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The Journal of Immunology, 2004, 172: 144-154.
Copyright © 2004 by The American Association of Immunologists

Analysis of Gene Expression and Ig Transcription in PU.1/Spi-B-Deficient Progenitor B Cell Lines1

Brock L. Schweitzer and Rodney P. DeKoter2

Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A number of presumptive target genes for the Ets-family transcription factor PU.1 have been identified in the B cell lineage. However, the precise function of PU.1 in B cells has not been studied because targeted null mutation of the PU.1 gene results in a block to lymphomyeloid development at an early developmental stage. In this study, we take advantage of recently developed PU.1-/-Spi-B-/- IL-7 and stromal cell-dependent progenitor B (pro-B) cell lines to analyze the function of PU.1 and Spi-B in B cell development. We show that contrary to previously published expectations, PU.1 and/or Spi-B are not required for Ig H chain (IgH) gene transcription in pro-B cells. In fact, PU.1-/-Spi-B-/- pro-B cells have increased levels of IgH transcription compared with wild-type pro-B cells. In addition, high levels of Ig{kappa} transcription are induced after IL-7 withdrawal of wild-type or PU.1-/-Spi-B-/- pro-B cells. In contrast, we found that Ig{lambda} transcription is reduced in PU.1-/-Spi-B-/- pro-B cells relative to wild-type pro-B cells after IL-7 withdrawal. These results suggest that Ig{lambda}, but not IgH or Ig{kappa}, transcription, is dependent on PU.1 and/or Spi-B. The PU.1-/-Spi-B-/- pro-B cells have other phenotypic changes relative to wild-type pro-B cells including increased proliferation, increased CD25 expression, decreased c-Kit expression, and decreased RAG-1 expression. Taken together, our observations suggest that reduction of PU.1 and/or Spi-B activity in pro-B cells promotes their differentiation to a stage intermediate between late pro-B cells and large pre-B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The development of B lymphocytes from hemopoietic stem cells is a multistep process, involving the ordered expression of intracellular and surface-located marker proteins, and stepwise rearrangement of gene segments in the Ig H and L chain gene loci (reviewed in Refs. 1, 2, 3, 4). Lymphoid progenitors, including the earliest detectable B cell progenitors, are found in a fraction of bone marrow cells which express the {alpha} subunit of the IL-7R (IL-7R{alpha}), low levels of c-Kit, and variable levels of terminal deoxynucleotidyl transferase (4, 5). The expression of the recombination activating genes RAG-1 and RAG-2, as well as DH-JH rearrangement of IgH genes, may also occur at this stage. It is generally agreed that acquisition of surface expression of both B220 and CD19 signifies commitment to the B cell lineage. These cells are generally referred to as progenitor B (pro-B)3 cells (4). Pro-B cells differentiate into pre-B cells when they complete VH to DHJH recombination and begin to express cytoplasmic IgH protein. Small amounts of functionally rearranged IgH protein can pair with the surrogate L chain components VPreB and {lambda}5 and be deposited on the cell surface as a pre-B cell receptor (pre-BCR) (1). Pre-BCR-expressing cells are termed large pre-B cells, because these cells undergo a burst of proliferation stimulated by combined signaling from the pre-BCR and IL-7R (reviewed in Ref. 6). Dilution of surrogate L chain proteins over a series of cell divisions, as well as down-regulation of IL-7R expression, results in a halt to VHDHJH recombination and a return to proliferative quiescence (1, 7). These quiescent cells shrink in size and are referred to as small pre-B cells (1, 3). Finally, reactivation of RAG gene transcription and rearrangement of the IgL genes are induced by this proliferative quiescence (1). Functional rearrangement of Ig{kappa} or Ig{lambda} and successful pairing with IgH proteins results in surface deposition of a functional BCR (1). BCR-expressing cells are considered to be immature B cells. There are at least three distinct classification schemes which define these stages (reviewed in Refs. 1, 2, 3, 4). In this paper, we will use the classification terminology recently reviewed by Lu and Osmond (3).

