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The Journal of Immunology, 1998, 161: 4760-4767.
Copyright © 1998 by The American Association of Immunologists

Regulation of Mouse CD72 Gene Expression During B Lymphocyte Development1

Han Ying*, James I. Healy{dagger}, Christopher C. Goodnow{dagger} and Jane R. Parnes2,*

* Division of Immunology and Rheumatology, Department of Medicine, and {dagger} Howard Hughes Medical Institute, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD72 is a 45-kDa transmembrane glycoprotein that is predominantly expressed on cells of the B lineage except plasma cells. Previously, we identified the 255-bp minimal mouse CD72 promoter capable of tissue-specific and developmental stage-specific expression. DNase I footprinting analysis of the 255-bp CD72 promoter revealed three protected elements, footprint (FP) I, FP II, and FP III. FP II, which extends from nucleotide -189 to -169 of the mouse CD72 promoter, exhibited both tissue-specific and developmental stage-specific activity that was reflective of the activity of the CD72 gene in vivo. In this report, we show that FP II is specifically recognized by the transcription factor B cell-specific activator protein (BSAP). Mutations eliminating the binding of BSAP in reporter constructs also eliminated the increase of reporter activity in B cells. In addition, cotransfections with reporter constructs plus different amounts of expression plasmids for BSAP showed that CD72 promoter activity was up-regulated by BSAP in plasmacytoma cells and T cells in a dose-dependent manner. Moreover, the expression level of CD72 decreased 10-fold on normal plasma cells. Compared with the presence of BSAP binding in mature B cells, the binding of BSAP was undetectable in those plasma cells. This study strongly suggests that BSAP-FP II interaction plays a critical role in determining the cell-type specificity of the CD72 promoter. The absence of positive factors such as BSAP accounts for at least part of the loss of mouse CD72 expression in plasma cells and thus might be common for the down-regulation of many molecules at the plasma cell stage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The development of B lymphocytes is a multistage process. This process, like any other developmental process, involves an ordered, sequential induction and extinction of specific sets of structural and regulatory gene products that are primarily controlled by a combination of tissue-specific and ubiquitous transcription factors. Therefore, regulation of cell type-specific gene expression is a central issue in B lymphopoiesis. Interestingly, there are many proteins that are expressed at all stages of B cell development, except terminally differentiated, Ab-producing plasma cells. These proteins include surface proteins such as Ig-{alpha} (1), Ig-ß (2), CD72 (3), CD40 (4), CD19 (5), CD20 (6), and CD24 (7); cytoplasmic proteins such as the tyrosine kinases Btk (8) and Blk (9); and nuclear proteins such as B cell-specific activator protein (BSAP)3 (10) and early B cell factor (11). Many studies have been performed to elucidate the roles of these proteins in B cell development and function, but our understanding of the mechanisms involved in regulating their tissue specificity and developmental stage specificity is rudimentary.

CD72 is among those proteins that are expressed on cells of the B lineage, except plasma cells (3, 12, 13, 14, 15, 16). Studies have shown that Abs specific for human or mouse CD72 can enhance the B cell proliferation induced by anti-IgM or Ag (17), induce the proliferation of B cells and synergize with IL-4 in the induction of Ag-specific B cells (18), partially rescue splenic B cells from the apoptosis induced by hypercross-linking of the B cell receptor (19), enhance MHC class II expression on activated B cells (20, 21), induce the mobilization of small amounts cytoplasmic-free calcium (21), and induce an increase in the metabolism of phosphatidylinositol in purified small splenic B cells (22). In addition, anti-CD72 mAb inhibits the production of IgG1 but not IgG2b or IgG3 in mouse splenic B cells cultured with LPS and IL-4 (23). Recently, CD72-/- mice were generated by targeted mutation (C. Pan and J.R.P., manuscript in preparation). Preliminary characterization of the mutant mice demonstrated that there is a significant decrease in the total number of B cells in the spleen and lymph nodes. These studies suggest that proper expression of the CD72 gene is essential for B cell development and function.

Previously, we have defined the 255-bp minimal CD72 promoter that is required for tissue-specific and developmental stage-specific expression (24). We also reported the identification of several cis-acting elements contributing to the tissue specificity and developmental stage specificity of the mouse CD72 promoter. One of the cis elements, encompassing -196 to -163 of the mouse CD72 promoter, yielded enhanced promoter activity only in pre-B and mature B cells but not in T cells or plasma cells, which is reflective of the activity of endogenous CD72 gene in vivo (24). Analysis of the DNA fragment -196 to -163 demonstrated that there is a highly conserved, putative BSAP-binding site in the DNA fragment -196 to -163. BSAP is a transcription factor expressed in the developing central nervous system, testis, and cells of B lymphocyte lineage except terminally differentiated plasma cells (25, 26). Therefore, the distribution pattern of BSAP in B cells correlates with that of CD72. The gene coding for BSAP is Pax 5, which belongs to the Pax gene family; members of this family share a common DNA-binding paired domain (26, 27). Pax 5-/- mice fail to produce small pre-B, B, and plasma cells because they have a complete arrest of B cell development at an early stage (28). BSAP-binding sites have been identified in genes encoding {lambda}5 and VpreB1 (29), which encode the pre-B cell-specific surrogate light chain complex; the promoter region of blk, which is a tyrosine kinase involved in B cell signaling (30, 31); the promoter region of mb-1, which encodes Ig {alpha}, a component of the B cell receptor complex (32); and the promoter of the gene coding for CD19, which is a costimulatory molecule associated with Ag receptor signaling (33). These studies suggest that BSAP is a transactivator for these genes. In contrast, BSAP confers a negative effect when binding to the J chain promoter (34) and Ig 3' {alpha} enhancer (35, 36, 37), suggesting that BSAP plays a dual regulatory role during B cell development.

In this report, we show that BSAP is a positive regulator for the CD72 gene. BSAP mediates its tissue-specific and developmental stage-specific activity by specifically interacting with the DNA fragment -196 to -163 of the mouse CD72 promoter. In addition, our data suggest that the loss of BSAP could at least in part account for the down-regulation of CD72 at the plasma cell stage. We anticipate that such interactions between BSAP and its target sequences contribute to the tissue-specific and developmental stage-specific regulation of many B cell-specific genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfection assays

The mouse L1–2 pre-B cell line (provided by Dr. I. Weissman, Stanford University), M12.4.1 B lymphoma cell line (provided by Dr. M. Lieber, Washington University, St. Louis, MO), MOPC315p plasmacytoma cell line (provided by Dr. M. Davis, Stanford University), and BW5147 thymoma cell line (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) supplemented with 5% FCS (Sigma, St. Louis, MO), 50 µM 2-ME (Sigma), and 25 µg/ml each of penicillin and streptomycin (Life Technologies).

