| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Molecular Genetics and Microbiology, Graduate Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA 01655
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RNA, BSAP has been hypothesized to be involved in
regulation of Ab class switch recombination. To directly examine the
effects of BSAP on isotype switching, we use a tetracycline-regulated
expression system to overexpress BSAP in the surface IgM+
I.29µ B cell line, a mouse cell line that can be induced to undergo
class switch recombination. We find that overexpression of BSAP
inhibits switching to IgA in I.29µ cells stimulated with LPS +
TGF-ß1 + nicotinamide, but enhances switching to IgE in
cells stimulated with LPS + IL-4 + nicotinamide. Parallel to its
effects on switching, overexpression of BSAP inhibits germline 
RNA
expression and the transcriptional activity of the germline
promoter, while enhancing activity of the germline promoter.
Proliferation of I.29µ cells is not affected in this system. The
possible mechanisms and significance of the effect of BSAP on isotype
switching are discussed. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. The intervening DNA is looped
out and deleted (1, 2).
Switch recombination to a particular isotype is preceded by
transcription of the corresponding, unrearranged CH gene,
producing what are termed germline transcripts. Transcription initiates
at the I exon located 5' to each S region and continues through the S
region and CH gene. Splicing then removes the S region
sequences and joins the I exon and CH exon. Both expression
of germline transcripts and subsequent switch recombination are
regulated by cytokines in concert with B cell activators (3, 4, 5, 6, 7). For
example, TGF-ß induces transcripts from the unrearranged C gene,
and subsequently directs switching to IgA in LPS-activated mouse B
cells (8, 9, 10), whereas IL-4 induces transcripts from the unrearranged
C
1 and C genes and directs switching to IgG1 and IgE in
LPS-activated B cells (3, 5, 11, 12, 13). Strict correlation between
expression of germline transcripts and subsequent switch recombination
led to the accessibility model of switch recombination, which proposes
that transcription opens the S region (14, 15). More recent data
suggested that the germline transcript itself may be required for
switch recombination (16, 17).
Despite great progress in the field, the molecular mechanism of switch
recombination and the protein factors involved in the process are still
largely unknown. Several proteins, only a few of which are B lineage
specific, have been implicated in the regulation of switch
recombination, which is a B cell-specific event. For example, the
NF-
B/Rel family (18, 19, 20, 21, 22, 23, 24, 25), the E2A gene product E47 (26),
LR1/nucleolin (27), Sµbp-2 (28, 29), Ku protein (30), DNA-dependent
protein kinase (31), poly(ADP-ribose) polymerase (Ref. 32, but see 33 , and BSAP/Pax-5 (34, 35, 36, 37) have all been implicated in switch
recombination, but evidence for their direct involvement in the process
is still missing or controversial (for review, see 6 . In an
ongoing endeavor to identify and delineate potential proteins involved
in switch recombination, we have chosen to analyze the effect of B
cell-specific activator protein (BSAP)/Pax-5 on isotype
switching.
BSAP, the product of the Pax-5 gene, is a member of a
vertebrate multigene family of transcription factors that share the
paired box DNA binding domain and are important regulators of early
development (38, 39). Within the adult animal, BSAP expression is
restricted to the B cell lineage, except for the testis. It is found in
pro-B, pre-B, and mature B cells, but not in terminally differentiated
plasma cells (34, 40, 41). BSAP is essential for the development of B
cells since pax-5 gene knockout mice have no mature B cells
and serum Igs, and B cell development in such mice is arrested at the
pro-B stage (42, 43). BSAP binding sites have been identified in the
regulatory sequences of a number of genes, including those encoding Ig
heavy chain genes (to be detailed below), the Ig-(mb-1)
subunit of B cell Ag receptor (44), the B cell Ag receptor coreceptor
CD19 (45, 46), the -light chain (47, 48, 49), the surrogate light chain
genes VpreB1 and 
5 (49, 50, 51), J chain (52), B cell-specific tyrosine
kinase Blk (53, 54), human X-box-binding protein-1 (55), the
mouse engrailed gene (56), and the tumor suppressor gene p53
(57), although the functional significance of many of these sites in
vivo is unknown (for reviews, see Refs. 58 and 59).
Several lines of evidence suggest that BSAP might be involved in
Ab class switching. First, BSAP binding sites have been found 5' to or
within almost all IgH S regions examined, including Sµ, S1,
S2a, S3, S, and S (34, 60, 61, 62), and mutating the BSAP
binding site in the germline C promoter decreases expression of this
promoter (25, 35, 36). Second, BSAP binding sites exist in the IgH 3'
enhancer (63, 64, 65), mutation of which leads to defective germline RNA
expression and isotype switching (66). Third, B cell proliferation is
required for class switching (67, 68), and overexpression of BSAP in
the CH12.LX B cell line augmented cell proliferation, whereas reducing
BSAP expression in mouse splenic B cells by an antisense oligo was
accompanied by reduced cell proliferation and decreased switching to
IgG1, IgG2a, and IgG3 (37).
In our study, a tetracycline-regulated expression system was used to overexpress BSAP in the mouse surface IgM+ I.29µ B lymphoma cell line. This cell line can be induced to undergo switch recombination from IgM to IgA, or much less frequently to IgE (69). By comparing the switching of cells induced to switch in the absence of tetracycline (BSAP overexpressed) with those induced in the presence of tetracycline (no BSAP overexpression), effects of BSAP on isotype switching could be assayed. Our results demonstrate that overexpression of BSAP inhibits switching to IgA, but enhances switching to IgE, and both effects are mediated, at least in part, through BSAP binding sites in the promoters of germline transcripts.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The pPCRII/BSAP plasmid containing full-length mouse BSAP cDNA
(65) was obtained from M. F. Neurath (National Institutes of
Health, Bethesda, MD). The tetracycline-regulated expression system,
which includes pTet-splice, pTet-TtAk, and pUHC13–3, has been
described (70, 71) and was a gift from P. Shockett and D. G.
