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The Journal of Immunology, 2001, 166: 6704-6710.
Copyright © 2001 by The American Association of Immunologists

Dissociation of Pax-5 from KI and KII Sites During {kappa}-Chain Gene Rearrangement Correlates with Its Association with the Underphosphorylated Form of Retinoblastoma1

Hiromu Sato, Dan Wang and Akira Kudo2

Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The KI and KII sites play a crucial role in {kappa}-chain gene rearrangement, which was investigated in mice deficient for these sites. Previously, we found that Pax-5 can bind to the KI and KII sites; however, the function of Pax-5 in {kappa}-chain gene rearrangement has not been investigated. Here, we have used an in vitro culture system in which differentiation from pre-B cells to immature B cells is induced by removing IL-7. We showed that, after the induction of differentiation, Pax-5 dissociated from the KI and KII revealed by EMSA analyses, and this dissociation occurred specifically at the KI and KII sites, but not at the Pax-5 binding site, in the CD19 promoter because of a lower binding affinity of Pax-5 for the KI and KII sites. During differentiation induced by removing IL-7, the underphosphorylated form of retinoblastoma preferentially associated with Pax-5, which caused dissociation of Pax-5 from KI and KII sites. These results suggest that the dissociation of Pax-5 from the KI and KII sites is important in the induction of {kappa}-chain gene rearrangement.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pax-5 is a member of the Pax family, which was identified on the basis of sequence homology with Drosophila segmentation genes and now consists of nine members (1). All Pax proteins contain a paired-box DNA-binding domain of 128 amino acids located at the amino terminal end (2, 3). This domain has been highly conserved during evolution and is found in Drosophila and in Pax genes of human, mouse, rat, chicken, quail, and zebrafish (1, 4). Pax-5 is a singly glycosylated polypeptide with a molecular mass of 50 kDa (5). It contains the DNA binding domain, an evolutionarily conserved octapeptide, a central region homologous to the half of the paired-type homeodomain (2), and a Ser/Thr/Pro-rich region at the C terminus of a transactivator (6). Eberhard and Busslinger reported that the partial homeodomain of Pax-5 is specifically bound by the underphosphorylated form of retinoblastoma (Rb)3 gene product and TATA-binding proteins (7).

Pax-5 expression is detected in many stages of B cell development, beginning with the very early commitment stage (8). During the early stages, Pax-5 exhibits a heterozygous monoallelic expression pattern, transcribing either of the two alleles in a stochastic, reversible manner that is independent of the parental origin. Interestingly, this monoallelic expression switches to biallelic expression at the immature B cell stage (9). In Pax-5 mutant mice, B cell development was arrested at the pre-BI stage, characterized by the large cell size and the surface expression of CD43 (8). CD19 was completely lost on the Pax-5 deficient cells, suggesting that Pax-5 is crucial for the expression of CD19. More recently, CD19 was shown to be a direct target for Pax-5 (10, 11). In Pax-5 mutant pre-BI cells, the first step of IgH gene rearrangement from germline to DHJH was normal. However, the frequency of rearrangement from DHJH to VHDHJH was reduced to 1/50 (8), suggesting that Pax-5 is involved in the machinery of Ig gene rearrangement during the differentiation from pre-BI cells to immature B cells.

In the early differentiation of B lineage, the pre-BI cells are large cell-cycling cells undergoing spontaneous DHJH rearrangement (12). Successful completion of VH-DHJH rearrangement is one of the checkpoints for the positive selection for the differentiation of B lineage cells into large pre-B cells, which transiently bear a complex of µ-chain coupled with the surrogate light chain (VpreB and {lambda}5) on the surface (13). This complex, known as pre-B cell receptor (pre-BCR) (14, 15, 16, 17, 18), was reported to trigger proliferation of pre-B cells (19), promote allelic exclusion at the IgH locus (20, 21), and induce differentiation to small pre-B cells, which then undergo Ig {kappa}-chain gene rearrangement (22, 23). Pax-5 might regulate H and {kappa}-chain gene rearrangement by a similar mechanism in which signals downstream of the pre-BCR may be involved.