Several transcription factors play important roles in B cell development, including PU.1, Ikaros, E2A, EBF, and Pax-5 (reviewed in Refs. 8 and 9). Mice homozygous for a null mutation in the PU.1 gene die during fetal development by 18.5 days postcoitum (d.p.c.) and lack B, T, and myeloid progenitors (10, 11). PU.1-/- fetal liver contains reduced numbers of multipotential lymphoid-myeloid progenitors (AA4.1+, Lin-). Furthermore, these mutant progenitors fail to proliferate and differentiate into pro-B cells in response to stromal cell contact and IL-7 (11). This is in part because PU.1-/- fetal liver progenitors fail to express the IL-7R{alpha} gene (12). An alternative knockout model of the PU.1 gene, generated by insertional mutation of the PU.1 DNA-binding domain, results in a different phenotype such that homozygous null mice survive until birth and generate small numbers of thymic T cells and neutrophil-like cells (13, 14). However, B cells and B cell progenitors are undetectable in both PU.1 mutant mouse lines, demonstrating that PU.1 is absolutely required for B cell development (10, 11, 13).

PU.1 is a member of a subfamily of Ets transcription factors that includes two other closely related factors, Spi-B and Spi-C. Spi-B exhibits 43% overall amino acid sequence identity to PU.1, but the Ets domain is 67% identical to PU.1 (15). Spi-C (also called Prf) shares 40% overall sequence identity with PU.1, and shares 59% identity in the DNA binding domain (16, 17). PU.1 and Spi-B function as strong transcriptional activators, while Spi-C has weak transcription activation ability (17). PU.1 is expressed in multiple hemopoietic lineages, including all stages of B cell development, while the expression of Spi-B is restricted to B cells and T cells (18, 19, 20). Targeted null mutation of Spi-B does not affect B cell development, but results in defective BCR-mediated responses (21, 22). Both PU.1 and Spi-B are expressed in the B cell lineage beginning at the pro-B cell stage and appear to interact with identical DNA binding sites (15). Therefore in developing B cells, the available evidence indicates that PU.1 and Spi-B are functionally interchangeable (22, 23).

A number of presumptive target genes for PU.1 and/or Spi-B have been identified in vitro in the B cell lineage. First, binding sites are found in a number of Ig promoters and enhancers, including Ig{kappa} V region promoters (24), the Ig{kappa} 3' enhancer (25), the Ig{lambda} 2–4 enhancer (26), the IgH intronic enhancer (27), and the Ig J-chain promoter (28). Other lymphoid and B cell-specific genes regulated by PU.1 and/or Spi-B include c-rel (29), CD20 (30), mb-1 (31), P2Y10 (23), Bruton’s tyrosine kinase (Btk) (32), germinal center nuclear-associated protein (33), and CD72 (34). These target genes have been identified by analysis of promoter and/or enhancer elements involved in driving their tissue-specific transcription. However, because mutation of the PU.1 gene results in complete loss of B cell development, it has been unclear whether PU.1 is required for transcription of any of these genes in B cells. We have recently shown that retroviral transduction of PU.1-/- or PU.1-/-Spi-B-/- progenitors with IL-7R{alpha} cDNA restores IL-7 and stromal cell-dependent proliferation. In addition, IL-7R{alpha} transduction promotes low-frequency differentiation into CD19+ pro-B cells (12). We have therefore been able to establish IL-7-dependent PU.1-/- and PU.1-/-Spi-B-/- pro-B cell lines and clones. The comparison of gene expression in wild-type and PU.1-/-Spi-B-/- pro-B cell lines represents a novel opportunity to investigate the target genes of PU.1 and/or Spi-B in the B lineage.

In this study, we compare wild-type and PU.1-/-Spi-B-/- pro-B cell lines to analyze the function of PU.1 and/or Spi-B in B cell development. We examined PU.1-/-Spi-B-/- pro-B cell lines due to possible functional redundancy of PU.1 and Spi-B in B cells. We show that contrary to previously published predictions, transcription of IgH is increased in PU.1-/-Spi-B-/- pro-B cells relative to wild-type pro-B cells. After IL-7 withdrawal, transcription of Ig{kappa}, but not Ig{lambda}, can be induced in PU.1-/-Spi-B-/- pro-B cells. Phenotypic characterization of PU.1-/-Spi-B-/- pro-B cell lines suggests that these cells are further advanced in their development than wild-type pro-B cell lines. We demonstrate that we can reproduce these results by interfering with PU.1 and/or Spi-B activity in wild-type cell lines using ectopic expression of an inactive mutant form of PU.1. Taken together, these results suggest that reduction of PU.1 and/or Spi-B activity in pro-B cells promotes their differentiation to a stage intermediate between late pro-B cells and large pre-B cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

The retroviral packaging cell lines Phoenix (35) and GP+E86 (36) were maintained in complete DMEM medium. S17 stromal cells (37) and J558-IL-7 cells (38) were maintained in complete RPMI 1640 medium. All complete medium contained 10% FBS (S17 stromal cells, 5%), 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, 5 x 10-5 M 2-ME, and 0.5 mM HEPES (Invitrogen, San Diego, CA).

