goat Abs in supershift experiments
with nuclear extracts from each tissue. Figure 1C shows that
the anti-Bright Ab reacted with the Bright motif-binding complex
from both LPS-stimulated spleen and fetal liver, while the preimmune
serum had no effect on the binding of those complexes. On the other
hand, little or no reactivity was observed with the Bright-site binding
activity from adult BM in three separate experiments.
To confirm that the mobility-shifted species observed in fetal liver
and stimulated spleen extracts interacted directly with the
Bright-binding site, we asked whether oligonucleotides containing only
the footprinted Bright motif (10) could compete for the binding
activity. The putative Bright complex from LPS-stimulated spleen was
totally competed away by the Bright oligonucleotides but was not
affected by the same molar excess of an unrelated oligonucleotide (Fig. 2A). Likewise, the
Bright-sized, mobility-shifted species from fetal liver was also
competed away with unlabeled Bright oligonucleotides. However, a higher
mobility-shifted species from the same extract was not affected (Fig. 2B). Similar studies with BM extracts also showed
competition of the putative Bright complex with the Bright motif (data
not shown). These data suggest that both LPS-stimulated spleen and
fetal liver contain Bright, but that BM may contain proteins
serologically different from Bright that also interact with the
Bright-binding site.
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Because Bright was expressed in LPS-activated spleen cells and in tissues that contain lymphocyte progenitors but was not expressed in unstimulated spleen, we asked whether Bright expression was limited to discrete stages of B cell differentiation. RNase protection studies (5) suggested that Bright mRNA was not an abundant transcript even in cell lines. Furthermore, the absolute numbers of B cell progenitors in the BM and fetal liver and activated mature B cells in normal spleen are relatively small. Therefore, we developed an RT-PCR assay to determine whether Bright mRNA was expressed within discrete subpopulations of B cells. Primers were designed that spanned 1.1 kb of the 2.2-kb cDNA to ensure spanning at least one exon-intron junction.
Adult spleen cells were separated into monocytic
(Mac-1+), T cell (CD3+), and B lymphocytic
(B220+, IgM+, CD3-) populations
using flow cytometry. B220+, CD3-, and
IgM- cells were also isolated to represent memory or
isotype-switched B cells. Additional fractionation included the most
immature splenic B lymphocytes (HSAhigh,
IgMhigh, IgDlow) (19) and an enriched
population of mature germinal center B lymphocytes (IgDlow,
CD19+, PNAhigh) (20, 21) (Fig. 3, A and B).
Bright mRNA was not expressed uniformly in all splenic B cells (Fig. 3C) and could not be detected at all in 10,000 total
spleen cells under the conditions used. However, RNA from the same
number of sorted IgM+ B cells did contain Bright mRNA.
Expression was much more abundant in B220+,
CD3- cells that had been depleted of IgM+
cells, suggesting that samples enriched for B cells expressing other Ig
isotypes and/or the more mature memory cells have increased Bright
levels. Alternatively, Bright may also be expressed in NK cells which
might also be found within this population. Bright expression was not
detected in CD3+ T lymphocytes or Mac-1+
monocyte lineage cells (Fig. 3C). Although these data
were not quantitative, they suggested that some populations of splenic
B cells might not express Bright or might express very low levels of
Bright mRNA. Indeed, the HSAhigh, IgM+
population of relatively immature B cells that had just arrived from
the BM (19) did not contain detectable Bright. Bright mRNA was abundant
in CD19+ cells enriched for PNAhigh expression
that may represent germinal center B cells (21) (Fig. 3D). These findings suggest that Bright expression
occurs predominantly in a small population of normal splenic B cells
that may represent final stages of differentiation.
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); however, adult BM also contains B lineage cells at earlier
stages of differentiation. To determine whether Bright could be
detected in BM B lineage cells in a stage-specific manner, we examined
precursor populations (Fig. 4) for Bright
transcripts by RT-PCR. Adult BM was also depleted of mature B
lymphocytes and fractionated into early B lineage precursors that are
CD45R+, CD43+ (pro-B cells). These cells were
further fractionated into three populations by the differential
expression of CD24 and BP-1 Ags according to Hardy et al. (16) as
previously described (22). Although Bright mRNA was not detected in the
very earliest fractions of pro-B cells (Fraction B), it was easily
detected in small pre-B cells (Fraction D) from adult BM, and was
present at low levels in large pre-B cells (Fraction C) (Fig. 4).