During {kappa} chain gene rearrangement, two sterile transcripts of 1.1 kb and 0.8 kb are detected before the V{kappa}->J{kappa} joining (24, 25). The 0.8-kb RNA transcript initiates immediately upstream of the mouse J{kappa}1 segment within a region that contains two binding sites (KI and KII) for Pax-5. Immediately adjacent and downstream of the KI/KII site, there is the recombination signal sequence where recombination-activating gene (RAG)1 and RAG2 bind (26). Importantly, specific mutations of the KI and KII sites did not affect germline {kappa} transcription in mice; however, they reduced the frequency of V{kappa}-J{kappa} rearrangement to 1/10 of the normal level (27). These data clearly demonstrate that the KI and KII sites are cis-acting recombination enhancing elements; however, the molecular mechanism of both sites in {kappa}-chain gene rearrangement has not been investigated. The recognition of these sites by Pax-5 implicates its function in the control of {kappa}-chain gene rearrangement (28).

Recently, we have established IL-7-dependent pre-BCR-positive pre-B cell lines. After removal of IL-7, the cells undergo differentiation into immature B cells because of enhanced {kappa}-chain gene rearrangement (29). This in vitro differentiation system is useful for studies of the molecular mechanism of {kappa}-chain gene rearrangement because the signals downstream of pre-BCR are active. In this study, a role of Pax-5 binding to the KI and KII sites in {kappa}-chain gene rearrangement has been investigated. The results demonstrate that the association of Pax-5 with Rb induces the dissociation of Pax-5 from the KI and KII sites during the induction of {kappa}-chain gene rearrangement, suggesting that Pax-5 binding to and dissociating from the KI and KII sites is important for {kappa}-chain gene rearrangement.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture

PreBR1 and -BR2 cells derived from bone marrow cells of BALB/c mice were maintained in the presence of IL-7 (100 U/ml) as described previously (29). For in vitro differentiation, cells were washed three times to remove IL-7 and then cultured for 3 days at 5 x 105-1 x 106 cells/ml. The immature B cell line, WEHI231, was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin-streptomycin, and 50 µM 2-ME.

Preparation of nuclear extracts

Nuclear extracts were prepared according to the method of Scrheiber et al. (30). Cells (1 x 107) were harvested and washed twice in PBS, then the cell pellet was resuspended in 400 µl buffer A (10 mM HEPES (pH 7.5), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM PMSF, 2 µg/ml aprotinin, 1 mM DTT, 400 µM Na3VO4, 2.5 µg/ml leupeptin, and 5 µg/ml pepstatin). After incubation for 15 min on ice, 25 µl 10% Nonidet P-40 was added followed by vortexing. Cells were centrifuged in a microcentrifuge at 15,000 rpm for 1 min at 4°C. The supernatants were discarded, the cell pellets were resuspended in 100 µl buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 2 µg/ml aprotinin, 1 mM DTT, 400 µM Na3VO4, 2.5 µg/ml leupeptin, and 5 µg/ml pepstatin), and then shaken for 15 min at 4°C. After centrifugation in a microcentrifuge at 15,000 rpm for 5 min at 4°C, the supernatant was transferred to a fresh Eppendorf tube. The protein concentration of the nuclear extracts was determined by the Bradford assay.

EMSA

DNA was end-labeled with 32P by the Megalabel kit (Takara Shuzo, Kyoto, Japan). Labeled DNA (1.5 x 104 cpm) was incubated with 2 µg poly(dI-dC) (Pharmacia Biotech, Uppsala, Sweden) and nuclear extracts in a total volume of 20 µl. After incubation at room temperature for 15 min, samples were loaded onto a 5% polyacrylamide gel (29:1, acrylamide:bis-acrylamide) which was prerun for 1 h at room temperature at 150 V in 1x TAE (Tris-acetate EDTA). Samples were electrophoresed at 150 V for 2 h. Gels were dried and exposed to x-ray film (Kodak, Rochester, NY). Competition experiments were performed by mixing competitor DNA oligonucleotides with the binding reaction mixture before adding nuclear extracts. For supershift experiments, nuclear extracts were incubated with 2 µg of a goat anti-mouse N-terminal 19 of Pax-5 Ab (N-19), a goat anti-mouse C-terminal 20 of Pax-5 Ab (C-20; Santa Cruz Biotechnology, Santa Cruz, CA), and a control goat IgG (Southern Biotechnology Associates, Birmingham, AL) for 20 min at room temperature, respectively, before the labeled DNA fragments were added.