Construction of retroviral vectors and packaging cell lines

MIG-PU.1 retrovirus containing PU.1 and an internal ribosomal entry site green fluorescent protein (IRES-GFP) has been described previously (39, 40). PU.1{Delta}N100 was generated as described previously (40). PU.1{Delta}N100 was ligated into the XhoI and EcoRI sites of the MIGR1 vector after digestion from the MSCV-EGFP vector using digestion with XhoI and EcoRI. To establish retroviral packaging cell lines, retrovirus produced by transient transfection of Phoenix cells was used to infect GP+E-86 cells after overnight treatment with tunicamycin. After 2 days, the brightest GFP-expressing cells were sorted using the BD Biosciences FACSVantage system (San Diego, CA) (Cincinnati Shriners Hospital, Cincinnati, OH) and expanded under gpt selection. Sorted cell lines were analyzed periodically to ensure continued GFP expression. Expression of PU.1 and PU.1{Delta}N100 were confirmed by Western blotting using an anti-C-terminal PU.1 Ab (Santa Cruz Biotechnology, Santa Cruz, CA).

Isolation of lineage-depleted fetal hemopoietic progenitors

PU.1+/- or PU.1+/-Spi-B-/- mice were mated to produce PU.1-/- and PU.1-/-Spi-B-/- fetuses as well as control littermates. The presence of a vaginal plug on the morning after matings was taken as 0.5 d.p.c. Genotyping of PU.1 alleles was performed by PCR as previously described (12). Lineage-depleted (Lin-) hemopoietic progenitors were isolated from 14.5 d.p.c. fetal liver suspensions also as described previously (40).

Infection and culture of lineage-depleted fetal hemopoietic progenitors

GP+E-86 lines were irradiated using 2000 rad from a Cesium-137 source and plated at 1.5 x 105 cells/well in a 24-well plate (Corning-Costar, Cambridge, MA) and incubated overnight. Lin- fetal liver progenitors (105) were added to each well and infected by coculture for 2 days in complete IMDM medium and 5% conditioned medium from J558-IL-7 cells. Infected cells were removed by gentle pipetting and transferred onto an monolayer of irradiated (2000 rad) S17 stromal cells. Cultures were fed with fresh IL-7-containing complete IMDM medium every 4 days and analyzed after 10–14 days.

Establishment of IL-7-dependent cell lines

Cells from 10–14 day cultures of PU.1-/- and PU.1-/-Spi-B-/- fetal hemopoietic progenitors rescued with IL-7R{alpha} cDNA were expanded and passaged by plating on monolayers of irradiated S17 stromal cells in IL-7 containing medium as described above. IL-7-dependent cell lines were passaged by plating 5 x 105 cells/ml every 3–4 days.

Infection and establishment of retrovirally infected pro-B cell lines

Irradiated (2000 rad) GP+E-86 packaging cell lines were plated into six-well plates at a concentration of 5 x 105 cells/3 ml. Wild-type IL-7-dependent pro-B cell lines (2 x 105) generated as described above were cocultured with packaging cell lines in IL-7-containing complete IMDM medium for 72 h. Following coculture, pro-B cells were harvested and washed three times in flow cytometry buffer (Dulbecco’s PBS (Life Technologies) supplemented with 0.5 mM EDTA and 0.5% BSA fraction V (Fisher, Pittsburgh, PA)) and sorted for the brightest 40% GFP-expressing cells. These cells were sorted into gentamicin-containing (10 µg/ml) IL-7-containing complete IMDM medium and plated into 25-cm2 flasks containing 7 x 105 irradiated (2000 rad) S17 stromal cells. Flow cytometric analysis was performed at each passage to ensure maintenance of GFP expression.

Affymetrix GeneChip analysis

Affymetrix GeneChip analysis (Santa Clara, CA) was performed by the Cincinnati Children’s Hospital Research Foundation Affymetrix core facility. Total RNA was extracted from IL-7-dependent pro-B cells using RNA-Bee (Tel-Test, Friendswood, TX). RNA was chloroform extracted a second time before submission for analysis. RNA was reverse-transcribed into cDNA and used to generate biotinylated cRNA. Biotinylated RNA was probed by the U74A version 2 Affymetrix GeneChip. Data was analyzed using MicroArray Suite 5.0. Probe sets were filtered based on presence call on array scan and a minimum of a 2-fold change was determined by using the signal log ratio of the PU.1-/-Spi-B-/- RNA samples. Comparison files were generated between PU.1-/-Spi-B-/- and wild-type pro-B cell lines by averaging the signal log ratios between experiments. Signal log ratio is a mathematical value calculated from differences in probe intensity between two sample arrays. Results are expressed as the mean and SE of n experiments. Gene lists indicating transcripts increased or decreased relative to wild-type controls were compiled among triplicate arrays (supplemental Tables I and II).