Schatz (Yale Medical School, New Haven, CT). Plasmid pBABEpuro (72)
containing the puromycin resistance gene (Puror) was
obtained from R. M. Gerstein (University of Massachusetts Medical
School, Worcester, MA). Plasmid pPGKNeobpA (73) containing the neomycin
resistance gene (Neor) was obtained from A. Bradley (Baylor
College of Medicine, Houston, TX). Reporter plasmids containing the
luciferase gene driven by the germline promoter -130/+14 segment
(74) (M. J. Shi and J. Stavnezer, in preparation) or by the
germline promoter -162/+53 segment (75) have been described. The
internal control plasmid containing the ß-galactosidase gene driven
by PGK promoter, pPGKß-gal (76), was obtained from P. Dobner
(University of Massachusetts Medical School). The pcDNA3 expression
vector was purchased from Invitrogen (San Diego, CA).
Standard molecular cloning techniques were followed in construction of
the following plasmids. pTet-Splice-Neo: A 1.62-kb
HindIII-XhoI fragment containing the
Neor gene driven by the PGK promoter and followed by the
bovine growth hormone polyadenylation signal was isolated from
pPGKNeobpA and cloned into the NotI site of pTet-Splice
after addition of NotI linkers. The resultant plasmid was
designated pTet-Splice-Neo (Fig. 1
). pTet-BSAP-Neo: A 1.2-kb
XhoI fragment containing full-length murine BSAP cDNA was
isolated from pPCRII/BSAP and cloned into the SalI site in
the polylinker region of pTet-Splice-Neo to generate pTet-BSAP-Neo
(Fig. 1). The same BSAP cDNA fragment was also cloned into the
XhoI site of pcDNA3 for use as a template for in vitro
transcription and translation. pTet-TtA-Puro: A 660-bp
HindIII-ClaI fragment containing the
Puror gene was isolated from pBABEpuro and cloned between
HindIII and XhoI sites of pcDNA3 after addition
of a XhoI linker to the blunt-ended ClaI
terminus. A 1.66-kb NruI-PvuII fragment
containing the CMV promoter, Puror gene, and bovine growth
hormone polyadenylation signal was then excised from the resultant
plasmid and cloned into the NotI site of pTet-TtAk after
addition of NotI linkers to produce pTet-TtA-Puro (Fig. 1).
|
22D, a subclone of the mouse B lymphoma cell line I.29µ (3, 69), was cultured at 37°C in an atmosphere of 8% CO2 in RPMI 1640 medium, as described (32).
To derive stably transfected lines, cells were washed three times in and resuspended in RPMI 1640 medium without supplements. A total of 2 x 107 cells in 1 ml vol was mixed with 20 to 50 µg plasmid DNA in a cuvette. Transfection was conducted by electroporation using Cell Zap II (Anderson Electronics, Brookline, MA) set at 1250 µF and 300 V (750 V/cm). Following electroporation, cells were rested at room temperature for 10 min, added to 50 ml complete medium containing tetracycline (1 µg/ml) (Sigma, St. Louis, MO), and cultured for 24 to 44 h. Cells were pelleted, resuspended in fresh medium containing appropriate antibiotics (1 µg/ml tetracycline, 1 µg/ml puromycin, and/or 400 µg/ml G418), and distributed into 96-well plates at 5 x 104 cells/200 µl/well. Selection was conducted for 10 to 20 days. Cells from select clones were sorted for IgM+/IgA- cells by flow cytometry, and after sorting, more than 99.5% cells were IgM+ and less than 0.1% were IgA+. Cells were cultured in the presence of tetracycline and puromycin and/or G418, except when the assays for function were performed, at which time the cells were washed three times in RPMI 1640 medium without supplements to remove tetracycline and selection drug(s), and then cultured with or without tetracycline for the indicated times.
Switching assay
To induce isotype switching, 105 cells/ml were cultured at 1 ml/well in 24-well plates. LPS (50 µg/ml) from Escherichia coli 055:B5 (Sigma), 10 mM nicotinamide (Sigma), and either 2 ng/ml human TGF-ß1 (R&D Systems, Minneapolis, MN) for IgA switching or 12,000 U/ml mouse rIL-4, made in a baculovirus expression system and a gift from W. E. Paul (National Institutes of Health) for IgE switching, were added on day 0. After 24 h, 0.8 ml supernatant was removed from each well and replaced with 1 ml of fresh medium. For IgA switching, TGF-ß1 was added again at day 1 and day 2, and the cells were analyzed at day 3. For IgE switching, rIL-4 was added again at days 1, 3, and 5. At days 3 and 5, cells were resuspended, and 0.8-ml cell suspension was removed and replaced with 0.8 ml of fresh medium with or without LPS (8.3 µg/ml) and 1.6 mM nicotinamide before addition of rIL-4. Cells were analyzed on day 7.
For immunofluorescence analysis by FACS, cells were pelleted and resuspended in 50 µl of PBS containing 1.5% FCS and 0.2% NaN3. For IgA switching analysis, they were stained with affinity-purified goat anti-mouse IgM-FITC and goat anti-mouse IgA-phycoerythrin (Southern Biotechnology, Birmingham, AL), washed, and subsequently fixed with 1.1% paraformaldehyde. For IgE switching analysis, cells were first incubated with rat anti-mouse IgE mAb (LO-ME-3; Serotec, Raleigh, NC) on ice for 30 min, washed three times, and then incubated with a mixture of goat anti-mouse IgM-FITC and F(ab')2 goat anti-rat IgG-phycoerythrin (Jackson ImmunoResearch, West Grove, PA), washed, and fixed as above. Staining withstood treatment with pH 4 acetate buffer (77), ensuring that membrane-bound Igs (mIgs) were not adventitiously bound to FcR. Cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA), and forward and side scatter were set to include live lymphocytes. Data were plotted using WinList 3.0 (Verity Software House, Topsham, ME). Inhibition of switching was calculated as: percentage of inhibition = 100 x (1 - fraction (or %) IgA+ cells without tetracycline/fraction (or %) IgA cells with tetracycline). The enhancement of switching was calculated as: fold = IgE+ cells without tetracycline/IgE+ cells with tetracycline.