Oligonucleotide pairs upstream of J{kappa}1 (KI, 5'-CTCTGTTCCTCTTCAGTGAGGAGGGTTTTTGTA-3'; KII, 5'- CCAAGCGCTTCCACGCATGCTTGGAGAGGGGGTTA-3'); CD19 promoter, 5'-CTAGACACACCCATGGTTGAGTACCCTCCAGT-3'; and Oct self-complementary loop (octamer), 5'-GCCTCATTTGCATGGACTTAGCTTGTCCATGCAAATGAGG-3') were used for EMSA. The octamer probe binds the ubiquitous Oct-1 factor (31).

Western blotting analysis

Each 10 µg of nuclear protein was applied to wells of a SDS-10% polyacrylamide gel. Separated protein was blotted onto a nitrocellulose membrane (Schleicher & Schüll, Dassel, Germany), and the membranes were blocked in 5% nonfat milk in TTBS (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20) at 4°C overnight. A goat anti-mouse N-terminal 19 of Pax-5 Ab, a goat anti-mouse C-terminal 20 of Pax-5 Ab, or a mouse anti-human Rb Ab (BD PharMingen, San Diego, CA) that cross-reacts to mouse Rb was incubated at a 1:500 dilution at room temperature for 1 h with shaking. Membranes were washed three times with TTBS and incubated at room temperature in a 1/1000 dilution of a HRP-conjugated rabbit anti-goat IgG Ab (Santa Cruz Biotechnology) or a HRP-conjugated goat anti-mouse IgG Ab (Bio-Rad, Hercules, CA) for 30 min. After washing membranes three times with TTBS, proteins were visualized with the chemiluminescence ECL kit according to the manufacture’s instruction (Amersham Pharmacia Biotech, Piscataway, NJ).

DNA content analysis of PreBR1 and PreBR2

For the DNA content analysis, 5 x 104–5 x 105 cells were suspended in 200 µl ice-cold PBS, mixed with 2 ml ice-cold solution consisting of 70% ethanol and 30% PBS, and then incubated for 30 min on ice. Cells were harvested by centrifugation, suspended in 940 µl PBS containing 20 µl RNase A (5 mg/ml) and 40 µl propidium iodide (PI) (1 mg/ml), and were incubated for 30 min at 37°C. DNA content was analyzed by measuring a peak of FL-2.

Flow cytometry

A FITC-conjugated mAb 1D3 (anti-mouse CD19) and a FITC-conjugated goat anti-mouse {kappa}-chain were purchased from BD PharMingen and Southern Biotechnology Associates, respectively. Flow cytometry using the FACSCalibur (BD Biosciences, Mountain View, CA) was performed as described (29).

Immunoprecipitation

Precleared PreBR1 and PreBR2 nuclear extracts (100 µg) from cells cultured 0 or 3 days after removal of IL-7 were incubated with a mouse anti-human Rb (2 µg) in buffer C on a rotator at 4°C overnight. The immune complexes were captured by the addition of 20 µl protein G-Sepharose (Amersham Pharmacia Biotech). After washing five times with buffer C before loading onto a SDS-polyacrylamide gel, the immunoprecipitates were suspended in a 2x SDS sample buffer, eluted from the beads by boiling, and separated by SDS-PAGE. After electrophoresis, the gel was transferred to a nitrocellulose membrane and blocked in 5% nonfat milk in TTBS overnight. Pax-5 was detected by Western blotting as described above.