PCR analysis

Total RNA was extracted from culture of IL-7-dependent pro-B cell lines using RNA-Bee (Tel-Test). Total RNA at 1 µg/µl was used as template for cDNA synthesis using a cDNA synthesis kit (Clontech Laboratories, Palo Alto, CA). This cDNA was analyzed using primers spanning intronic sequence for {beta}-actin to verify lack of DNA contamination and equal cDNA amplification from various template RNAs. Primers for various genes were designed to span introns or splice junctions using Stratagene PCR primer designer. For real-time PCR, 25 µl PCR consisted of 100 pmol of primers, 1 U Taq polymerase (GeneChoice, Frederick, MD), 0.5x of 50,000x stock of SYBR Green I (Molecular Probes, Eugene, OR), 100 pmol of MgCl, 1x standard PCR buffer, and nuclease-free deionized water. All amplification protocols used a 1-min melting step of 90°C followed by 45 cycles of amplification. Each cycle for various primer sets used an initial melt of 95°C for 15 s, an annealing step of 7 s at optimized temperature, followed by an optimized extension step of 10–20 s at an appropriate temperature, followed by fluorescence measurement. Product quality was tested using a 3-min melting curve cycle at which the signal was measured every second as the temperature was increased incrementally from extension temperature to 95°C. The presence of a single exponential decrease in signal was considered to be a single product. Reactions were run on agarose gels and stained with ethidium bromide to confirm the product. Threshold cycle (Ct) values were determined automatically by the Smartcycler software (Cepheid, Sunnyvale, CA) based on a 10x SD of baseline signal. These values were used to calculate relative expression of various genes relative to G6PDH control expression using the {Delta}{Delta}Ct method (41). Primer sequences used for real-time PCR are listed as follows: Iµ (5'-ACCTGGGAATGTATGGTTGTGGCTT-3' and 5'-ATGCAGATCTCTGTTTTTGCCTCC-3'), {kappa}° (5'-TGGACCAGAGACTTCAAGGCA-3' and 5'-ACTGCTCACTGGATGGTGGGA-3'), G6PDH (5'-GAACATCATCCCTGCATCCA-3' and 5'-CCAGTGAGCTTCCCGTTCA-3'), RAG-1 (5'-CATTCTAGCACTCTGGCCGGG-3' and 5'-GGCAAGGAGGCAGCCATGTTG-3'), BLNK (5'-TCCAAGTCATCTTTGCCTGCC-3' and 5'-TGCATTCGGTAGGGGAGGAAC-3'). Primer sequences used for other PCR analyses in these studies are listed as follows: {beta}-actin (5'-CCTAAGGCCAACCGTGAAAAG-3' and 5'-TCTTCATGGTGCTAGGAGCCA-3'), {lambda}1 V-JC rearrangement (5'-CGCGAATTCTCAGGCTCCCTGATTGGAGACAAGG-3' and 5'-GACCTAGGAACAGTCAGCACGGG-3'), {lambda}2 germline (5'-GCTGTGAGAGAACAGGACCA-3' and 5'-CTCGGGGAAAAGTTGGAAAT-3'), Fc{gamma}RIIb (5'-CCCAAGTCCAGCAGGTCTTTACC-3' and 5'-CCCAATGCCAAGGGAGACTAAAT-3').

Determination of doubling time

S17 stromal cells were irradiated (2000 rad) and 5 x 104 cells/well were plated into 24-well plates (Corning-Costar) containing 1 ml of complete RPMI 1640. Pro-B cells were then plated at 2 x 104 cells/well in 1 ml of IL-7 medium and grown for 96 h. After growth, cells were resuspended in a well by gentle pipetting and 10 µl were removed for counting with trypan blue using a hemacytometer. Each cell line was independently grown and counted in quadruplicate per experiment.