Cells were transfected by electroporation (Bio-Rad Gene Pulser, Hercules, CA). A total of 1 x 107/ml cells were harvested and resuspended in 0.4 ml cytomix buffer (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4 (pH 7.6), 25 mM HEPES (pH 7.6), 2 mM EGTA (pH 7.6), and 5 mM MgCl2 (pH adjusted by KOH)) (38) containing 10 µg of the luciferase reporter plasmid and 5 µg of plasmid pON405 containing LacZ driven by an immediate early CMV promoter (provided by Ed Mocarski, Stanford University). Electroporation was performed in a 0.4-cm cuvette (Invitrogen, La Jolla, CA) using the following parameters: M12.4.1 at 280 V and 960 µF capacitance, MOPC315p at 260 V and 960 µF capacitance, BW5147 at 320 V and 960 µF capacitance, and NIH-3T3 cells at 260 V and 960 µF capacitance.

After 24-h, transfected cells were harvested for luciferase and ß-galactosidase (ß-gal) assays. Luciferase activity was measured from 50 µl of the cell extract with the luciferase reagents as described by the supplier (Analytical Luminescence Laboratory, San Diego, CA). Light emission was measured with a Monolight 2010 instrument (Analytical Luminescence Laboratory), reading relative light for 10 s. Luciferase activities were normalized for transfection efficiency as determined by ß-gal activity. The ß-gal assay was performed as described previously (39).

Plasmid constructions and in vitro mutagenesis

The general strategy for making luciferase reporter gene constructs carrying mouse CD72 5' flanking sequence fragments has been described previously (24). In particular, the luciferase reporter constructs -63, -131, -162, -196, and -255 used in this article were generated by inserting fragments -63 to -6, -131 to -6, -162 to -6, -196 to -6, and -255 to -6 into the HindIII site immediately upstream of the luciferase reporter gene in the enhancerless, promoterless luciferase vector pSVOAL{Delta}5' (40). Therefore, all of these inserts have identical 3' ends that were generated by cleavage of the BstXI site which is just upstream of the ATG translation initiation site of the CD72 gene. All constructs were analyzed by both restriction enzyme digestion analysis and sequencing of the pertinent DNA junctions to verify copy number and orientation of inserts.

The mutated BSAP site was generated using oligonucleotides carrying point mutations in the BSAP-binding site. As the consensus sequence for BSAP consists of two distinct half sites, oligonucleotides were designed to carry two point mutations (indicated as underlined) in each half site, replacing the two most critical nucleotides for BSAP binding in each half site. Mutant luciferase constructs were generated by PCR according to standard protocols and were confirmed by sequence analysis.

The two (reverse complementary) oligonucleotides used for site-specific mutagenesis were BSAPmut1, 5'-CCCAAGGACCTCTCTAATTCATGAAGTCCATCT-3' and BSAPmut2, 5'-AGATGGACTTCATGAATTAGAGAGGTCCTTGGG-3'.

Electrophoretic mobility shift assay (EMSA)

Nuclear proteins were prepared from cultured cells as described previously (41). The double-stranded oligonucleotides were end-labeled with [{gamma}-32P]ATP (Amersham, Arlington Heights, IL). A total of 1 to 3 fmol of the probe was incubated with 15 µg of nuclear protein extract and 1 µg of poly(dI-dC) in a final volume of 30 µl of a buffer consisting of 8 mM HEPES (pH 7.9), 2.5 mM Tris-HCl (pH 7.9), 60 mM NaCl, 1 mM DTT, 10% glycerol, 1 mM EDTA, and 2.5 mM MgCl2 for 30 min at 20°C. Samples were analyzed on a 4% native polyacrylamide gel.

The following duplex oligonucleotides were used for direct binding or competition studies: oligo918–950, 5'-CCCAAGGACCTCTCTGCTTCATTGAGTCCATCT-3'; oligo950–918, 3'-GGGTTCCTGGAGAGACGAAGTAACTCAGGTAGA-5'; H2A2.2, 5'-TTGTGACGCAGCGGTGGGTGACGACTGT-3' and 3'-AACACTGCGTCGCCACCCACTGCTGACA-5'; H2A2.2mut, 5'-TTGTGACGCAGCGGTTGGTGACGACTGT-3' and 3'-AACACTGCGTCGCCAACCACTGCTGACA-5'; and 33 to 34, 5'-GATCCAGGCAGTTTTATTGAAATA-3' and 3'-CTAGGTCCGTCAAAATAACTTTAT-5'. The nucleotides in boldface represent the mutations.

Splenic B cell preparation and LPS culture

Single-cell suspensions of splenocytes from naive anti-hen egg white lysozyme (HEL) Ig transgenic mice (42) were generated by gently extruding cells from the splenic capsule and passing them through a sterile sieve. After lysing E with Tris-ammonium chloride, leukocytes were cultured in RPMI 1640 medium (Biofluids, Rockville, MD) with 10% FCS (HyClone Laboratories, Logan, UT), 20 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Irvine Scientific, Santa Ana, CA), 5 x 10-5 M 2-ME (Sigma), and 20 µg/ml LPS from Escherichia coli (0111:B4) (Difco, Detroit, MI). Phorbol-12,13-dibutyrate (PDBu) (Calbiochem, La Jolla, CA) was included at 2 ng/ml as indicated. Cultures were harvested after 96 to 108 h. Resting, mature, naive B cells were purified from the spleen by lysing E (as above) and depleting non-B cells with sheep anti-fluorescein magnetic beads (PerSeptive Diagnostics, Cambridge, MA) and fluorescein-conjugated Abs to CD4, CD8, Thy-1, and Mac-1 (Caltag, San Francisco, CA). Flow cytometric analysis of the pan-B cell marker B220 (Caltag) indicated that both the resting and LPS-stimulated B cells were >90% purity.

Flow cytometry

Approximately 5 x 105 cells of each type were stained with biotinylated mAb to CD72 (K10.6). After removing unbound Ab by washing, cells were incubated with fluorescein-conjugated streptavidin. Before cytometric analysis, propidium iodide (1 µg/ml) was added to the final cell suspension so that gates could be set to exclude nonviable cells. Immunofluorescence was determined by analysis on a modified FACS II (Becton Dickison, Mountain View, CA).