Preparation of GST fusion protein and GST pull-down assay

GST-Rb 379–928(379–928) was a gift from Dr. W. Kaelin (Dana Farber Cancer Institute, Boston, MA). Escherichia coli BL21 cells transformed with pGEX 2T and pGEX 2T-Rb 379–928(379–928) were inoculated overnight and then diluted. Diluted cultures were incubated with 0.2 mM isopropyl-{beta}-D-thiogalactopyranoside for 4 h. The cells were centrifuged and suspended in 2.5 ml of a lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM PMSF, 2 µg/ml aprotinin, 1 mM DTT, 400 µM Na3VO4, 2.5 µg/ml leupeptin, 5 µg/ml pepstatin, and 0.2 mg/ml lysozyme). After rotating for 30 min at 4°C, 277 µl 10% Triton X-100 was added to the cell lysate, and the cultures were incubated for further 20 min at 4°C. The solutions were centrifuged at 3000 rpm for 30 min at 4°C, and the remaining supernatants were incubated with 50 µl glutathione beads at 4°C overnight. Glutathione beads containing 3 µg GST or GST-fusion protein were washed with the buffer C and incubated with precleared nuclear proteins (100 µg) of PreBR1 and PreBR2 cells cultured in the presence of IL-7 for 1 h at 4°C. Where indicated, 100 µg/ml ethidium bromide (EtBr) was included in the reaction buffer. After washing five times with buffer C, glutathione beads were suspended in 2x SDS sample buffer, and protein was eluted from the beads by boiling and separated by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane, and Pax-5 was detected by using a goat anti-mouse N-terminal 19 of Pax-5 Ab. Abs were revealed by an ECL Western blot detection system as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pax-5 in PreBR cells bound to KI and KII sites at a low affinity

PreBR1 and PreBR2 are IL-7-dependent preBCR+ pre-B cell lines; thus, they express IgH (µ-chain) together with the surrogate L chain (VpreB and {lambda}5), Ig{alpha}-, and Ig{beta}-chain. Three days after removal of IL-7, pre-B cells differentiated into IgM+ (µ and {kappa}) immature B cells, with smaller sizes shown in Fig. 1Go.



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  		FIGURE 1. In vitro differentiation of PreBR1 and PreBR2. Expression of {kappa}-chain by PreBR1 and PreBR2 cells cultured in the presence (left panel) or the absence (right panel) of IL-7 for 3 days. Immunofluorescence and forward scatter (FSC) were examined by flow cytometry (FACSCalibur; BD Biosciences). Cells were stained with FITC-conjugated goat anti-mouse {kappa}-chain.

 
To examine Pax-5 binding to KI and KII sites upstream of the J{kappa}1 region, gel shift analyses were performed by using KI- and KII-specific oligonucleotide probes (Fig. 2Go, A and B). Two different anti-Pax-5 Abs were used in supershift experiments because Pax-5 has two splicing variants, Pax-5A and Pax-5B, that differ only in their use of exon 1A or exon 1B. One Ab is specific for the N-terminal 19 amino acids of Pax-5A, whereas the other is specific for the C-terminal 20 amino acids that both Pax-5A and Pax-5B contain. The shifted band (lane 2) was competed out with the unlabeled KI and KII probes and was supershifted by both N-19 and C-20 Abs. In the case of N-19, a band of about half intensity remained because Pax-5B was not supershifted. The results demonstrate that Pax-5 in PreBR cells bound to KI and KII sites.



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  		FIGURE 2. Binding activity of Pax-5 to KI and KII sequences. A, Mobility shift analysis using KI probe. 32P-labled double-strand oligonucleotide probes of KI sequences were incubated with 3 µg of nuclear extracts from pre-B cell lines (PreBR1 and PreBR2) before an analysis by EMSA. Nuclear protein was extracted from PreBR1 and PreBR2 cultured in the presence of IL-7 (100 U/ml). Lane 1, without nuclear extract; lanes 2 and 6, with nuclear extracts from PreBR1 or PreBR2. Complexes of the KI probe and nuclear proteins from PreBR1 and PreBR2 were competed with 100-fold molar excess of the unlabeled KI probe (lanes 3 and 7). The supershifted band of Pax-5-DNA Ab complexes resulting from the addition of control goat IgG (lanes 4 and 9), anti-Pax-5 Ab N-19, (lanes 5 and 10), and anti-Pax-5 Ab C-20, (lanes 6 and 11) are indicated by an arrow. B, Mobility shift analysis using the KII probe. The same nuclear extracts in A (indicated as the same lane number) were analyzed for binding to the radiolabeled KII probe.