Flow cytometric analysis

Pro-B cells (0.5 x 106) were washed three times in flow cytometry wash buffer. Following the third wash, cells were suspended in 100 µl of a 1/100 dilution of the appropriate Ab and stained for 20 min on ice. Cells were spun out of Ab solution and washed three times. Secondary Ab staining was done at a 1/250 dilution for 20 min on ice. Cells were spun out, washed three times, and finally diluted into 300 µl of 748 mM propidium iodide solution. Cells were analyzed using a BD Biosciences FACSCalibur system by gating on forward and side scatter characteristics for optimal size and shape of cells. Secondary gating was done based on propidium iodide staining for viable cells. The final gating procedure involved either staining from PE or allophycocyanin based on the presence of endogenous GFP expression.

Western blotting

Lysates were generated from pro-B cells and S17 stromal cells using Laemlli buffer according to standard methods. Lysates were run on SDS-PAGE gel using the Mighty Small Electrophoresis System (Hoeffer, San Francisco, CA). Proteins were transferred to nitrocellulose and stained with anti-IgM or anti-PU.1 using standard procedures. Anti-{beta}-actin was used as control for lysate concentration. HRP secondary Abs were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and used to expose x-ray film (Kodak, Melville, NY).

Statistics

All statistics reported are the mean and SE of n experiments. Experiments were performed on separate sets of cell lines when possible. The figures show results of representative experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotypic characterization of PU.1-/-Spi-B-/- pro-B cell lines

PU.1 and Spi-B are thought to directly regulate a number of target genes in the B cell lineage. However, homozygous null mutation of the PU.1 gene results in an early block to B cell development. We have previously shown that retroviral transduction of PU.1-/-Spi-B-/- fetal liver progenitors with IL-7R{alpha} cDNA, followed by culture in IL-7 on S17 stromal cells, results in differentiation into CD19+ pro-B cells (12). These pro-B cells can be established as IL-7-dependent cell lines. We showed that by several criteria, such as expression of B cell-specific genes and active DH-JH DNA rearrangement, these cell lines appear to represent normal late pro-B cells (12). To investigate whether PU.1-/-Spi-B-/- pro-B cells are phenotypically identical to wild-type pro-B cells, we characterized two sets of these cell lines further.

First, cell counting was used as a method of measuring the doubling time of wild-type and mutant cell lines. PU.1-/-Spi-B-/- pro-B cell lines had a significantly reduced doubling time than wild-type cell lines, indicating an increased proliferation rate (16.5 ± 0.6 h, n = 3; compared with 20.2 ± 1.1 h, n = 3) (Fig. 1A). Next, flow cytometric analysis was used to characterize wild-type and mutant cells. Expression of cell surface markers has been extensively used to classify distinct stages of B cell development (reviewed in Ref. 2). PU.1-/-Spi-B-/- pro-B cells had previously been shown to express CD19 (12). CD19 is a B cell-specific surface Ag that is expressed upon commitment to the B cell lineage and functions as an amplifier of BCR signaling (42). As expected, all PU.1-/-Spi-B-/- and wild-type pro-B cell lines expressed similar levels of CD19 (Fig. 1B, upper panels). In contrast, the low-affinity IgG FcR Fc{gamma}RIIb, which is suspected to be a target gene of PU.1 (43), was expressed on wild-type pro-B cell lines but was reduced or undetectable on PU.1-/-Spi-B-/- cell lines (Fig. 1B, lower panels). Analysis of surface H chain expression showed that extracellular IgH levels were low on PU.1-/-Spi-B-/- (1.4 ± 1.4%, n = 4) and wild-type (1.5 ± 1.4%, n = 6) pro-B cells (Fig. 1C, first row). CD43 (sialophorin) is an early B cell developmental marker that is lost in the transition from early large pre-B to late large pre-B cells, coinciding with surface expression of the pre-BCR (44). We found that CD43 was expressed at similar levels in wild-type and PU.1-/-Spi-B-/- cell lines (Fig. 1C, second row). The receptor for stem cell factor, c-Kit, is expressed throughout early B cell development but is lost during the transition from pro-B to the large pre-B stage (reviewed in Ref. 1). Although wild-type pro-B cells were positive for c-Kit (mean fluorescence (MF) 109 ± 7.0, n = 4), PU.1-/-Spi-B-/- pro-B cells had reduced levels of expression (MF 5 ± 1.4, n = 4) (Fig. 1C, third row). Despite retroviral infection with IL-7R{alpha} cDNA, the PU.1-/-Spi-B-/- pro-B cell lines had greatly reduced expression (MF 7 ± 1.7, n = 3) compared with wild-type pro-B cells (MF 33 ± 1.7, n = 3) (Fig. 1C, fourth row). Finally, we examined expression of CD25 (IL-2R{alpha}, Tac), which is a marker for differentiation to the pre-B cell stage (45). PU.1-/-Spi-B-/- cell lines expressed significantly higher levels of CD25 (MF 53 ± 3.9, n = 3) than did wild-type cell lines (MF = 14 ± 0.9, n = 3) (Fig. 1C, bottom row). In summary, PU.1-/-Spi-B-/- pro-B cell lines proliferate at a greater rate; yet express lower levels of c-Kit and IL-7R{alpha} than wild-type pro-B cell lines. Other surface markers, such as CD19, CD43, and H chain, are comparable to wild-type pro-B cell lines. Expression of CD25 on PU.1-/-Spi-B-/- cell lines suggests that these cells may be further advanced in their differentiation than wild-type cell lines.