Nuclear protein preparation from splenic B cells

A total of 107 B cells of each type were chilled on ice, centrifuged at 600 x g at 3°C, and resuspended in ice-cold hypotonic buffer Hx (containing 10 mM HEPES (pH 7.6), 5 mM NaCl, 40 µg/ml each aprotinin and leupeptin, 1 mM PMSF, 1 mM DTT, and 2 mM EDTA). An equal volume of Hx with 0.8% Nonidet P-40 was added. After 2 min on ice and centrifugation at 700 x g, the supernatant was removed; nuclei were rinsed once in buffer Hx. Rinsed nuclei were resuspended in Hx containing 200 mM NaCl and incubated for 20 min on ice with intermittent mixing. After centrifugation at 70,000 x g in a Beckman airfuge (Palo Alto, CA), the supernatant was stored at -80°C. Protein concentrations were normalized according to their OD value at 280 nm. Nuclear extracts from ~106 cells were used in each gel mobility shift assay.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The -196 to -163 fragment is bound by a B cell-specific protein complex

Previously, we have identified several cell type-specific cis-acting elements by deletional analysis of the mouse CD72 promoter (24). Among them, the DNA fragment from nucleotide (nt) -196 to -163 (the translation start site ATG was considered to be +1) increased luciferase activity by fourfold in M12.4.1 cells, representing the mature B stage, but caused little change in luciferase activity in MOPC315p cells, representing the plasma cell stage, or in BW5147, representing thymic T cells (24) (also see Fig. 5Go, plasmid -162 vs plasmid -196). Thus, this fragment may contain a regulatory element contributing to the B cell-specific and developmental stage-specific activity of the mouse CD72 promoter. DNase I footprinting analysis of the CD72 minimal promoter revealed three protected elements footprint (FP) I, FP II, and FP III. The FP II element, which encompasses -190 to -168 of the CD72 promoter, lies within the fragment -196 to -163. These studies suggest that FP II may be recognized by transcription factors that are expressed specifically in B cells, and that this interaction might account for the cell type-specific activity of the minimal CD72 promoter.



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  		FIGURE 5. Mutation of the BSAP-binding site in luciferase reporter constructs diminishes CD72 promoter activity in B cell lines. Site-specific mutagenesis was performed on the luciferase plasmid -196 to generate the single BSAPmut or the doublemut (see Materials and Methods). wt and mutant luciferase constructs were transfected into M12.4.1 (A), MOPC315p (B), and BW5147 (C) cells (note the difference in scale among A, B, and C). Luciferase analysis was performed as described in Materials and Methods. Relative luciferase activity is expressed as fold activity above the background conferred by the promoterless control plasmid. Each histogram represents the value of luciferase activity ± SD (error bar) of three independent experiments.

 
We consequently performed EMSAs using the fragment -196 to -163 as a probe and identified two shifted bands as shown in Figure 1Go, lane 3. The upper band was present in the lanes in which the probe was incubated with nuclear extract from the mature B cell line L10A6.2 (Fig. 1Go, lane 2) and purified splenic B cells from C57BL/6 mice (Fig. 1Go, lane 3) but not in the lanes in which the probe was incubated with nuclear extract from the plasmacytoma line MOPC315p (Fig. 1Go, lane 4) or from the T lymphoma line BW5147 (Fig. 1Go, lane 5). In Figure 1Go, there are two upper bands visible in lane 2 in contrast to lane 3. The two upper bands often appeared as one band when the same experiment was performed multiple times. It is unclear whether the band representing the highest m.w. is just multimerization of the same protein-DNA complex represented by the lower band. Also in Figure 1Go, the lower band was present in lanes 2 and 3 and to a much lesser extent, in lanes 4 and 5. We have tested the specificity of these shifted bands by competing with unlabeled probe. All of the bands shown could be competed by an excess of unlabeled probe except the lowest one, which was not always present when the same experiment was repeated, suggesting that the bands represent a specific protein-DNA interaction (data not shown). This specific binding activity could also be detected in nuclear extracts from HAFTL1.clone6 (pre-B), L1–2 (pre-B), M12.4.1 (mature B), BAL17 (mature B), L10A6 (mature B), BCL1 (activated B), and A20 (mature B), but not from Ag8653 (plasmacytoma), J558 (plasmacytoma), S194 (myeloma), P388D1 (monocyte-macrophage), PU5-1 (macrophage), MEL745 (erythroleukemia), or NIH-3T3 cells (data not shown). EMSAs using an oligonucleotide probe recognized by Oct-1 were performed in parallel to controls for the quality of the protein extracts used in the assays. The above protein extracts contain functional Oct-1 that bound to the Oct-1 oligonucleotide probe (data not shown). These results suggest that the fragment -196 to -163 is bound by nuclear protein(s) whose expression is restricted to early and mature B cells.



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  		FIGURE 1. The -196 to -163 fragment of the CD72 promoter is bound by a B cell-specific protein complex. EMSAs were performed as described in Materials and Methods. A double-stranded oligonucleotide corresponding to nt -196 to -163 of the CD72 promoter region was radiolabeled and incubated in the absence (lane 1) or presence of 15 µg of nuclear protein extract from L10A6.2 (lane 2), splenic B cells from a C57BL/6 mouse (lane 3), MOPC315p (lane 4), or BW5147 (lane 5). Samples were analyzed on a 4% native polyacrylamide gel.

 
The transcription factor BSAP binds to the fragment -196 to -163

Examination of this sequence element revealed that there is a consensus binding site for the B cell-specific transcription factor BSAP (Fig. 2Go). To determine whether BSAP binds to the fragment -196 to -163, mobility shift competition assays were performed with unlabeled double-stranded oligonucleotides (Fig. 3Go). Oligonucleotide 33 to 34 does not contain a BSAP-binding site; therefore, this oligonucleotide was used as a nonspecific competitor. Oligonucleotide H2A2.2 (25) contains a known BSAP-binding site and was used as a specific competitor. The oligonucleotide H2A2.2mut has a point mutation at position 16 where G-C was replaced by T-A (see Materials and Methods). This mutation greatly reduced the affinity of the interaction between BSAP and H2A2.2mut (27). Therefore, H2A2.2mut was used as a specificity control. In assays performed with a pre-B cell line (HAFTL1.clone6) nuclear extract, the protein-DNA complex was specifically inhibited by H2A2.2 but not by 33 to 34. H2A2.2mut exhibited a slight inhibition, as the shifted bands shown in Figure 3Go were less intense in lanes 6 and 7 than in lane 2; however, the degree of inhibition was greatly reduced due to the low affinity as compared with wild-type (wt) H2A2.2.