 
By comparing sequences of the KI/KII sites and the Pax-5 binding site in the CD19 promoter to the consensus recognition sequence of Pax-5, we found that the CD19 promoter site was much closer to the consensus sequence. To verify the functional consequences of the higher homology of the CD19 promoter site (termed CD19 site), we used unlabeled CD19 and KI/KII site oligonucleotides in an EMSA competition experiment with the labeled CD19 site probe (Fig. 3Go). As the amount of competitor CD19 oligonucleotide site increased, CD19 site-Pax-5 binding was efficiently competed, resulting in a reduction of the DNA-Pax-5 shifted band. However, competition using unlabeled KI and KII oligonucleotides was not as efficient, resulting in only a slight reduction in the intensity of the DNA-Pax-5 shifted band. The result of this EMSA experiment indicates that Pax-5 has a much lower binding affinity for the KI or KII site than for the CD19 site, and there is also a subtle difference in that the affinity for the KII site is higher than for the KI site.



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  		FIGURE 3. Competition analyses of Pax-5-CD19 promoter binding by KI and KII oligonucleotides. Mobility shift analysis using nuclear extracts of PreBR1 (A) or PreBR2 (B) cells cultured in the presence of IL-7 (100 U/ml). Before performing EMSA, a 32P-labled double-strand CD19 promoter oligonucleotide probe was incubated without nuclear extract (lane 1), with 3 µg PreBR1 nuclear extract (lane 2) in the presence of the unlabeled CD19 promoter competition oligonucleotides at 1-, 5-, and 25-fold molar excess (lanes 3, 4, and 5, respectively), KI site oligonucleotide at 1-, 5-, and 25-fold molar excess (lanes 5, 6, and 7, respectively), or KII site oligonucleotides at 1-, 5-, and 25-fold molar excess (lanes 9, 10, and 11, respectively). An arrow indicates the position of the Pax-5-DNA complex.

 
Pax-5 specifically dissociates from the KI and KII sites during differentiation

PreBR1 and PreBR2 cell lines differentiate into immature B cells within 3 days after removal of IL-7. Pax-5 binding to the KI and KII sites was analyzed by EMSA during this differentiation process (Fig. 4Go, A and B). The ubiquitous Oct-1 transcription factor was used as a control for the equivalence of the nuclear extracts. The signal of the shifted KI-Pax-5 and KII-Pax-5 bands decreased by 2 days after removal of IL-7, a reduction that was even more pronounced by day 3, whereas the Oct-1 signal was unchanged (Fig. 4GoC), indicating that the removal of IL-7 did not result in nonspecific degradation of nuclear proteins. Three days after removal of IL-7, pre-B cell differentiation was almost complete. These results demonstrate that Pax-5 dissociates from the KI and KII sites during differentiation from pre-B to immature B cells.



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  		FIGURE 4. Dissociation of Pax-5 from the KI and KII binding sites during differentiation. Mobility shift analysis using KI, KII, and octamer probes. Nuclear extracts for EMSA were obtained from PreBR1 and PreBR2 cells cultured in the presence or absence of IL-7 (100 U/ml) for 1, 2, and 3 days to induce differentiation. Before an analysis by EMSA, 32P-labeled double-strand KI site oligonucleotide probe (A), KII site (B), or the octamer (C) was incubated without nuclear extract (lane 1) or with 3 µg PreBR1 (lanes 2, 3, 4, and 5) and PreBR2 (lanes 6, 7, 8, and 9) nuclear extracts. An arrow indicates the position of the Pax-5-DNA complex. These nuclear proteins contained similar amounts of the ubiquitous Oct-1 transcription factor, confirming their relative equivalence and integrity.

 
We performed several experiments to rule out the possibility that Pax-5 expression is simply down-regulated during differentiation. First, we examined the expression of Pax-5 protein by Western blot analysis 1, 2, and 3 days after removal of IL-7 from PreBR1 and PreBR2 cells. The mouse immature B cell line WEHI 231 was used as a positive control. Both Abs N-19 and C-20 revealed that the levels of Pax-5 protein were unchanged by IL-7 withdrawal (Fig. 5Go). Second, we examined Pax-5 binding to the CD19 site during differentiation (Fig. 6GoA). The result showed that the intensity of the CD19 site-Pax-5 shifted band was unchanged during differentiation 1, 2, and 3 days after removal of IL-7. Finally, because CD19 expression is totally dependent on the Pax-5 expression and binding, CD19 expression on the surface of PreBR1 and PreBR2 cells was examined by flow cytometry before or 3 days after removal of IL-7 (Fig. 6GoB). The levels of CD19 expression on the surface of PreBR1 and PreBR2 cells were identical in both cases. These results demonstrate decreased Pax-5 binding to KI and KII sites during differentiation even though the expression level of Pax-5 protein is unchanged, suggesting that Pax-5 is compulsorily absorbed with another molecule or is post-translationally modified such that it cannot bind KI and KII sites but can bind to the CD19 site.