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  		FIGURE 1. Phenotypic analysis of PU.1-/-Spi-B-/- pro-B cells. A, Growth rate analysis of pro-B cells. PU.1-/-Spi-B-/- or wild-type pro-B cells (5 x 104) were grown in IL-7 medium on S17 stromal cells for 96 h. Means of absolute cell counts are presented with doubling time, as calculated based on trypan blue counting. B and C, Representative histograms for flow cytometric analysis of surface marker expression on pro-B cells. B, PU.1-/-Spi-B-/- and wild-type pro-B cells were analyzed for surface expression of CD19 (top row) and Fc{gamma}RII/III (bottom row). C, PU.1-/-Spi-B-/- and wild-type pro-B cells were analyzed for surface expression of IgH (first row), CD43 (second row), c-Kit (third row), IL-7R{alpha} (fourth row), and CD25 (bottom row).

 
Comparison of gene expression profiles of wild-type and PU.1-/-Spi-B-/-pro-B cell lines

Affymetrix GeneChip analysis was used to comprehensively compare patterns of gene expression in PU.1-/-Spi-B-/- and wild-type pro-B cells. Total RNA was prepared from two sets of independently derived wild-type and PU.1-/-Spi-B-/- pro-B cell lines. One RNA set was probed once and the other twice using the U74Av2 GeneChip (Affymetrix). The data was analyzed using MicroArray Suite 5.0 software (Affymetrix). Data from the three independent analyses were compiled into lists of genes that were either increased or decreased at least 2-fold in the PU.1-/-Spi-B-/- pro-B cells relative to the wild-type (see full list in supplemental Tables I and II). Genes were broken down into classifications using GeneSpring software (Silicon Genetics, Redwood City, CA). Of the genes increased in expression in PU.1-/-Spi-B-/- pro-B cell lines, the greatest proportion was comprised of genes encoding enzymes (28%) and signal transduction molecules (30%). Genes encoding DNA binding proteins such as transcription factors made up 5% of those increased, whereas genes encoding cell surface Ags and receptors also made up 5%. Increased unassigned expressed sequence tags made up a large proportion (13%). Genes encoding cell cycle factors and other various proteins made up only a small fraction (5%). The most unexpected finding in the increased gene list was the percentage of immunity related proteins (14%), which included multiple IgH transcripts (11 probe sets) (supplemental Table I and Table I).


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  		Table I. Fold changes of B-cell-specific transcripts expressed in PU.1-/-Spi-B-/- pro-B cell lines relative to wild-type cell lines

 
Genes decreased in expression may represent genes that require PU.1 or Spi-B function either directly or indirectly. Similar to those increased in PU.1-/-Spi-B-/- pro-B cells, the majority of the genes decreased in expression in PU.1-/-Spi-B-/- cell lines were those encoding enzymes (34%) and signal transduction molecules (27%). More genes encoding cell surface proteins were decreased (15%) than increased (5%). The proportions of genes encoding DNA binding proteins (5%) and cell cycle components (1%) decreased in expression were similar to those increased. The number of unassigned expressed sequence tags found to be decreased in expression (12%) was very similar to those increased. Despite the lower percentage of immunity related molecules (6%) decreased relative to those increased, an interesting result was that multiple Ig{kappa} L chain transcripts were significantly decreased in expression (nine probe sets) (supplemental Table II). Table I lists several genes that have B cell developmental relevance, which were selected for further analysis.