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  		FIGURE 2. Comparisons of the CD72 promoter with BSAP-binding sequences. The CD72 promoter sequences (top) shown extend from nt -191 to -163. The BSAP-binding sites shown are from sea urchin histone H2A-2.2 promoter (25) (bottom) and a consensus BSAP recognition sequence (27) (middle).

 


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  		FIGURE 3. Interaction of BSAP with the mouse CD72 promoter. EMSAs were performed using a radiolabeled, double-stranded oligonucleotide encompassing nt -196 to -163 of the mouse CD72 promoter. The probe was incubated in the absence (lane 1) or presence of 15 µg of nuclear protein extract from the cell line HAFTL1.clone6 (pre-B) (lanes 2–7). Unlabeled, double-stranded oligonucleotide competitors were added in the following sequence: no competitor (lanes 1 and 2); 100 times excess of 33 to 34 (lane 3); 100 times excess of H2A-2.2 (lane 4); 200 times excess of H2A-2.2 (lane 5); 100 times excess of H2A-2.2mut (lane 6); and 200 times excess of H2A-2.2mut (lane 7). The shifted band representing the BSAP complex is indicated.

 
To further determine whether the protein we detected was indeed BSAP rather than another protein with the same binding specificity, anti-BSAP antiserum directed against the DNA-binding domain was added to block the formation of the B cell-specific complex. As shown in Figure 4Go, EMSAs were performed using end-labeled fragment -196 to -163 (lanes 1–6 and lane 11) and H2A2.2 (lanes 6–10) as probes. Probes were incubated with nuclear extract from L10A6.2 (mature B cell line), and samples were analyzed on a native polyacrylamide gel. When anti-BSAP antiserum was added, the specific BSAP-DNA complex was inhibited (Fig. 4Go, lanes 4, 5, 9, and 10). In contrast, formation of the complex was not disturbed by rabbit IgG. The shifted band in Figure 4Go, lane 11 is an antiserum-DNA complex (no nuclear extract was present); this band, which may represent a nonspecific serum protein-DNA complex, was also present, although much less intensively, in lanes 3, 4, 9, and 10. Similar results were seen using nuclear extracts from splenic B cells and M12.4.1 cells (data not shown). We have performed experiments with anti-BSAP antiserum plus nuclear extract from MOPC315 or BW5147. We did not see any inhibition of the formation of the shifted bands shown in lanes 4 and 5 of Figure 1Go. These studies suggest that BSAP indeed binds to the fragment -196 to -163 in the mouse CD72 promoter.



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  		FIGURE 4. Anti-BSAP antiserum specifically inhibits binding of BSAP to the CD72 promoter. EMSAs were conducted using an end-labeled, double-stranded oligonucleotide encompassing nt -196 to -163 of the mouse CD72 promoter (lanes 1–5) or using the end-labeled, double-stranded oligonucleotide H2A-2.2 (lanes 6–11). Probes were incubated in the absence (lanes 1 and 6) or presence of nuclear protein extract from L10A6.2 (lanes 2–5 and 7–10). Anti-BSAP antiserum was added in lanes 4 (1 µl), 5 (2 µl), 9 (1 µl), 10 (2 µl), and 11 (1 µl). Rabbit IgG was added in lanes 3 and 8 as an isotype control. The shifted band representing the BSAP complex and the band representing the DNA-antiserum complex are indicated. Similar results were obtained in experiments using rabbit serum as a control (data not shown).

 
Mutational analysis of the BSAP-binding site in the mouse CD72 promoter

To understand whether the binding of BSAP to the fragment -196 to -163 is responsible for the increase of luciferase activity seen by inclusion of this fragment in the luciferase reporter gene construct in M12.4.1 cells, the BSAP site in the reporter construct -196 was mutated by site-specific mutagenesis (see Materials and Methods). The binding of BSAP to the mutated site could not be detected in EMSAs using the double-stranded oligonucleotide containing the mutated BSAP site as a probe (data not shown). Luciferase analysis comparing the luciferase activity of wt reporter constructs with the mutant constructs showed that knocking out the BSAP site in the reporter construct -196 completely eliminated the increase of luciferase activity in the M12.4.1 B cell line (Fig. 5GoA). In contrast, mutations of the BSAP site in the reporter construct had no effect on the reporter gene activity in MOPC315p plasmacytoma cells (Fig. 5GoB) or BW5147 thymoma cells (Fig. 5GoC).

Knocking out both the BSAP- and PU.1-binding sites in the reporter construct -196 decreased the luciferase activity to background level in M12.4.1 cells, suggesting that both BSAP and PU.1 contribute to the activity of the CD72 promoter (Fig. 5GoA). Unlike the BSAP site mutant (BSAPmut) construct, the BSAP and PU.1 double mutant (doublemut) construct demonstrated decreased luciferase activity in MOPC315p cells, implying a role of PU.1 rather than BSAP, which is not expressed in plasma cells, in regulating CD72 promoter activity in plasma cells. However, CD72 is not expressed in MOPC315p cells, suggesting that PU.1 alone cannot fully activate the CD72 promoter and that other factors such as BSAP are required for CD72 promoter activity in CD72-expressing cells.

CD72 promoter activity is enhanced in plasma cells and T cells transfected with expression plasmids containing BSAP

To determine further the effect of BSAP on the CD72 promoter, cotransfections were performed using expression vectors for BSAP together with the reporter construct -196 (Fig. 6GoA, lanes 1–5). MOPC315p cells were transfected by electroporation, and luciferase activity was determined at 24 h after transfection. As shown in Figure 6GoA, lanes 1–5, BSAP up-regulated CD72 promoter activity in a dose-dependent manner. In contrast, there was no increase in luciferase activity when -196 was cotransfected with the plasmid containing the BSAP cDNA in the antisense orientation (Fig. 6Go, lane 6). Cotransfections were also conducted using expression vectors containing the BSAP cDNA together with the reporter construct -255 containing the CD72 gene sequence from nt -255 to -6. As shown in Figure 6Go, lanes 9 (-255 alone) and 10 (-255 plus 10 µg of the expression vectors for BSAP), BSAP up-regulated CD72 promoter activity by ~21-fold. Interestingly, the reporter activity of -196 cotransfected with 10 µg of the expression plasmid for BSAP (525.4 ± 31.2) was even higher than the activity of the reporter construct -196 in M12.4.1 cells (63.2 ± 1.8). This finding could be explained by the fact that BSAP was overexpressed in MOPC315p cells and the expression level was likely higher than that seen in M12.4.1 cells.