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  		FIGURE 5. Constitutive Pax-5 expression during differentiation. Western blot analysis of Pax-5 was performed using nuclear extracts from PreBR1 and PreBR2 cells cultured in the presence or absence of IL-7 (100 U/ml) for 1, 2, and 3 days. Nuclear extracts (10 µg/lane) were analyzed with anti-Pax-5 Ab N-19 (top panel) and anti-Pax-5 Ab C-20 (bottom panel), respectively. The immature B cell line WEHI-231 was used as a positive control.

 


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  		FIGURE 6. Constitutive CD19 expression during differentiation. A, Mobility shift analysis using the CD19 promoter oligonucleotide as a probe. Nuclear proteins of PreBR1 and PreBR2 were extracted from cells cultured in the presence or absence of IL-7 (100 U/ml) for 1, 2, and 3 days. An arrow indicates the position of the Pax-5-CD19 promoter complex. B, Flow cytometry of CD19 expression by PreBR1 and PreBR2 cells before (top panel) and after (bottom panel) 3 days of culture without IL-7. A stippled line indicates the negative control (no Ab).

 
Pax-5 associates with underphosphorylated Rb during differentiation

After induction of differentiation, the size of differentiated PreBR cells becomes smaller, suggesting that cells enter the resting stage of the cell cycle. To examine this possibility, flow cytometry was performed. The DNA content of PreBR1 and PreBR2 cells was analyzed by staining cells with propidium iodide before and 3 days after removal of IL-7. In this experiment, only living cells were gated. PreBR1 and PreBR2 growing on IL-7 contained 56 and 52% in S/G2/M phases of the cell cycle, whereas only 33 and 38% of the cells were in S/G2/M phases 3 days after removal of IL-7 (Fig. 7GoA). This result shows that differentiated PreBR cells existed more in G0/G1 phases, indicating the resting stage of cell cycle, and it prompted us to examine the possible Rb involvement in the differentiation process. Because the underphosphorylated Rb can associate with Pax-5, we examined the phosphorylation of Rb during the differentiation process (Fig. 7GoB). The analysis revealed that Rb in the PreBR cells growing in the presence of IL-7 was hyperphosphorylated but was underphosphorylated 3 days after removal of IL-7.



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  		FIGURE 7. Changes in cell cycle and Rb phosphorylation during differentiation of PreBR cells. A, DNA content analysis of PreBR1 and PreBR2 cells before (top panel) and after (bottom panel) 3 days of culture without IL-7. PreBR1 or PreBR2 cells growing with IL-7 (100 U/ml) contained 56 or 52% of cells in S/G2/M phases of the cell cycle, whereas the small lymphocytes, after 3 days of IL-7 deprivation, were resting, because 33 or 38% of cells were in S/G2/M. B, Western blot analysis of Rb in PreBR1 and PreBR2 cells before and after 3 days of culture without IL-7. Nuclear proteins (10 µg/lane) were analyzed by anti-Rb Ab. Nuclear proteins of PreBR1 and PreBR2 were extracted from cells 0 or 3 days after removal of IL-7. Arrows indicate the positions of underphosphorylated (Rb) and hyperphosphorylated Rb (P-Rb).