Northern blot and Southern blot hybridization
Total RNA was isolated from cultured cell lines using the
Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX),
according to the manufacturer’s protocol. Genomic DNA was isolated
using the proteinase K digestion/phenol extraction method, as
previously described (69). Northern blot hybridization (10) and
Southern blot hybridization (69) were performed as described. The I,
C, and GAPDH probes (10) were described previously. The BSAP probe
is a 1.2-kb XhoI cDNA fragment isolated from pCRII/BSAP, and
the Neo probe is a 1.62-kb XhoI fragment isolated from
pPGKNeobpA. Probes were labeled by random priming. Quantitation of
hybridization was performed using a Fluor-S MultiImager system
(Bio-Rad, Richmond, CA).
Western blot analysis
A total of 3 to 5 µg nuclear extract was mixed with SDS sample buffer in a final volume of 10 µl and boiled for 5 min before loading onto a 10% SDS-polyacrylamide minigel. The samples were electrophoresed at 200 V for 30 to 60 min and transferred onto an Immobilon P membrane (Millipore, Bedford, MA) at 100 V for 1 h. The membranes were blocked for 2 to 4 h in 5% nonfat milk dissolved in PBS containing 0.1% Tween-20. A 1/10,000 dilution of rabbit anti-human BSAP antiserum directed to the DNA binding domain of BSAP (41) (from M. Busslinger, Research Institute of Molecular Pathology, Vienna, Austria) was incubated with blocked membranes at 4°C overnight with continuous shaking. Membranes were washed three times in PBS/0.1% Tween-20, incubated at room temperature in a 1/2000 dilution of a horseradish peroxidase-conjugated goat anti-rabbit IgG Ab (Santa Cruz Biotechnology, Santa Cruz, CA), washed again, and developed using an ECL kit (Amersham, Arlington Heights, IL). Densitometry was performed using a Molecular Dynamics (Sunnyvale, CA) Personal Densitometer SI and analyzed by Bio-Rad MultiAnalyst.
Nuclear extract preparation and in vitro translation
Nuclear extracts from cell lines were prepared using the method of Schreiber et al. (78). Protein concentration was determined by the Bradford assay (Bio-Rad). In vitro translation of BSAP protein was conducted using a TNT T7-coupled reticulocyte lysate system (Promega, Madison, MI), according to the manufacturer’s protocol with the BclI-linearized pcDNA3 plasmid with or without BSAP cDNA insert as template.
Electrophoretic mobility shift assay (EMSA)
The sequences of the top strand of double-stranded oligonucleotide probes used in EMSAs are: CD19, 5'-GAATGGGGCACTGAGGCGTGACCACCGC-3'; IRF-1, 5'-GGAAGCGAAAATGAAATTGACT-3'.
The dsCD19 binding site was produced by mixing complementary
single-strand oligonucleotides at 0.1 µM each in 100 mM NaCl, 10 mM
Tris-HCl, pH 8, and 1 mM EDTA, and incubating at 95°C for 10 min,
followed by incubation at 10°C below the melting temperature
(Tm) for 1 h and slow cooling to
room temperature. The annealed oligonucleotides were purified on
polyacrylamide gels, ethanol precipitated, and dissolved. The dsIRF-1
oligo, containing a binding site for IFN-regulatory factor-1, purchased
from Santa Cruz Biotechnology, was used directly. Oligonucleotides were
end labeled with [-32P]ATP using T4 polynucleotide
kinase. The probes for the I promoter region were generated by PCR
using the germline promoter luciferase reporter plasmid as
template.
DNA-binding reactions were performed in 16 µl vol containing 0.3 to 0.5 ng (30,000–50,000 cpm) probe, 1 to 10 µg nuclear extract, and 4 µg poly(dI-dC) (Pharmacia, Piscataway, NJ). For reactions using in vitro translation products, 0.1 µg poly(dI-dC) was used. The final concentration of NaCl in each reaction was adjusted to 100 mM by adding buffer C (78). The reaction mixtures were incubated at room temperature for 30 min and then loaded onto a 5% native polyacrylamide gel. The gels were electrophoresed in recirculating 0.5x TBE buffer at 140 V for 3 to 4 h. Antiserum (1 µl) or competitors were added to nuclear extracts before other components were added. The probe was always added last.
Transient transfection, luciferase assay, and ß-galactosidase assay
Transient transfections were performed in essentially the same way as for derivation of stably transfected cell lines, except that cells were precultured with or without tetracycline for 2 days before transfection, and 5 x 107 cells were mixed with 50 µg each of the reporter plasmid and pPGKß-gal before electroporation. Cells were then cultured at 1.25 x 106/ml with or without various stimuli for 24 h and assayed for luciferase (79) and ß-galactosidase (80) activity, as described. The ß-galactosidase activity was used as an internal control for variation in transfection efficiency.
[3H]TdR incorporation
Cells were cultured in a final volume of 200 µl under the conditions used for assaying switching for 3 days, and [3H]thymidine (1 µCi) (Amersham) was added to the cultures for the final 6 h. Cells were harvested onto glass fiber filters with an automatic cell harvester, and incorporation of [3H]thymidine was quantitated.