Consistent with the immature B cells found in the spleen, Fraction E
immature BM B cells did not express detectable Bright mRNA. However,
Fraction F cells, which contained the more mature and recirculating B
lymphocytes, did express Bright mRNA (Fig. 4). Thus, Bright mRNA
expression occurs in B cell progenitors, is down-regulated in the
immature B cell stage, and is up-regulated again at later stages of B
lymphocyte differentiation.
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The fetal liver is another site of hemopoiesis, and our mobility
shift studies indicated that Bright expression also occurs there. Early
on during gestation, fetal liver should not contain activated B
lymphocytes that are similar to those that might be found in adult BM.
RT-PCR analyses of total RNA obtained from fetal liver cells enriched
for lymphocyte progenitors indicated that full-length Bright was
expressed as early as day 12 of gestation (Fig. 5A). It was abundant at
day 19 and was still present in neonatal liver by day 2 after birth
(data not shown). However, adult liver that no longer produced B
lymphocytes did not contain detectable levels of Bright mRNA (Fig. 5A). Thus, Bright expression also occurs in fetal
lymphocytes at about the time that the rearrangement of Ig heavy chain
genes is first evident (23).
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, A and B) confirmed the existence
of multiple PCR products, suggesting that the products result from
alternatively spliced transcripts rather than PCR artifacts.
Embryonic tissues often express genes that exhibit more limited
patterns of expression in the adult. Indeed, D. dri, which
is the protein most closely related to Bright to date, is expressed
ubiquitously in all nuclei until after gastrulation, at which point its
expression becomes restricted to discrete tissues (6). Therefore, we
asked whether Bright expression might also occur in fetal tissues other
than the liver. Bright-hybridizing PCR products occurred in the thymus
and brain but not in the kidney (Fig. 5B).
Smaller-sized products were observed in the heart and lung. Thus, while
Bright was not expressed uniformly in all day 16 embryonic tissues,
expression did occur in multiple organs. In addition, fetal tissues
contained several amplification products of varying sizes that
hybridized with Bright cDNA. While some of the amplified products could
result from related genes, cloning and sequencing suggest the existence
of splice variants in fetal liver. Additional experiments will be
required to isolate full-length transcripts that are representative of
each of the observed species.
Bright is not expressed by T cells in the adult thymus
In contrast to our RT-PCR analyses of splenic T cells, the
expression of Bright mRNA in fetal thymus led us to ask whether Bright
might also be expressed in adult thymus. Indeed, EMSAs of extracts
obtained from adult thymus showed a Bright motif-binding complex of
just slightly faster mobility than in vitro-translated Bright (Fig. 6). This complex was also competed away
with an oligo containing the Bright-binding site but did not shift with
our anti-Bright Abs or with Abs prepared against the complete
Bright protein (data not shown). Nor did the complex react with
antisera against CDP/Cux (data not shown), a component of the NFµNR
complex that was observed in T cell lines (R. Scheuermann, and our
unpublished observations). Thus, the adult thymus produced a protein
complex that interacted with Bright motifs, but it is unlikely that
this complex contains Bright.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a means of gaining insight into the function of Bright, we defined which stages of B lymphocyte development express Bright. Several important observations have resulted from these studies. First, Bright mRNA expression occurs in a developmentally regulated fashion at two distinct stages of B lymphocyte development, the pre-B cell and activated mature B lymphocyte. In addition, Bright expression occurred early during ontogeny in fetal hemopoietic tissues. Furthermore, these experiments present the first evidence suggesting the existence of alternatively spliced transcripts of Bright mRNA. Finally, a novel mobility-shifted species was identified from adult thymus extracts that bound to Bright DNA-sequence motifs. This complex, although similar in size to Bright, did not react with any anti-Bright Abs and could represent a novel T cell-specific protein that interacts with Bright motifs. Interestingly, NFµNR, the mobility-shifted species commonly observed in extracts from monocytic and T cell lines, was not present in normal spleen or thymic extracts. Collectively, these data suggest that Bright activity is tightly regulated.
These studies analyzed normal tissues for Bright mRNA by RT-PCR and for
Bright DNA-binding activity by EMSA. Although it was not technically
feasible to perform EMSAs with small numbers of sorted subpopulations
of B lymphocytes, the RNA and mobility-shift data were generally in
agreement. While Bright-binding activity was not detected in extracts
from unstimulated spleen cells, it was present in LPS-activated spleen
cells. This observation is consistent with the finding that the more
mature, activated spleen cell populations expressed Bright mRNA even in
unstimulated spleens. Likewise, while we did not observe identifiable
Bright DNA-binding activity in extracts from whole BM, the fraction of
pre-B cells in which Bright transcripts were detected represents only
10% of total BM. Thus, it is possible that the EMSA was not
sensitive enough to detect a supershifting complex in that case.