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  		FIGURE 6. Overexpression of BSAP in MOPC315p and BW5147 cells increases CD72 promoter activity. The reporter construct -196 was transiently transfected into MOPC315p (A) or BW5147 cells (B). Appropriate samples were cotransfected with 0 µg (lane 1), 0.5 µg (lane 2), 2 µg (lane 3), 5 µg (lane 4), and 10 µg (lane 5) of the expression vector pcßAmp containing BSAP cDNA (53) or with 10 µg of the expression vector pcßAmp containing antisense BSAP cDNA (53) (lane 6). The reporter construct BSAPmut containing the mutated BSAP site was transfected alone (lane 7) or together with 2 µg of the expression plasmid for BSAP (lane 8). The last two lanes represent luciferase activity of the reporter construct -255 (lane 9), which contains the 5' flanking sequence of the CD72 promoter extending to nt -255, and -255 cotransfected with 10 µg of BSAP expression construct (lane 10). Relative luciferase activity is presented relative to the plasmid -196 alone after correction for transfection efficiency. A represents luciferase analyses in MOPC315p cells; B represents luciferase activity in BW5147 cells.

 
To gain more insight into the nature of the BSAP mutant reporter construct, which showed decreased luciferase activity in B cells, cotransfections were performed using expression plasmids containing BSAP cDNA together with the BSAP mutant reporter construct. As shown in Fig. 6Go, lanes 7 (BSAPmut alone) and 8 (BSAPmut plus 2 µg of expression plasmid containing BSAP), BSAP up-regulated the activity of the mutant construct. However, the increase in luciferase activity was much less than that observed with the wt construct (Fig. 6Go, lane 8 vs lane 3). It is conceivable that the mutagenesis generated a low-affinity BSAP-binding site in either the reporter construct whose affinity was undetectable in gel retardation assays (data not shown) or the luciferase analysis in the M12.4.1 cells which produce physiologic levels of BSAP (Fig. 5GoA) but whose interaction with BSAP was significantly magnified when BSAP was overexpressed (Fig. 6GoA).

Similar findings were observed with cotransfections of the BSAP expression vector and the reporter construct -196 into BW5147 cells (Fig. 6GoB). However, the relative increase in luciferase activity in T cells (47.3 ± 5.3) (Fig. 6GoB, lane 5) was much less than in plasma cells (525.4 ± 31.2) (Fig. 6GoA, lane 5). The difference could be due to the presence of PU.1 in plasma cells but not in T cells, suggesting that both PU.1 and BSAP are important in regulating the activity of the CD72 promoter.

The expression pattern of BSAP correlates with that of CD72 on B cells in vivo

Previous studies have shown that neither the expression nor DNA-binding activity of BSAP can be detected in plasmacytoma cell lines (25, 26). To gain insight into the expression of CD72 and BSAP on normal plasma cells, purified splenic B cells from anti-HEL Ig transgenic mice (42) were cultured in the presence of LPS or LPS plus PDBu. Cells were harvested at day 4.5 and analyzed for surface markers by flow cytometry and for cytoplasmic markers by Western blotting. It was determined that 75 to 80% of the cells cultured with LPS displayed the plasma cell phenotype (Syndecan+, B220low, J chain+, Blimp-1+). Under the electron microscope, these cells demonstrated enlarged Golgi complexes and increased numbers of granules, which are typical of plasma cells (data not shown). This relatively pure plasma cell population from the culture of Ig transgenic splenic B cells enabled us to perform in vitro protein-binding assays without further purification. Consequently, we took advantage of this system to study the expression of CD72 and BSAP in this specific plasma cell population. The CD72 level was 10 times lower on the plasma cells as compared with the level on resting B cells (Fig. 7Go). Concomitantly, the DNA-binding activity of BSAP was dramatically decreased, as shown in gel retardation assays (Fig. 8Go). The BSAP-DNA complex was almost undetectable in the cells cultured with LPS (Fig. 8Go, lane 3).



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  		FIGURE 7. Surface CD72 decreases upon differentiation of B cells into plasma cells. Splenic B cells from anti-HEL transgenic mice were prepared and treated with LPS or LPS plus PDBu as described in Materials and Methods. Cells were recovered at day 4.5. The level of surface CD72 on untreated splenic B cells, LPS-treated cells, and cells treated with LPS plus PDBu was determined by immunofluorescence staining followed by flow cytometry (see Materials and Methods).

 


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  		FIGURE 8. The binding of BSAP to the CD72 promoter is lost upon B cell differentiation into plasma cells. EMSAs were performed using a radiolabeled, double-stranded oligonucleotide encompassing -196 to -163 of the mouse CD72 promoter as a probe. The probe was incubated with nuclear protein extract from resting B cells (lanes 1 and 2), 4.5-day LPS-treated B cells (lane 3), and B cells treated for 4.5 days with LPS plus PDBu. Anti-BSAP antiserum was added in lane 2. The shifted band representing the BSAP complex is indicated.

 
In contrast to the effect of LPS alone, the phorbol ester PDBu has been shown to inhibit the differentiation of B cells into plasma cells when mouse splenic B cells are cultured with both LPS and PDBu (43). We found that B cells treated with both LPS and PDBu displayed the phenotype of activated B blasts (Syndecan-, B220high) and were capable of proliferating (data not shown). The surface CD72 level on these cells remained the same as on resting B cells (Fig. 7Go), and BSAP-binding activity was maintained as well (Fig. 8Go, lane 4).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated that BSAP binds to the DNA fragment encompassing nt -196 to -163 of the mouse CD72 promoter. This interaction plays a critical role in regulating the B cell-specific and developmental stage-specific activity of the CD72 promoter.

Both BSAP and CD72 are constitutively expressed during the early stages of B cell development. As mentioned previously, failure to express either protein results in abnormal B lymphopoiesis. The expression of BSAP and CD72 is increased after B cell activation. The level of CD72 was increased fivefold on mouse splenic B cells cultured for 24 h in the presence of mAbs specific for IgM, and the level peaked at 48 h (J.-F. Chang, C. Pan, and J.R.P., unpublished observations). It is conceivable that the increased expression of CD72 is important for its regulatory function in B cell activation and proliferation. Interestingly, BSAP-binding activity also increased at 24 and 48 h after the splenic B cells were stimulated with LPS, CD40 ligand, or anti-IgD (44). Furthermore, overexpression of BSAP in splenic cells or de novo expression of BSAP in the plasmacytoma cell line MOPC315 stimulates proliferation (45). The suppression of BSAP expression by antisense oligonucleotides reduced the LPS-induced proliferation of splenic B cells and the proliferation of the B cell lymphoma line CH12.LX (44). These studies provide an impressive correlation between CD72 and BSAP both at the quantitative expression level and at the functional level, further supporting our data on the positive regulatory function of BSAP on the native CD72 promoter.