PU.1-/-Spi-B-/-cell lines have increased levels of IgH transcription and protein expression

Functional rearrangement and expression of the IgH locus is required to generate a functional BCR. Steady state levels of IgH transcripts were unexpectedly increased in PU.1-/-Spi-B-/- cell lines relative to wild-type cell lines. The increase in IgH transcripts in the PU.1-/-Spi-B-/- cell lines was observed with probe sets representing transcription from multiple regions of the IgH locus including those initiating in V regions and the DH-JH regions (Table I). The largest fold increase was seen in probe set 100361_f_at (53.1 ± 5.7-fold) representing germline IgH transcripts initiating in the DH-JH3 region. To confirm increased levels of H chain transcript expression, we used the {Delta}{Delta}Ct real-time RT-PCR method (41) to quantitate Iµ transcripts, which initiate within the IgH intronic enhancer (46). We found that Iµ transcripts were increased 23.5 ± 2.5-fold (n = 3) in PU.1-/-Spi-B-/- cell lines relative to wild-type cell lines (Fig. 2A). We also performed Western blotting to investigate whether increased concentrations of IgH protein were detectable in PU.1-/-Spi-B-/- cell lines. Cytoplasmic IgH protein is normally detectable before assembly with surrogate L-chain components to form a functional pre-BCR (1). The results shown in Fig. 2B show that these cell lines express high concentrations of full-length IgH protein, whereas wild-type pro-B cells expressed low concentrations. Because most PU.1-/-Spi-B-/- cells do not express detectable IgH on their surface (see Fig. 1C, first row) this likely represents protein accumulated in the cytoplasm of these cells. In summary, PU.1-/-Spi-B-/- cell lines have increased levels of IgH transcription and cytoplasmic IgH protein compared with wild-type cell lines. These results indicate that PU.1 and/or Spi-B are not required for IgH transcription in pro-B cell lines.



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  		FIGURE 2. Analysis of gene expression and H chain protein levels in PU.1-/-Spi-B-/- pro-B cells. A, Real-time RT-PCR analysis of gene transcripts in pro-B cells. Total RNA from PU.1-/-Spi-B-/- and wild-type pro-B cells was used to generate cDNA that was analyzed by real-time RT-PCR. cDNA concentrations were normalized to G6PDH expression. Fold change of H chain (Iµ) germline, {kappa}0 germline, RAG-1, and BLNK transcripts in PU.1-/-Spi-B-/- pro-B cells are expressed relative to wild-type pro-B cells using the {Delta}{Delta}Ct method (41 ). B, RT-PCR analysis of Fc{gamma}RIIb gene transcripts in pro-B cells. Total RNA from PU.1-/-Spi-B-/- and wild-type pro-B cells was used to generate cDNA that was analyzed by RT-PCR. Upper panel, Fc{gamma}RIIb transcripts; lower panel, {beta}-actin transcripts. C, Western blot analysis of Ig H chain in pro-B cells. Western blot analysis was performed on lysates from wild-type pro-B cells, PU.1-/-Spi-B-/- pro-B cells, and S17 stromal cells as a control. Blots were probed with anti-IgM and anti-{beta}-actin as control.

 
PU.1-/-Spi-B-/-cell lines have decreased levels of Ig{kappa} transcription

Results from nine independent probe sets on the U74A arrays indicated that steady state levels of Ig{kappa} transcripts were decreased in PU.1-/-Spi-B-/- pro-B cells relative to wild-type cells (Table I). To confirm this result, real-time PCR was performed to quantitate levels of Ig{kappa} germline transcripts. {kappa}0 transcripts initiate at a site 5' to the germline J{kappa}1 gene (47, 48). We found that {kappa}0 transcript levels were decreased by 2.9-fold in PU.1-/-Spi-B-/- pro-B cells relative to wild-type cells (Fig. 2A). Although there are two probe sets representing Ig{lambda} transcripts on the U74A arrays, these transcripts were scored as absent in all cell lines and also could not be detected by RT-PCR (data not shown). In summary, Ig{kappa} germline transcription is decreased in PU.1-/-Spi-B-/- pro-B cells relative to wild-type cells, suggesting that PU.1 and/or Spi-B might be required for optimal levels of Ig{kappa} transcription in IL-7-dependent pro-B cells.