Alternatively, it is also possible that the presence of Bright mRNA or
protein is not sufficient to produce Bright DNA-binding activity.
Our studies confirm earlier findings suggesting that Bright is not
required for the maintenance of Ig expression (10). Indeed, Bright
expression was down-regulated in immature B cells in both the BM and
spleen, but these cells express Ig on their surfaces. Although Bright
expression occurs in many cell populations that are activated and
presumably dividing, it is also expressed relatively abundantly in
nondividing, small pre-B cells (Fig. 4). Although Bright may have a
different function when expressed in the early lymphocyte progenitors
of the BM, fetal liver, and thymus compared with when it is expressed
at later stages of B cell differentiation, one might speculate that it
could be involved in signaling pathways through the pre-B and mature B
cell receptors. Immature B cells that do not express Bright are often
not responsive to signaling through the IgR. It is not known whether
different splice variants dominate at individual stages.
Earlier studies failed to detect Bright expression in normal adult
tissues other than testes (5). However, a related DNA-binding protein,
D. dri, was expressed ubiquitously in the early stages of
Drosophila development before becoming localized to specific
organs in the adult. Similarly, PCR products of the expected size for
Bright were observed in fetal brain, heart, lung, thymus, and liver
(Fig. 5). Bright mRNA has not been observed in any of these tissues in
the adult (Ref. 5 and our unpublished observations) except at very low
levels in CD4low cells from adult thymus. While we cannot
explain this expression pattern, it is possible that Bright or related
proteins might be required early during development in nonhemopoietic
tissues.
Our data also suggest that alternatively spliced forms of Bright may exist in several tissues, although we have not yet confirmed their existence by cDNA library screening. Sequence analyses of our RT-PCR products suggested that some of these alternatively spliced products lack the putative activation domain. Because Bright is thought to bind DNA as a tetramer (5), alternatively spliced products might participate in the tetramerization process and could act as negative regulators of Bright function. Several transcription factors have been shown to be expressed as alternatively spliced transcripts, and the resulting protein products can act as dominant negative regulators in some cases (31, 32, 33, 34). Studies are underway to isolate full-length cDNAs that are representative of each of these splice variants for further characterization.
Although EMSAs (Fig. 6) showed prominent DNA-binding complexes of
approximately the same size as Bright in the adult thymus, these
complexes did not interact with polyclonal anti-Bright Abs, and we
were not able to show any evidence that Bright mRNA was expressed in
mature T cell populations. Thus, thymocytes produce a fairly abundant
protein with similar binding specificities to Bright. This protein was
not detected in normal splenic extracts, in which a large proportion of
the proteins was derived from T cells. In fact, we were surprised that
the NFµNR mobility-shifted species found in nuclear extracts from a
broad panel of T cell and non-B lineage cell lines (5, 18) was not
detected in unstimulated splenic extracts. It has been speculated that
NFµNR acts as a negative repressor of the intronic heavy chain
enhancer in non-B cells, so that the Ig locus is not rearranged by
RAG-1 and RAG-2 when they are expressed in T cells (15). These data
emphasize the need for the examination of the expression of proteins in
normal tissues as well as in cell lines.
Many new transcription factors have been described in the last few years, and some of them appear to be lymphocyte-specific (35, 36). There is evidence that some of these factors are absolutely required for the progression of lymphocyte differentiation. Although some of them bind directly to the Ig heavy chain locus, others do not, and the relevant binding sites that give rise to phenotypic blocks in B cell differentiation are unknown. While Bright binds directly to regions within the Ig heavy chain locus that have been suggested to play important roles in the expression of that locus (30, 37, 38), it is also possible that it interacts with other B cell-specific genes as well. Understanding how Bright functions may ultimately lead to a better understanding of how B cell differentiation occurs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Carol F. Webb, Oklahoma Medical Research Foundation, Immunobiology and Cancer Research Program, 825 N.E. 13th Street, Oklahoma City, OK 73104. E-mail address:
3 Abbreviations used in this paper: Bright, B cell regulator of Ig heavy chain transcription; BM, bone marrow; EMSA, electrophoretic mobility shift assay; RAG, recombinase-activating gene; bp, base pair; kb, kilobase; sIgM, surface IgM; MARs, matrix-attachment regions; HSA, heat stable antigen; PNA, peanut agglutinin; PE, phycoerythrin.