Both mouse CD72 and BSAP are undetectable in plasmacytoma cell lines and are down-regulated in normal plasma cells. Luciferase activity of the reporter construct -196, which contains the BSAP-binding site, was lower in plasmacytoma cell lines than in mature B cell lines. In B cell lines, mutations that eliminated the binding of BSAP to the CD72 promoter also eliminated the increase in luciferase activity. A previous analysis of the B lymphoma cell line BCL1 had shown that BCL1 cells can be induced to secrete IgM by IL-2 stimulation (46). To some extent, this in vitro stimulation system mimics early plasma cell differentiation. We found that the level of surface CD72 decreased by ~60 to 70% on BCL1 cells cultured for 3 days in the presence of either IL-2 or IL-2 plus IL-5 (our unpublished observations). Moreover, the level of BSAP protein and mRNA dropped by ~50% (34). These results strongly suggest that a loss of BSAP at the plasma cell stage removes a positive regulatory signal for the CD72 promoter activity, which may at least in part account for the down-regulation of CD72 expression at the plasma cell stage.

The previously published analyses of J chain gene regulation provide an interesting contrast to CD72 gene regulation. Unlike CD72, J chain is expressed after Ag stimulation and binding of IL-2 to its receptor on activated B cells (46). The minimal cell type-specific J chain promoter contains a repressor element, Jc, which is specifically recognized by BSAP. Overexpression of BSAP in plasmacytoma cells, which enhances CD72 promoter activity, inhibits expression of the endogenous J chain gene (34). Alternatively, down-regulation of BSAP in IL-2-stimulated or IL-2 plus IL-5-stimulated BCL1 cells correlates perfectly with the down-regulation of CD72 expression and up-regulation of J chain expression (47). Our analyses of CD72 gene regulation in conjunction with the analyses of J chain expression suggest that BSAP plays a dual regulatory role during B cell development by activating the transcription of genes that are expressed on early and mature B cells, such as CD72 and CD19, but repressing the transcription of genes that are involved in plasma cell differentiation and function, such as J chain. Alternatively, the down-regulation of BSAP at the plasma cell stage removes a positive signal that is essential for expressions of early B cell-specific genes, which contributes to the loss of expression of those genes at the plasma cell stage, and relieves a repression signal for genes that are highly expressed at the plasma cell stage, thus facilitating plasma cell differentiation and high level Ig production.

Although BSAP is a critical factor for CD72 gene expression, mutations in the BSAP-binding site in the mouse CD72 promoter only partially abolish the B cell-specific activity of the promoter, suggesting that BSAP is not the only factor involved. Previous studies have demonstrated that there are three sequence elements contributing to the cell type-specific activity of the CD72 promoter. One sequence element encompasses nt -162 to -132, is adjacent to the BSAP-binding site, and is bound by PU.1, a macrophage- and B cell-specific transcription factor (48, 49). Mutation of the PU.1 site in this DNA fragment eliminated the B cell-specific activity of this cis element, suggesting that PU.1 is essential for the B cell-specific activity of the mouse CD72 promoter (24). To analyze further the roles of BSAP and PU.1 in regulating the cell type-specific activity of the mouse CD72 promoter, the relative luciferase activity of the reporter construct -196 (63.2 ± 1.8) in M12.4.1 cells was compared with that of the BSAPmut construct (16.3 ± 0.5) and the doublemut construct (2.7 ± 0.5). In addition, the relative luciferase activity of the reporter construct -255 (94.3 ± 3.9) in M12.4.1 cells was compared with that of BSAPmut (22.3 ± 0.7), the PU.1 single mutant (17.1 ± 0.8), and the doublemut (4.3 ± 0.2). This analysis demonstrated that PU.1 and BSAP synergistically contribute to the cell type-specific activity of the CD72 promoter.

Since the PU.1 protein and mRNA levels remain relatively constant from the pro-B to the plasma cell stage, it is unclear how PU.1 contributes to the developmental stage-specific expression of CD72. Unlike BSAP, PU.1 is expressed in B cells, macrophages, monocytes, and, to a lesser extent, erythroid cells (48, 50). Targeted disruption of the PU.1/Spi-1 gene is lethal to the PU.1 mutant mice. The mutant embryos present multilineage defects that are characterized by a defective development of progenitors of monocytes, granulocytes, T cells, and B cells as well as a variable impairment of erythroid maturation (51). In addition, target genes for PU.1 are found in every lineage in which PU.1 is expressed. The above observations suggest that PU.1 is necessary but not sufficient for lineage commitment. This finding may explain why the PU.1 mRNA level is similar between different lineages and at different developmental stages.

Strikingly, the J chain promoter also contains a PU.1-binding site that is adjacent to the negative element recognized by BSAP. PU.1 alone confers a positive activity on the J chain promoter (52). Our results, in conjunction with studies on J chain gene regulation, have provided new insights into the dramatic picture of plasma cell differentiation, which involves the induction of certain gene products, such as J chain and Blimp-1, and the extinction of certain gene products, such as CD72 and BSAP. Our studies suggest that the dual regulatory molecule BSAP mediates its function by gradual changes in its protein concentration during plasma cell differentiation. By contrast, the level of PU.1 protein remains constant in this process, which correlates with its essential role in maintaining the hemopoietic system. In agreement with the above notion, the full function of the CD72 promoter requires a concerted action of both BSAP and PU.1.

Although our results support the hypothesis that the absence of a positive signal is responsible for the down-regulation of early B cell-specific genes at the plasma cell stage, they do not exclude the possibility of the presence of an inhibitory signal at this stage. Our preliminary analyses of the expression of CD72, BSAP, and Blimp-1 at the post-B cell activation stage indicated that CD72 and BSAP are only expressed in cells in which the Blimp-1 protein level is low or absent and vice versa (H. Y., D. Mack, J.I.H., and J.R.P., unpublished observations). In addition, the transfection of Blimp-1 into the mature B cell line BAL17 promotes differentiation into the plasma cell phenotype (45). It is conceivable that Blimp-1 may down-regulate CD72 expression by either directly binding to an unknown target sequence(s) in the CD72 gene or indirectly turning off the expression of positive regulators of CD72 expression, such as BSAP. Continuing efforts are needed to elucidate such mechanisms.


   Acknowledgments
 
We thank Dr. Meinrad Busslinger (Research Institute of Molecular Pathology, Vienna, Austria) for providing the antiserum specific for BSAP and Dr. Laurie Glimcher (Harvard Medical School, Boston, MA) for providing expression plasmids for BSAP.