Digestion-circularization PCR (DC-PCR)
DC-PCR was performed as described (81). Briefly, genomic DNA was
isolated from cells after 7 days of culture under conditions for
switching to IgE. DNA was digested with EcoRI and ligated
under dilute conditions (81) to produce circles. After determination of
the effective template concentration (see below), detection of
Sµ-S switch recombination was performed by PCR amplification of a
648-bp fragment with a primer complementary to sequences 5' to Sµ
(5'-GGAGACCAATAATCAGAGGGAAG-3') (81) and a primer derived from
sequences in the C membrane exon region
(5'-GCAGAGCATCCTCACATACA-3') (82). PCR was performed using the
Expand High Fidelity PCR System (Boehringer Mannheim, Indianapolis,
IN), as follows: reaction mixture was heated at 95°C for 10 min
before addition of enzyme (hot start); 5 cycles of 94°C for 45
s, 58°C for 60 s, and 72°C for 90 s were then conducted,
followed by 25 cycles of 94°C for 45 s, 62°C for 60 s,
and 72°C for 90 s; the final incubation was at 72°C for 10
min. A portion (20 µl) of PCR product was analyzed by Southern blot
hybridization with an end-labeled internal oligonucleotide probe
derived from the C membrane exon region
(5'-TAGGTGCGATGCCAGCAC-3') (82). As a control for variation in
amounts of DNA, efficiency of digestion, and ligation, primers from an
EcoRI fragment of the mouse nicotinic acetylcholine receptor
ß subunit (nAChR) (5'-GACTGCTGTGGGTTTCACCCAG-3' and
5'-AGGCGCGCACTGACACCACTAAG-3') (81) were used to amplify a 753-bp
fragment. PCR conditions were the same as for Sµ-S, except that
the annealing temperature was 65°C for the first 5 cycles and 68°C
for another 25 cycles. PCR products were analyzed by Southern blot
hybridization with an end-labeled internal oligonucleotide probe
(5'-CCAGCCCTGTTTGCCTAAGC-3'). The template concentration in the
ligation product of each DNA sample was determined using a competitive
substrate method (81) by titrating varying amounts of p2AO plasmid,
obtained from E. E. Max (Food and Drug Administration, Bethesda,
MD), into a constant amount of ligation products before amplification
of nAChR fragment. Preliminary experiments established that under the
conditions detailed above, the PCR reaction was unsaturated.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To analyze the effect of BSAP on Ab class switching, we
overexpressed BSAP using a tetracycline-regulated expression system
(70) consisting of two plasmids, pTet-TtA-Puro and pTet-BSAP-Neo (Fig. 1
). This approach was chosen because it
eliminates variability due to site of integration and intrinsic
differences among subclones of the I.29µ B cell line (3) (G. Q.,
laboratory observations). Furthermore, the approach of overexpression
seemed feasible because BSAP levels appear to be limiting, as a
heterozygous knockout mouse has a phenotype (42), and constitutive
overexpression of BSAP in two B cell lines was shown to suppress Ig
secretion (83).
We first stably transfected the plasmid encoding the
tetracycline-inhibitable TtAk transactivator, pTet-TtA-Puro, into the
22D subclone of I.29µ. Fewer than 5% of the wells from the
transfection showed colony growth, making it statistically highly
likely that cells from an individual well were derived from a single
clone, and thus, the cells were not further subcloned. The 22 clones
obtained were screened for expression of the TtAk transactivator in the
absence of tetracycline, as assayed by their abilities to induce
luciferase expression from a transiently transfected reporter plasmid
(pUHC13-3) (71). We also screened for their abilities to be induced to
switch to IgA by LPS and TGF-ß1. One clone, designated TtA/22D, was
found to best fulfill these two criteria. As shown in Table I, in the presence of tetracycline,
luciferase activity in this clone is as low as in untransfected 22D
cells, and removing tetracycline induces luciferase activity from the
reporter plasmid by 45- to 67-fold. Switching to IgA in 22D cells or in
TtA/22D cells is not affected by tetracycline (see below).
|
The Western blotting analyses of nuclear extracts from these four
clones (Fig. 2B), cultured in
the presence or absence of tetracycline, demonstrate the overexpression
of BSAP in the four clones, in comparison with 22D cells expressing
only the transactivator (TtA), and 22D/TtA cells transfected with the
empty vector pTet-Splice-Neo (TtA/Splice) and untransfected 22D cells.
Similar levels of BSAP protein are detected in the control clones and
in 22D cells and in the BSAP-transfected clones cultured in the
presence of tetracycline. Presumably, this level represents endogenous
BSAP. Densitometry analysis of this blot indicated that BSAP levels are
induced by two- to fourfold in the TtA/BSAP clones 1–3 in the absence
of tetracycline (see legend). Clone 4 appears to have a greater
induction due to the very low level of BSAP in the presence of
tetracycline, when this same nuclear extract was analyzed in Figure 2C, a 4.5-fold induction was observed. Southern blot
hybridization of genomic DNA isolated from these clones using BSAP and
Neo probes showed a single specific band whose size differed among the
clones, confirming their clonality and indicating their independence
from each other (data not shown).
|
A) showed
that a 2.4-kb band, as expected for BSAP mRNA transcribed from the
transfected plasmid, was detected only in cells transfected with the
BSAP expression plasmid (TtA/BSAP 1–4) cultured in the absence of
tetracycline, but not in the same cells cultured in the presence of
tetracycline, nor in parental 22D/TtA cells, nor in 22D/TtA cells
transfected with the empty vector (TtA/Splice), nor in untransfected
22D cells. Hybridization of the same blot with a GAPDH probe showed
that similar amounts of RNA were loaded in each lane. It should be
noted that endogenous BSAP mRNA is 10 kb in length (83) and barely
detectable under the conditions we used (data not shown). We conclude
that expression of the TtA/BSAP plasmids is highly inducible, but the
effective induction of BSAP relative to endogenous BSAP ranges from 2-
to 4.5-fold in the clones examined.
To determine whether the overexpressed BSAP results in enhanced BSAP
DNA-binding activity, the nuclear extracts used for the Western blots
in Figure 2B were analyzed by EMSA using a double-stranded
oligonucleotide probe derived from the mouse CD19 promoter, which
contains a high affinity BSAP binding site (84). As shown in Figure 2C, a predominant shifted complex, which is induced by the
removal of tetracycline, is detected in the four clones. This complex
comigrates with a complex in 22D cells that was demonstrated to bind
specifically by competition with an unlabeled CD19 probe, but not by an
irrelevant oligo (IRF) (Fig. 2C). The complex was also
inhibited by an anti-BSAP antiserum specific for the DNA binding
domain and previously shown to inhibit DNA binding by BSAP (41), thus
demonstrating that the complex contains BSAP. Thus, the rBSAP expressed
from the transfected plasmid is capable of binding to its recognition
site, and therefore should be functional.