 
It was reported previously that the underphosphorylated form of Rb specifically binds the partial homeodomain of Pax-5 (7). To test the specific association between Pax-5 and underphosphorylated Rb, immunoprecipitation by anti-Rb Ab followed by Western blot analysis with anti-Pax-5 Ab N-19 was performed (Fig. 8GoA). The result indicated that Pax-5 and Rb were associated in the differentiated PreBR cells 3 days after removal of IL-7. To examine Rb-Pax-5 association further, the pull-down analysis was performed using the GST-Rb fusion protein. The GST-Rb fusion protein could pull down Pax-5 from cell lysates of PreBR cells (Fig. 8GoB), confirming the physical association of Pax-5 with Rb. Moreover, to test whether the Pax-5 binding to the KI or KII site but not the CD19 site is competed by the GST-Rb fusion protein, GST-Rb was preincubated with nuclear extracts before adding oligonucleotides, and then EMSA was performed (Fig. 9Go). The result clearly reveals that incubation with GST-Rb but not GST alone remarkably diminished the signal of Pax-5-KI site; however, Pax-5-CD19 site binding was not significantly changed in the same condition. Thus, we conclude that the underphosphorylated form of Rb preferentially associates with Pax-5 compared with the KI site.



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  		FIGURE 8. Association of Pax-5 with the underphosphorylated form of Rb. A, PreBR1 and PreBR2 nuclear proteins (100 µg) before and after 3 days of culture without IL-7 were subjected to immunoprecipitation with 2 µg anti-Rb Ab. The immune complexes were resolved on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and blotted with anti-Pax-5 Ab N-19. B, GST pull-down assay. Nuclear protein (250 µg) of PreBR1 and PreBR2 cells cultured with IL-7 (100 U/ml) were incubated with 3 µg GST (lanes 3 and 5) or GST-Rb (379–928) (lanes 4 and 6) fusion protein attached to glutathione-Sepharose beads. After washing, bound proteins were eluted by boiling, separated by SDS-PAGE, and Pax-5 was revealed by Western blot analysis using anti-Pax-5 Ab N-19. Lanes 1 and 2 contain 10 µg PreBR1 and PreBR2 nuclear proteins.

 


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  		FIGURE 9. Competition analysis of Pax-5-KI site or Pax-5-CD19 site binding with GST-Rb. Mobility shift analyses were performed using the KI site (A) and the CD19 site (B) as probes. Nuclear proteins were extracted from PreBR1 and PreBR2 cells cultured in the presence of IL-7 (100 U/ml). Lane 1, Without nuclear extract. Before performing EMSA, PreBR1 or PreBR2 nuclear extract (1 µg) was incubated with 100 ng GST (lanes 2 and 4) or GST-Rb (lanes 3 and 5) at room temperature for 20 min, then oligonucleotides were added and incubated at room temperature for 15 min. An arrow indicates the position of the Pax-5-KI (A) or Pax-5-CD19 (B) complex.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrate here the involvement of Pax-5 in {kappa}-chain gene rearrangement. Pax-5 dissociates from KI and KII binding sites after associating with Rb during differentiation from pre-B cells to immature B cells in vitro. The severe disruption of rearrangement from DHJH to VHDHJH in Pax-5-deficient mice indicates that Pax-5 may be involved in Ig H chain gene rearrangement (8) as well and probably regulates Ig gene rearrangement generally. In our previous studies, we demonstrated that Pax-5 binds to KI and KII sites located upstream of J{kappa}1 and close to the recombination signal sequences (28). The importance of the KI and KII sites for {kappa}-chain gene rearrangement was confirmed by experiments with mice deficient for KI and KII sites. In the present study, we investigated the dissociation of Pax-5 from KI and KII sites during induction of {kappa}-chain gene rearrangement in vitro. During the differentiation from pre-BI cells to immature B cells, the following successive events occur: 1) IL-7 receptor signals cease, 2) Rb becomes unphosphorylated, 3) Pax-5 becomes associated with unphosphorylated Rb, and 4) Pax-5 is dissociated from KI and KII binding sites.