   Footnotes
 
1 This work is supported by National Institutes of Health Grant CA68675 (to J.R.P.). H.Y. was supported in part by U.S. Public Health Service Training Grant CA09302 awarded by the National Cancer Institute, Department of Health and Human Services. Back

2 Address correspondence and reprint requests to Dr. Jane R. Parnes, Division of Immunology and Rheumatology, MSLS P-306, Stanford University School of Medicine, Stanford, CA 94305-5487. Back

3 Abbreviations used in this paper: BSAP, B cell-specific activator protein; HEL, hen egg white lysozyme; PDBu, phorbol-12,13-dibutyrate; EMSA, electrophoretic mobility shift assay; wt, wild type; ß-gal, ß-galactosidase; nt, nucleotide(s); FP, footprint; BSAPmut, BSAP site mutant; doublemut, BSAP and PU.1 double mutant. Back

Received for publication January 15, 1998. Accepted for publication June 24, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hombach, J., T. Tsubata, L. Leclercq, H. Stappert, M. Reth. 1990. Molecular components of the B-cell antigen receptor complex of the IgM class. Nature 343:760.[Medline]
  2. Hermanson, G. G., D. Eisenberg, P. W. Kincade, R. Wall. 1988. B29: a member of the immunoglobulin gene superfamily exclusively expressed on ß-lineage cells. Proc. Natl. Acad. Sci. USA 85:6890.[Abstract/Free Full Text]
  3. Nakayama, E., I. von Hoegen, J. R. Parnes. 1989. Sequence of the Lyb-2 B-cell differentiation antigen defines a gene superfamily of receptors with inverted membrane orientation. Proc. Natl. Acad. Sci. USA 86:1352.[Abstract/Free Full Text]
  4. Stamenkovic, I., E. A. Clark, B. Seed. 1989. A B-lymphocyte activation molecule related to the nerve growth factor receptor and induced by cytokines in carcinomas. EMBO J. 8:1403.[Medline]
  5. Tedder, T. F., C. M. Isaacs. 1989. Isolation of cDNAs encoding the CD19 antigen of human and mouse B lymphocytes: a new member of the Ig superfamily. J. Immunol. 143:712.[Abstract]
  6. Tedder, T. F., G. Klejman, S. F. Schlossman, H. Saito. 1989. Structure of the gene encoding the human B lymphocyte differentiation antigen CD20 (B1). J. Immunol. 142:2560.[Abstract]
  7. Kay, R., P. M. Rosten, R. K. Humphries. 1991. CD24, a signal transducer modulating B cell activation responses, is a very short peptide with a glycosyl phosphatidylinositol membrane anchor. J. Immunol. 147:1412.[Abstract]
  8. Rawlings, D. J., O. N. Witte. 1995. The Btk subfamily of cytoplasmic tyrosine kinases: structure, regulation, and function. Semin. Immunol. 7:237.[Medline]
  9. Dymecki, S. M., J. E. Niederhuber, S. V. Desiderio. 1990. Specific expression of a tyrosine kinase gene, blk, in B lymphoid cells. Science 247:332.[Abstract/Free Full Text]
  10. Busslinger, M., P. Urbánek. 1995. The role of BSAP (Pax-5) in B-cell development. Curr. Opin. Genet. Dev. 5:595.[Medline]
  11. Hagman, J., C. Belanger, A. Travis, C. W. Turck, R. Grosschedl. 1993. Cloning and functional characterization of early B-cell factor, a regulator of lymphocyte-specific gene expression. Genes Dev. 7:760.[Abstract/Free Full Text]
  12. Sato, H., E. A. Boyse. 1976. A new alloantigen expressed selectively on B cells: the Lyb-2 system. Immunogenetics 3:565.
  13. Tung, J.-S., H. Michaelson, H. Sato, E. S. Vitetta, E. A. Boyse. 1977. Properties of the Lyb-2 molecule. Immunogenetics 5:485.
  14. Shen, F.-W., M. Spanodis, E. A. Boyse. 1977. Multiple alleles of the Lyb-2 locus. Immunogenetics 5:481.
  15. Von Hoegen, I., E. Nakayama, J. R. Parnes. 1990. Identification of a human protein homologous to the mouse Lyb-2 B cell differentiation antigen and sequence of the corresponding cDNA. J. Immunol. 144:4870.[Abstract]
  16. Yakura, H., F.-W. Shen, E. A. Boyse, L. Tang. 1980. The Lyb-2 phenotype of hemolytic PFC. J. Immunol. 10:603.
  17. Gordon, J.. 1994. B-cell signalling via the C-type lectins CD23 and CD72. Immunol. Today 15:411.[Medline]
  18. Yakura, H., I. Kawabata, T. Ashida, F. W. Shen, M. Katagiri. 1986. A role for Lyb-2 in B cell activation mediated by a B cell stimulatory factor. J. Immunol. 137:1475.[Abstract]
  19. Nomura, T., H. Han, M. C. Howard, H. Yagita, H. Yakura, T. Honjo, T. Tsubata. 1996. Antigen receptor-mediated B cell death is blocked by signaling via CD72 or treatment with dextran sulfate and is defective in autoimmunity-prone mice. Int. Immunol. 8:867.[Abstract/Free Full Text]
  20. Polla, B. S., J. Ohara, W. E. Paul, N. Nabavi, A. Myer, H. C. Liou, F. W. Shen, S. Gillis, J. V. Bonventre, L. H. Glimcher. 1988. Differential induction of class II gene expression in murine pre-B-cell lines by B-cell stimulatory factor-1 and by antibodies to B-cell surface antigens. J. Mol. Cell. Immunol. 3:363.[Medline]
  21. Subbarao, B., J. Morris, A. R. Baluyut. 1988. Properties of anti-Lyb-2-mediated B-cell activation and the relationship between Lyb-2 molecules and receptors for B-cell stimulatory factor-1 on murine B lymphocytes. Cell. Immunol. 112:329.[Medline]
  22. Grupp, S. A., J. A. Harmony, A. R. Baluyut, B. Subbarao. 1987. Early events in B-cell activation: anti-Lyb2, but not BSF-1, induces a phosphatidylinositol response in murine B cells. Cell. Immunol. 110:131.[Medline]
  23. Yakura, H., I. Kawabata, T. Ashida, M. Katagiri. 1988. Differential regulation by Ly-5 and Lyb-2 of IgG production induced by lipopolysaccharide and B cell stimulatory factor-1 (IL-4). J. Immunol. 141:875.[Abstract]
  24. Ying, H., J.-F. Chang, J. R. Parnes. 1998. PU.1/Spi-1 is essential for the B cell-specific activity of the mouse CD72 promoter. J. Immunol. 160:2287.[Abstract/Free Full Text]