Overexpression of BSAP inhibits switching to IgA
The effect of BSAP overexpression on isotype switching was
assessed by stimulation of the transfected clones with inducers of
class switching in the presence or absence of tetracycline. Cells were
stimulated with LPS, TGF-ß1, and nicotinamide to induce IgA
switching. Nicotinamide was demonstrated previously to increase switch
recombination in I.29µ cells due to its ability to inhibit
poly(ADP-ribose) polymerase (32). The levels of switching were
quantitated by flow-cytometric analysis of mIgM and mIgA. As shown in
Figures 3 and
4, switching to IgA by the four
TtA/BSAP clones is reduced by 50 to 80% when cells are
stimulated in the absence of tetracycline, whereas switching by TtA,
TtA/Splice, and 22D cells is not affected under the same conditions.
These data indicate that inhibition is not due to enhancement of
switching by tetracycline, nor to the expression of the TtAk
transactivator, but rather due to the overexpression of BSAP. The
inhibition is inversely correlated with the concentration of
tetracycline (Fig. 4); therefore, the inhibition increases as the
levels of BSAP increase.
|
|
). Although
[3H]TdR incorporation is slightly reduced in the presence
of LPS, TGF-ß1, and nicotinamide, it is not affected by the presence
or absence of tetracycline. These data indicate that inhibition of
switching to IgA in the transfected clones is not due to the effect of
BSAP on cell proliferation.
|
transcripts and inhibits the germline promoter
The inability of BSAP overexpression to affect cell proliferation
prompted us to search for other possible mechanisms for its inhibitory
effect on IgA switching. Since switching is regulated by induction of
germline transcripts, we tested the effect of BSAP overexpression on
the expression of germline transcripts. As shown in Figure 6, the levels of germline RNA
detected by an I probe on Northern blots in cells treated under the
same conditions used to induce class switching are reduced by 35 to
80% when the TtA/BSAP clones are cultured in the absence of
tetracycline, as compared with clones cultured in the presence of
tetracycline. Germline RNA levels in TtA/Splice, TtA, or 22D cells
show little or no inhibition under the same conditions. Figure 6B shows the quantitation of the I signals, normalizing
with the GAPDH signals obtained from rehybridization of the same blots.
The percentage of inhibition of IgA switching by BSAP overexpression is
similar to, or slightly greater than, the inhibition of germline
transcript levels during induction of class switching in the
transfected clones and is inversely correlated with the levels of BSAP
induced in these clones.
|
transcripts by BSAP, we examined its effect on expression of a
transiently transfected luciferase reporter plasmid driven by the
germline RNA promoter (74). Figure 7
shows that luciferase activity was reduced in the absence of
tetracycline in the TtA/BSAP clones that were tested, but not in
TtA/Splice or 22D cells. The inhibition of luciferase activity was
greater in cells stimulated with LPS plus TGF-ß1 than in unstimulated
cells. These data suggest that inhibition of IgA switching by
overexpressed BSAP results, at least in part, from its inhibitory
effect on the promoter for germline transcripts.
|
promoter
Although BSAP was found previously to bind to two sites located
immediately 5' to the S region, but which are 3' to the I exon
and thus not in the promoter of the reporter construct, inhibition of
the germline promoter activity by overexpressed BSAP suggests that
BSAP might also bind to the germline promoter region. To attempt to
localize the BSAP binding site(s) in the promoter segment (-130 to
+14) in the reporter plasmid, EMSAs were performed using various
fragments of the germline promoter as probes and nuclear extracts
isolated from one TtA/BSAP clone (TtA/BSAP 4). When the promoter
segment (-130/+14) is used as probe, two retarded complexes, which are
enhanced by removing tetracycline from the culture media, are visible
(Fig. 8A). These two complexes
are competed by the CD19 oligo, but not by the IRF-1 oligo, and
inhibited by anti-BSAP antiserum, and hence are BSAP-containing
complexes. Although the predominant lower complex is competed entirely
by the CD19 oligo (at both 10- and 100-fold excess, Fig. 8A
and data not shown), it could only be partially competed by a 100-fold
excess of unlabeled probe (Fig. 8A), suggesting that the
binding to the germline promoter is of very low affinity.
|
To determine whether BSAP could bind to DNA in the absence of other
components present in I.29µ nuclear extracts, we tested whether in
vitro translated BSAP protein would bind to the promoter. In vitro
translated BSAP forms a complex with the -130/+14 segment that can be
competed by the CD19 oligo, but is competed poorly by the unlabeled
probe (Fig. 8B). This complex is inhibited by anti-BSAP
antiserum (data not shown). (The BSAP-containing complex migrates just
slightly faster than a complex that also forms with the product of mock
translation using an empty plasmid, pcDNA3, as template.) Similar to
results obtained using nuclear extracts, the -71/+14 probe forms a
complex with in vitro translated BSAP (upper part of doublet), which is
competed by the CD19 oligo, but only partially by excess unlabeled
probe (Fig. 8B).
Since the -94/-30 segment does not bind the inducible complex (data
not shown), the EMSA data suggest that a binding site for BSAP resides
between -30 and +14. A sequence that partially matches (12 of 15
nucleotides) a consensus BSAP binding site (84, 85) is located at
-29/-13 in the germline promoter (Fig. 8, C and
D).