To address the question of why Pax-5 binding to KI and KII sites is dissociated although Pax-5 binding to CD19 promoter remains, we conclude that the binding affinity to KI and KII sites is much lower than that of CD19 promoter or Rb-Pax-5 association; thus KI and KII probes cannot take away Pax-5 from the Rb-Pax-5 complex in cell lysates. Wallin et al. (32) examined Pax-5 binding affinity to two activator elements, CD19 (10, 11) and germline transcription of the {epsilon} gene (33, 34, 35, 36), and two repressor elements, the J chain gene promoter (37) and the H chain 3' {alpha} enhancer (38, 39, 40, 41), by using competitive gel-shift assays. The results demonstrated that the Pax-5 site with positive regulatory activity had 20 times the affinity for the factor as those sites with repressor activity. They concluded that the difference in affinity indicated that the activator sites could outcompete the repressor sites when the supply of Pax-5 was limiting. Thus, we and Wallin et al. obtained similar results and conclusions. Using in vivo footprinting analyses, Shaffer et al. (31) also observed the dissociation of Pax-5 from its binding site in the 3' {kappa}-chain gene enhancer during the differentiation from pre-BI cells to immature B cells. They suggested that signals from pre-BCR result in a decrease in Pax-5 binding. The nature of this repression is quite similar to that in KI and KII sites, where we also found binding and releasing during differentiation.

In our experiments, Pax-5 dissociation from KI and KII sites is found in pre-BCR-positive cells; however, the signals from IL-7 removal dissociate Pax-5 from the KI and KII sites more efficiently than those from the pre-BCR. After Pax-5 is removed, RAG1 and RAG2 can bind to the recombination signal sequences because the KI site starts one base upstream of the recombination signal sequence nonamer, and RAG proteins possibly cover or penetrate the KI site (42), suggesting that Pax-5 may interfere with RAG binding. In the IgH 3' {alpha} enhancer, a similar function was revealed in that Pax-5 binding blocks the positive regulator for IgH {alpha}-chain transcription (38, 39, 40, 41). We will further investigate the correlation between Pax-5 binding and RAG binding by in vivo footprinting. In contrast, to investigate the function of Pax-5 at KI and KII sites during differentiation, the constitutive expression of Pax-5 was performed by retroviral transfection of the Pax-5 gene into pre-B cells. Our results demonstrated that a high expression of Pax-5 brought cell death (data not shown), illustrating the difficulty of such a experiment in vitro, because the Pax-5 amount might be very strictly regulated in B lineage cells (9).

Itoh et al. (43) reported that, by using IL-7-stimulated B precursor cells, Rb was phosphorylated by anti-cyclin-dependent kinase (CDK)4 immunoprecipitants in vitro, indicating that CDK4, assembled with cyclin D3, in the regulation of G1/S transition was activated under this condition. Rb is known to be regulated by the CDK4-cyclin D complex (44); thus, this machinery functions in the pre-B cell line. In vivo, this system is functioned in the pre-BI cell stage; therefore, during transition from the pre-BI to immature B cell stage, Pax-5 is initially released for the activation of the {kappa} locus, and then signals from pre-BCR may induce the rearrangement. Recently, lipid raft formation has been suggested to be important for signaling from the pre-BCR (45) because it is autonomously involved in the lipid raft. Thus, another possibility is that IL-7 removal induces the activation of lipid rafts, and signals from pre-BCR are transduced from the raft structure.

Besides the Rb association, there are several possibilities for dissociation of Pax-5 from the KI and KII sites. First, in terms of post-translational modification, although we do not deny this possibility, it seems unlikely because no reports have appeared for the modification of Pax-5. Second, protein-protein interaction except for Pax-5-Rb possibly occurs because Pax-5 potentially associates with other proteins. This second case is likely. Pax-5 may associate with other proteins whose expression is regulated in a differentiation-dependent manner during {kappa}-chain rearrangement; thus, Pax-5 loses the binding affinity to the KI and KII sites.

Finally, the fact that Rb and Pax-5 interact together in B lineage cells suggests that Rb has a common regulation in T cells for TCR gene rearrangement, because IL-7 is a common growth factor for both B and T cell progenitors.


   Acknowledgments
 
We thank Dr. W. Kaelin for providing Rb-GST and Dr. P. Burrows for critical review of the manuscript.


   Footnotes
 
1 This work was supported by a grant-in-aid for scientific research from the Japan Ministry of Education, Science, Sports, and Culture. Back

2 Address correspondence and reprint requests to Dr. Akira Kudo, Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501 Japan. E-mail address: akudo{at}bio.titech.ac.jp Back

3 Abbreviations used in this paper: Rb, retinoblastoma; pre-BCR, pre-B cell receptor; RAG, recombination-activating gene; CDK, cyclin-dependent kinase. Back

Received for publication January 2, 2001. Accepted for publication March 23, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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