  25. Barberis, A., K. Widenhorn, L. Vitelli, M. Busslinger. 1990. A novel B-cell lineage-specific transcription factor present at early but not late stages of differentiation. Genes Dev. 4:849.[Abstract/Free Full Text]
  26. Adams, B., P. Dörfler, A. Aguzzi, Z. Kozmik, P. Urbánek, I. Maurer-Fogy, M. Busslinger. 1992. Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes, the developing CNS, and adult testis. Genes Dev. 6:1589.[Abstract/Free Full Text]
  27. Czerny, T., G. Schaffner, M. Busslinger. 1993. DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site. Genes Dev. 7:2048.[Abstract/Free Full Text]
  28. Urbánek, P., Z. Q. Wang, I. Fetka, E. F. Wagner, M. Busslinger. 1994. Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP. Cell 79:901.[Medline]
  29. Okabe, T., T. Watanabe, A. Kudo. 1992. A pre-B- and B cell-specific DNA-binding protein, EBB-1, which binds to the promoter of the VpreB1 gene. Eur. J. Immunol. 22:37.[Medline]
  30. Lin, Y. H., E. J. Shin, M. J. Campbell, J. E. Niederhuber. 1995. Transcription of the blk gene in human B lymphocytes is controlled by two promoters. J. Biol. Chem. 270:25968.[Abstract/Free Full Text]
  31. Zwollo, P., S. Desiderio. 1994. Specific recognition of the blk promoter by the B-lymphoid transcription factor B-cell-specific activator protein. J. Biol. Chem. 269:15310.[Abstract/Free Full Text]
  32. Fitzsimmons, D., W. Hodsdon, W. Wheat, S. M. Maira, B. Wasylyk, J. Hagman. 1996. Pax-5 (BSAP) recruits Ets proto-oncogene family proteins to form functional ternary complexes on a B-cell-specific promoter. Genes Dev. 10:2198.[Abstract/Free Full Text]
  33. Kozmik, Z., S. Wang, P. Dörfler, B. Adams, M. Busslinger. 1992. The promoter of the CD19 gene is a target for the B-cell-specific transcription factor BSAP. Mol. Cell. Biol. 12:2662.[Abstract/Free Full Text]
  34. Rinkenberger, J. L., J. J. Wallin, K. W. Johnson, M. E. Koshland.. 1996. An interleukin-2 signal relieves BSAP (Pax5)-mediated repression of the immunoglobulin J chain gene. Immunity 5:377.[Medline]
  35. Singh, M., B. K. Birshtein. 1996. Concerted repression of an immunoglobulin heavy-chain enhancer, 3' {alpha} E(hs1, 2). Proc. Natl. Acad. Sci. USA 93:4392.[Abstract/Free Full Text]
  36. Neurath, M. F., W. Strober, Y. Wakatsuki. 1994. The murine Ig 3' {alpha} enhancer is a target site with repressor function for the B cell lineage-specific transcription factor BSAP (NF-HB, S {alpha}-BP). J. Immunol. 153:730.[Abstract]
  37. Neurath, M. F., E. E. Max, W. Strober. 1995. Pax5 (BSAP) regulates the murine immunoglobulin 3' {alpha} enhancer by suppressing binding of NF-{alpha}P, a protein that controls heavy chain transcription. Proc. Natl. Acad. Sci. USA 92:5336.[Abstract/Free Full Text]
  38. van den Hoff, M. J., A. F. Moorman, W. H. Lamers. 1992. Electroporation in "intracellular" buffer increases cell survival. Nucleic Acids Res. 20:2902.[Free Full Text]
  39. Jain, V. K., I. T. Magrath. 1991. A chemiluminescent assay for quantitation of ß-galactosidase in the femtogram range: application to quantitation of ß-galactosidase in lacZ-transfected cells. Anal. Biochem. 199:119.[Medline]
  40. de Wet, J. R., K. V. Wood, M. DeLuca, D. R. Helinski, S. Subramani. 1987. Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7:725.[Abstract/Free Full Text]
  41. Dignam, J. D., R. M. Lebovitz, R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475.[Abstract/Free Full Text]
  42. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, et al 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334:676.[Medline]
  43. Simpson, L. G., P. C. Isakson. 1985. Phorbol esters specifically inhibit induction of Ig secretion in a murine B cell leukemia. J. Immunol. 134:2759.[Abstract]
  44. Wakatsuki, Y., M. F. Neurath, E. E. Max, W. Strober. 1994. The B cell-specific transcription factor BSAP regulates B cell proliferation. J. Exp. Med. 179:1099.[Abstract/Free Full Text]
  45. Jr Turner, C. A., D. H. Mack, M. M. Davis. 1994. Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells. Cell 77:297.[Medline]
  46. Koshland, M. E.. 1985. The coming of age of the immunoglobulin J chain. Annu. Rev. Immunol. 3:425.[Medline]
  47. Wallin, J. J., E. R. Gackstetter, M. E. Koshland. 1998. Dependence of BSAP repressor and activator functions on BSAP concentration. Science 279:1961.[Abstract/Free Full Text]
  48. Klemsz, M. J., S. R. McKercher, A. Celada, C. Van Beveren, R. A. Maki. 1990. The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene. Cell 61:113.[Medline]
  49. Moreau-Gachelin, F.. 1994. Spi-1/PU.1: an oncogene of the Ets family. Biochim. Biophys. Acta. 1198:149.[Medline]
  50. Hromas, R., A. Orazi, R. S. Neiman, R. Maki, C. Van Beveran, J. Moore, M. Klemsz. 1993. Hematopoietic lineage- and stage-restricted expression of the ETS oncogene family member PU.1. Blood 82:2998.[Abstract/Free Full Text]
  51. Scott, E. W., M. C. Simon, J. Anastasi, H. Singh. 1994. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 265:1573.[Abstract/Free Full Text]
  52. Shin, M. K., M. E. Koshland. 1993. Ets-related protein PU.1 regulates expression of the immunoglobulin J-chain gene through a novel Ets-binding element. Genes Dev. 7:2006.[Abstract/Free Full Text]
  53. Reimold, A. M., P. D. Ponath, Y. S. Li, R. R. Hardy, C. S. David, J. L. Strominger, L. H. Glimcher. 1996. Transcription factor B cell lineage-specific activator protein regulates the gene for human X-box binding protein 1. J. Exp. Med. 183:393.[Abstract/Free Full Text]



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