Overexpression of BSAP enhances activity of the germline
promoter and switching to IgE
BSAP binds to a high affinity binding site in the germline
promoter region, mutation of which reduces the activity of the promoter
in transfection assays (25, 35, 36, 75, 84). However, the effect of
BSAP on switching to IgE has not been directly addressed. We first
tested the effect of overexpression of BSAP on the activity of the
germline promoter using the reporter gene assay. As shown in Figure 9, A and B, the
luciferase activity obtained upon transient transfection of the
reporter plasmid containing the germline promoter into two TtA/BSAP
clones is increased an average of 1.7-fold in cells cultured in the
absence of tetracycline. Mutations in the BSAP binding site
(LSM-25/-15 and LSM-48/-30) abrogate or greatly reduce this
enhancement. Control cells (TtA/Splice and 22D) show no enhancement of
the germline promoter activity in the absence of
tetracycline.
|
A, the percentage of
mIgE+ cells was increased when TtA/BSAP cells were induced
to switch in the absence of tetracycline relative to the presence of
tetracycline. Control cells (TtA/Splice, TtA, and 22D) showed no
increase in mIgE+ cells under the same condition. The
enhancement varied from 3.5- to 9.2-fold, depending on the clones
tested (Fig. 10B). In this experiment, we tested a new
clone, TtA/BSAP 5, which was also inhibited from switching to IgA in
the absence of tetracycline (data not shown). The percentage of
mIgE+ cells was not affected by treating cells with acid
(77), and thus was not due to binding of exogenous IgE to FcR (data not
shown). Time-course experiments indicate that mIgE+ cells
are detectable after 7 days of stimulation, but not after only 3 or 5
days (data not shown).
|
-heavy
chain mRNA in TtA/BSAP clones stimulated in the absence of
tetracycline, but this mRNA was not detectable in control cells
cultured under the same conditions (Fig. 11A).
|
recombination, a DC-PCR (81, 82) analysis of genomic DNA was performed.
This analysis demonstrated that recombination between Sµ and S
segments was enhanced in the TtA/BSAP clones stimulated with IL-4 + LPS
+ nicotinamide and cultured in the absence of tetracycline, relative to
the clones cultured in the presence of tetracycline and to control
cells stimulated in the presence or absence of tetracycline (Fig. 11B). Taken together, these experiments demonstrate that
overexpression of BSAP enhances transcription of the germline
promoter and switch recombination to IgE in 22D cells. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RNA, but enhances germline RNA promoter activities.
The fold increase in BSAP levels correlated quite well with the levels
of inhibition of IgA switching and germline transcripts. Although
the increase observed in activity of the germline promoter was
slightly less than the fold increase in BSAP levels, the effect on
switching to IgE appeared greater. It is possible that BSAP affects
switching at additional levels besides regulating germline transcripts.
BSAP has long been suspected to play a role in switch
recombination due to the presence of BSAP binding sites 5' to and also
within the S regions of almost all IgH genes, its expression within B
cells, and absence from plasma cells, which do not normally undergo
class switch recombination, and due to its previously known effects on
the promoter for germline RNA. The effect of BSAP on class switch
recombination has not been directly examined previously, except by
Wakatsuki et al. (37), who demonstrated that an antisense
oligonucleotide for BSAP mRNA decreased switching to IgG1 in in vitro
cultured mouse spleen B cells. However, since cell proliferation was
also inhibited in their experiments, they were unable to conclude that
BSAP has a direct role in switch recombination. A major difficulty
impeding progress in this direction is that mice lacking BSAP do not
develop B cells (42, 43).
Therefore, we decided to analyze the effects of BSAP on class switching in a B cell line. The I.29µ B cell line was chosen, as it can switch from IgM to IgA or IgE in vitro by stimulation with LPS plus the appropriate cytokines. We encountered much difficulty in our initial efforts to assess the effects of overexpressed BSAP using a constitutive expression vector because different clones had vastly different abilities to be induced to switch, some entirely losing their switching capability, making it impossible to interpret the results. This difficulty was overcome by the use of an inducible expression system, allowing the effect of overexpression of BSAP to be tested in individual clones under well-controlled conditions. We used the tetracycline-regulated expression system developed by Shockett et al. (70) because of its low leakage and its minimal interference in cellular processes.
It has been well established that BSAP is involved in regulation of cell proliferation during B cell development and differentiation (86). Because cell proliferation appears to be required for switch recombination, it might well be one of the mechanisms by which BSAP regulates switch recombination (37). However, this effect of BSAP cannot fully explain its effects on switch recombination, because we found that cell proliferation was not affected by BSAP overexpression in 22D cells, and because BSAP overexpression has opposite effects on class switching to different isotypes, i.e., inhibiting IgA while enhancing IgE switching. The reason overexpressed BSAP does not affect proliferation of I.29µ cells may be because the endogenous level of BSAP may not be limiting for proliferation. I.29µ cells proliferate rapidly, doubling every 16 to 20 h (data not shown). It is also possible that enhanced proliferation by overexpressed BSAP may be masked by an inhibitory effect of the TtA transactivator, which is also induced in these clones by removal of tetracycline. It has been shown that prolonged induction or constitutive expression of TtA protein has cytotoxic effect (70 and references therein). Whatever the reason, our results clearly indicate that the effect of BSAP on switch recombination cannot be accounted for solely by its effects on cell proliferation.
BSAP binds both the germline and promoters and regulates
their activity
The binding site for BSAP in the germline promoter is
centered at approximately -30 nucleotides 5' to the first RNA
initiation site in the mouse germline promoter (35, 75, 84), and
approximately 10 nucleotides 5' to the first RNA initiation site in the
human promoter (36). Both mouse and human sites have good homology
with the consensus BSAP-binding sequence and are of high affinity (35, 36, 84).
The previously mapped BSAP binding sites in the mouse C locus are
located 3' to the I exon (34), and thus are not present in the
germline promoter segment studied in this work. In the current
study, we have provided evidence for a low affinity BSAP binding site
in the -30/+14 germline promoter segment. Visual inspection
identified a sequence resembling a BSAP consensus binding site centered
at -23 relative to the first RNA initiation site. Similar to results
with the germline promoter (35), we detected two BSAP-containing
complexes binding to the germline promoter segment. By analogy with
the -binding complexes, the faster migrating complex may contain
BSAP alone, whereas the upper complex may contain BSAP plus one or more
unknown proteins. Both complexes formed with the germline promoter
are completely competed by an oligo containing a high affinity BSAP
binding site and are inhibited by an antiserum directed against BSAP
DNA binding domain, suggesting they indeed include BSAP. Nevertheless,
the fact that the complexes are only partially competed by a large
excess of the unlabeled germline promoter probe makes it difficult
to definitively localize the binding site(s) for these complexes. Low
affinity binding seems a plausible but possibly oversimplified
explanation for the inability to compete.