Received for publication September 16, 1997. Accepted for publication January 16, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 antibodies on immunoglobulin isotype expression in lipopolysaccharide-stimulated cultures of mouse spleen cells. Eur. J. Immunol. 13:556.[Medline]
) is a transdominant negative inhibitor of C/EBP family transcriptional activators. Nucleic Acids Res. 23:4371.This article has been cited by other articles:
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  J. C. Nixon, S. Ferrell, C. Miner, A. L. Oldham, U. Hochgeschwender, and C. F. Webb Transgenic Mice Expressing Dominant-Negative Bright Exhibit Defects in B1 B Cells J. Immunol., November 15, 2008; 181(10): 6913 - 6922. [Abstract] [Full Text] [PDF]
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 D. Kim, L. Probst, C. Das, and P. W. Tucker REKLES Is an ARID3-restricted Multifunctional Domain J. Biol. Chem., May 25, 2007; 282(21): 15768 - 15777. [Abstract] [Full Text] [PDF]
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M. Shankar, J. C. Nixon, S. Maier, J. Workman, A. D. Farris, and C. F. Webb Anti-Nuclear Antibody Production and Autoimmunity in Transgenic Mice That Overexpress the Transcription Factor Bright J. Immunol., March 1, 2007; 178(5): 2996 - 3006. [Abstract] [Full Text] [PDF]
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C. M. Johnston, A. L. Wood, D. J. Bolland, and A. E. Corcoran Complete Sequence Assembly and Characterization of the C57BL/6 Mouse Ig Heavy Chain V Region J. Immunol., April 1, 2006; 176(7): 4221 - 4234. [Abstract] [Full Text] [PDF]
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 J. Rajaiya, M. Hatfield, J. C. Nixon, D. J. Rawlings, and C. F. Webb Bruton's Tyrosine Kinase Regulates Immunoglobulin Promoter Activation in Association with the Transcription Factor Bright Mol. Cell. Biol., March 15, 2005; 25(6): 2073 - 2084. [Abstract] [Full Text] [PDF]
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J. C. Nixon, J. Rajaiya, and C. F. Webb Mutations in the DNA-binding Domain of the Transcription Factor Bright Act as Dominant Negative Proteins and Interfere with Immunoglobulin Transactivation J. Biol. Chem., December 10, 2004; 279(50): 52465 - 52472. [Abstract] [Full Text] [PDF]
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 K. Ma, K. Araki, S. J.A. Ichwan, T. Suganuma, M. Tamamori-Adachi, and M.-A. Ikeda E2FBP1/DRIL1, an AT-Rich Interaction Domain-Family Transcription Factor, Is Regulated by p53 Mol. Cancer Res., April 1, 2003; 1(6): 438 - 444. [Abstract] [Full Text] [PDF]
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P. Goebel, A. Montalbano, N. Ayers, E. Kompfner, L. Dickinson, C. F. Webb, and A. J. Feeney High Frequency of Matrix Attachment Regions and Cut-Like Protein x/CCAAT-Displacement Protein and B Cell Regulator of IgH Transcription Binding Sites Flanking Ig V Region Genes J. Immunol., September 1, 2002; 169(5): 2477 - 2487. [Abstract] [Full Text] [PDF]
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 D. Wilsker, A. Patsialou, P. B. Dallas, and E. Moran ARID Proteins: A Diverse Family of DNA Binding Proteins Implicated in the Control of Cell Growth, Differentiation, and Development Cell Growth Differ., March 1, 2002; 13(3): 95 - 106. [Abstract] [Full Text] [PDF]
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C. F. Webb, Y. Yamashita, N. Ayers, S. Evetts, Y. Paulin, M. E. Conley, and E. A. Smith The Transcription Factor Bright Associates with Bruton's Tyrosine Kinase, the Defective Protein in Immunodeficiency Disease J. Immunol., December 15, 2000; 165(12): 6956 - 6965. [Abstract] [Full Text] [PDF]
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 C. WEBB, R.-T. ZONG, D. LIN, Z. WANG, M. KAPLAN, Y. PAULIN, E. SMITH, L. PROBST, J. BRYANT, A. GOLDSTEIN, et al. Differential Regulation of Immunoglobulin Gene Transcription via Nuclear Matrix-associated Regions Cold Spring Harb Symp Quant Biol, January 1, 1999; 64(0): 109 - 118. [Abstract] [PDF]
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M. H. Kaplan, R.-T. Zong, R. F. Herrscher, R. H. Scheuermann, and P. W. Tucker Transcriptional Activation by a Matrix Associating Region-binding Protein. CONTEXTUAL REQUIREMENTS FOR THE FUNCTION OF BRIGHT J. Biol. Chem., June 8, 2001; 276(24): 21325 - 21330. [Abstract] [Full Text] [PDF]
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