Another mechanism by which BSAP may exert its effect on isotype
switching is via its binding to the IgH 3' enhancer. The IgH 3'
enhancer is composed of at least four DNase I hypersensitive (hs)
segments. There are two well-defined BSAP binding sites in the
3'E(hs1,2) segment, and BSAP binding to these two sites is
inhibitory to enhancer activity in transfection assays (63, 65).
Another BSAP binding site is located in the hs4 segment, and its effect
on enhancer activity is not yet known (64). The four segments together
make up a locus control region (87). Insertion of a Neor
gene driven by the PGK promoter into the segment comprising hs1,2 by
homologous recombination has been shown to lead to defective isotype
switching and defective germline RNA expression (66).
Although we have not measured the effect of overexpressed BSAP on the
activity of the IgH 3' enhancer, the smaller effect of BSAP
overexpression on the activity of the germline promoters relative to
its greater effects on class switching suggests that additional
mechanism(s) may be involved. In addition to the possible effect of the
IgH 3' enhancer, it is possible that the two additional BSAP binding
sites located 3' to the I exon may also contribute to effects on IgA
switching (34).
BSAP has both stimulatory and inhibitory effects on transcription
The opposite effects of BSAP overexpression on transcriptional
activities of germline and promoters raise the interesting
question of how this is achieved. This result indicates that the BSAP
binding sites in the two germline promoters function differently. It
has previously been reported that BSAP can activate or inhibit
transcription, depending on the particular promoter or enhancer at
which it binds. The mechanism for these opposite effects is unknown,
although it has been demonstrated that BSAP contains both a
transcriptional stimulatory and inhibitory domain (88). BSAP inhibits
the J chain (52) and p53 promoters (57), but activates the CD19 (45, 46) and blk promoters (53, 54). Recent data suggest that the
context of the binding site determines whether it will activate or
inhibit transcription (89). Consistent with this, the binding of BSAP
recruits the Ets family factors to the Ig-(mb-1)
promoter, resulting in transcriptional activation (44), but appears to
inhibit the IgH 3' (hs1, 2) enhancer activity by competition for
binding with Ets family factors (90) and/or by interaction with Oct
proteins and a protein binding a G-rich sequence (91).
As B cells differentiate to become capable of secreting high levels of
Igs at the plasma cell stage, BSAP levels are down-regulated. Since
germline transcripts are required for class switching, our results
suggest that switching to IgE might occur preferentially in less
differentiated B cells having high levels of BSAP, whereas IgA
switching might occur more readily in highly activated B cells poised
to begin secreting high levels of Ab. IgA switching occurs in Peyer’s
patches in which B cells are highly activated by the Ags and mitogens
transcytosed into the Peyer’s patches from the small intestine. IgE
switching is not known to occur at these sites and presumably occurs
within other secondary lymphoid organs. It is also possible that the
low affinity of the BSAP binding site(s) in the germline promoter
may allow IgA switching in cells expressing levels of BSAP that
stimulate transcription of the germline RNA, due to the higher
affinity of the binding site in the germline promoter. It has been
suggested that sites at which BSAP stimulates transcription are of
higher affinity than the sites at which BSAP inhibits transcription
(89).
Our data suggest that inhibition of BSAP expression in B cells as they are stimulated to undergo class switching should reduce switching to IgE and increase switching to IgA. If one could use this means to shift the balance of class switching from IgE to IgA, this might be useful clinically. Since IgA is secreted at mucosal surfaces and IgE is present on mast cells at mucosal surfaces, one could imagine that an increased production of IgA at mucosal surfaces might be able to compete with IgE for Ag binding, potentially resulting in a reduction of allergic responses.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Janet Stavnezer, Department of Molecular Genetics and Microbiology, Graduate Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA 01655-0122. E-mail address:
3 Abbreviations used in this paper: S region, switch region; BSAP, B cell-specific activator protein; DC-PCR, digestion-circulation PCR; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hs, hypersensitive; IRF, IFN-regulatory factor; LSM, linker-scanning mutation; mIg, membrane-bound Ig; Neor, neomycin resistance gene; Puror, puromycin resistance gene.
Received for publication March 18, 1998. Accepted for publication May 11, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2b transcripts. Mol. Cell. Biol. 8:1849. germline transcripts in the B lymphoma I.29µ: transforming growth factor-ß activates transcription of the unrearranged C gene. J. Immunol. 147:4374.[Abstract]
transcripts in B lymphoid cells. J. Exp. Med. 168:2385.3 DNA-binding proteins. J. Exp. Med. 176:339. tandem repeat. J. Immunol. 151:4718.[Abstract]
B leads to multifocal defects in immune responses. Cell 80:321.[Medline]
B knockout mice have selective defects in proliferation, differentiation, germ-line CH transcription, and Ig class switching. J. Immunol. 156:183.[Abstract]
B/Rel by CD40 engagement induces the mouse germ line immunoglobulin C1 promoter. Mol. Cell. Biol. 16:4591.[Abstract]
germline transcripts in B cells via activation of NF-B: synergy with IL-4 induction. J. Immunol. 158:4769.[Abstract]
-specific DNA binding protein SNAP is related to the helix-loop-helix transcription factor E47. Int. Immunol. 9:1021. heavy chain class switching. Immunity 5:319.[Medline]
tandem repeats is regulated during differentiation. Mol. Cell. Biol. 9:5594. transcription. J. Immunol. 152:2904.[Abstract]
germline promoter. J. Immunol. 158:5874.[Abstract]
3' enhancer. Mol. Cell. Biol. 16:3138.[Abstract]
light chain enhancers in primary pro- and pre-B cells: a model for locus activation. Immunity 6:131.[Medline]
5 promoters as well as the KI and KII sites upstream of the J genes. Eur. J. Immunol. 27:750.[Medline]
5 gene. J. Immunol. 155:2498.[Abstract]
transcript. Mol. Cell. Biol. 11:5551. enhancer at early stages of B-cell differentiation. Mol. Cell. Biol. 13:3611.B-binding proteins. J. Immunol. 156:2828.[Abstract]
enhancer is a target site with repressor function for the B cell lineage-specific transcription factor BSAP (NF-HB, S-BP). J. Immunol. 153:730.[Abstract]
constant region gene by ATF element and by novel transforming growth factor-ß1-responsive elements. J. Immunol. 149:2914.[Abstract]
promoter: regulation by NF-IL-4, a C/EBP family member and NF-B/p50. J. Exp. Med. 181:181.1 heavy chain switch region recombination by a digestion-circularization polymerase chain reaction method. Proc. Natl. Acad. Sci. USA 89:6978. represses germline transcription and subsequently down-regulates switch recombination to . Int. Immunol. 4:515.
enhancer by suppressing binding of NF-P, a protein that controls heavy chain transcription. Proc. Natl. Acad. Sci. USA 92:5336.E(hs1, 2). Proc. Natl. Acad. Sci. USA 93:4392. heavy-chain RNA and its location on polysomes. Mol. Cell. Biol. 10:382.This article has been cited by other articles:
|
|
 |
|
  G. A. Robichaud, J.-P. Perreault, and R. J. Ouellette Development of an isoform-specific gene suppression system: the study of the human Pax-5B transcriptional element Nucleic Acids Res., August 1, 2008; 36(14): 4609 - 4620. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kishida, Y. Hiromura, M. Shin-Ya, H. Asada, H. Kuriyama, M. Sugai, A. Shimizu, Y. Yokota, T. Hama, J. Imanishi, et al. IL-21 Induces Inhibitor of Differentiation 2 and Leads to Complete Abrogation of Anaphylaxis in Mice J. Immunol., December 15, 2007; 179(12): 8554 - 8561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Bradley, D. A. Kaminski, A. H. F. M. Peters, T. Jenuwein, and J. Stavnezer The Histone Methyltransferase Suv39h1 Increases Class Switch Recombination Specifically to IgA J. Immunol., July 15, 2006; 177(2): 1179 - 1188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Pawlitzky, C. V. Angeles, A. M. Siegel, M. L. Stanton, R. Riblet, and P. H. Brodeur Identification of a Candidate Regulatory Element within the 5' Flanking Region of the Mouse Igh Locus Defined by Pro-B Cell-Specific Hypersensitivity Associated with Binding of PU.1, Pax5, and E2A. J. Immunol., June 1, 2006; 176(11): 6839 - 6851. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Oppezzo, G. Dumas, A. I. Lalanne, B. Payelle-Brogard, C. Magnac, O. Pritsch, G. Dighiero, and F. Vuillier Different isoforms of BSAP regulate expression of AID in normal and chronic lymphocytic leukemia B cells Blood, March 15, 2005; 105(6): 2495 - 2503. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Gonda, M. Sugai, Y. Nambu, T. Katakai, Y. Agata, K. J. Mori, Y. Yokota, and A. Shimizu The Balance Between Pax5 and Id2 Activities Is the Key to AID Gene Expression J. Exp. Med., November 3, 2003; 198(9): 1427 - 1437. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Schaffer, E. C. Kim, X. Wu, H. Zan, L. Testoni, S. Salamon, A. Cerutti, and P. Casali Selective Inhibition of Class Switching to IgG and IgE by Recruitment of the HoxC4 and Oct-1 Homeodomain Proteins and Ku70/Ku86 to Newly Identified ATTT cis-Elements J. Biol. Chem., June 13, 2003; 278(25): 23141 - 23150. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
A. Suto, H. Nakajima, K. Hirose, K. Suzuki, S.-i. Kagami, Y. Seto, A. Hoshimoto, Y. Saito, D. C. Foster, and I. Iwamoto Interleukin 21 prevents antigen-induced IgE production by inhibiting germ line Cepsilon transcription of IL-4-stimulated B cells Blood, December 15, 2002; 100(13): 4565 - 4573. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-I Lin, C. Angelin-Duclos, T. C. Kuo, and K. Calame Blimp-1-Dependent Repression of Pax-5 Is Required for Differentiation of B Cells to Immunoglobulin M-Secreting Plasma Cells Mol. Cell. Biol., July 1, 2002; 22(13): 4771 - 4780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. E. Drouin, C. E. Schrader, J. Stavnezer, and U. Hansen The Ubiquitously Expressed DNA-Binding Protein Late SV40 Factor Binds Ig Switch Regions and Represses Class Switching to IgA J. Immunol., March 15, 2002; 168(6): 2847 - 2856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 M.-J. Shi, S.-R. Park, P.-H. Kim, and J. Stavnezer Roles of Ets proteins, NF-{{kappa}}B and nocodazole in regulating induction of transcription of mouse germline Ig {{alpha}} RNA by transforming growth factor-{beta}1 Int. Immunol., June 1, 2001; 13(6): 733 - 746. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Tell, A. Zecca, L. Pellizzari, P. Spessotto, A. Colombatti, M. R. Kelley, G. Damante, and C. Pucillo An environment to nucleus' signaling system operates in B lymphocytes: redox status modulates BSAP/Pax-5 activation through Ref-1 nuclear translocation Nucleic Acids Res., March 1, 2000; 28(5): 1099 - 1105. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Stavnezer, S. P. Bradley, N. Rousseau, T. Pearson, A. Shanmugam, D. J. Waite, P. R. Rogers, and A. L. Kenter Switch Recombination in a Transfected Plasmid Occurs Preferentially in a B Cell Line That Undergoes Switch Recombination of Its Chromosomal Ig Heavy Chain Genes J. Immunol., August 15, 1999; 163(4): 2028 - 2040. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. G. Soro, P. Morales-A., J. A. Martinez-M., S. Morales-A., S. G. Copin, M. A. R. Marcos, and M.-L. Gaspar Differential Involvement of the Transcription Factor Blimp-1 in T Cell-Independent and -Dependent B Cell Differentiation to Plasma Cells J. Immunol., July 15, 1999; 163(2): 611 - 617. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-J. Shi and J. Stavnezer CBF{alpha}3 (AML2) Is Induced by TGF-{beta}1 to Bind and Activate the Mouse Germline Ig {alpha} Promoter J. Immunol., December 15, 1998; 161(12): 6751 - 6760. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