PU.1-/-Spi-B-/-cell lines have decreased levels of RAG-1, Btk, and B cell linker protein (BLNK) transcription

Besides Ig{kappa}, several lymphocyte-restricted genes were decreased in expression in PU.1-/-Spi-B-/- pro-B cells. As suggested by the flow cytometric analysis and the large fold change using Affymetrix GeneChip analysis, Fc{gamma}RIIb was expressed in wild-type pro-B cell lines but was undetectable in PU.1-/-Spi-B-/- cell lines (Fig. 2B). Rearrangement of the Ig loci requires the action of the recombinase enzymes RAG-1 and RAG-2 (reviewed in Ref. 2). As measured on the U74A arrays, steady state levels of both RAG-1 and RAG-2 transcripts were reduced in the PU.1-/-Spi-B-/- cell lines. Using real-time RT-PCR, we found that RAG-1 transcripts were decreased by 4.1-fold in PU.1-/-Spi-B-/- relative to wild-type cell lines (Fig. 2A). Btk and BLNK transcripts were reduced by 4.5 ± 1.9-fold (one probe set, n = 3) and 4.8 ± 1.4 (two probe sets, n = 3) respectively, in PU.1-/-Spi-B-/- cells. Using real-time RT-PCR, we found that levels of BLNK transcription were decreased by 4.0-fold in PU.1-/-Spi-B-/- cells relative to wild-type cells (Fig. 2A). These results suggest that PU.1 and/or Spi-B might be required for optimal transcription of the Fc{gamma}RIIb, RAG-1, btk, and BLNK genes in IL-7-dependent pro-B cells.

Ig{kappa} but not Ig{lambda} transcription and rearrangement is increased in PU.1-/-Spi-B-/-cells after IL-7 withdrawal

Culture in IL-7 arrests developing B cells at the late pro-B cell stage (49). When IL-7-dependent cell lines are cultured on stromal cells in the absence of IL-7, they initiate apoptotic cell death but also initiate differentiation, by rearrangement of L chain genes, into surface Ig-expressing B cells (49, 50). Therefore, we withdrew IL-7 from culture media of wild-type and PU.1-/-Spi-B-/- pro-B cells for 72 h to test the ability of these cells to successfully rearrange and express Ig{kappa} L chain on their surface relative to wild-type pro-B cells. Ig{kappa} expression was measured using flow cytometry or using real-time RT-PCR for {kappa}0 transcripts. In the presence of IL-7, few wild-type (0.3 ± 0.1%, n = 3) or PU.1-/-Spi-B-/- cells (0.9 ± 0.2%, n = 3) expressed Ig{kappa} on their surface (Fig. 3A, upper panels). After IL-7 withdrawal, an increased fraction (2.5 ± 0.8%, n = 3) of wild-type pro-B cells expressed Ig{kappa} on their surface, similar to what has previously been reported (49). In contrast, a larger proportion of PU.1-/-Spi-B-/- cells rearranged and expressed Ig{kappa} on their surface (10.9 ± 1.5%, n = 3) compared with wild-type cell lines (Fig. 3A, lower panels). These results demonstrate that PU.1-/-Spi-B-/- pro-B cells have an increased ability to rearrange and express IgH and Ig{kappa} genes after IL-7 withdrawal.



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  		FIGURE 3. Induction of Ig{kappa} in pro-B cells after IL-7 withdrawal. To induce differentiation of pro-B cells, wild-type, and PU.1-/-Spi-B-/- pro-B cells were cultured in the presence or absence of IL-7 for 72 h. A, Representative histograms for flow cytometric analysis of pro-B cells cultured in the presence or absence of IL-7. Wild-type and PU.1-/-Spi-B-/- pro-B cells were analyzed for surface Ig{kappa} expression before and after culture in the presence or absence of IL-7 for 72 h. B, RT-PCR analysis for Ig{kappa} and Ig{lambda} germline transcripts. cDNA generated from total RNA extracted from pro-B cells cultured in the presence or absence of IL-7 was analyzed for expression of {kappa}0, {lambda}1 V-JC, or {lambda}2 germline transcripts and normalized to {beta}-actin over 30 cycles of amplification. C, Real-time RT-PCR analysis of {kappa}0 transcripts. Fold change of {kappa}0 germline transcripts was determined in pro-B cells cultured in the absence of IL-7 relative to pro-B cells grown in the presence of IL-7 by using cDNA generated in (B), normalized to G6PDH, and calculated using the {Delta}{Delta}Ct method (41 ).

 
Germline ("sterile") transcription of the Ig{kappa} and Ig{lambda} genes has been shown to be increased in pro-B cells after IL-7 withdrawal (51). Therefore, we performed standard RT-PCR and relative quantitation real-time RT-PCR to visualize the changes in Ig{kappa} and Ig{lambda} transcription after IL-7 withdrawal. As suggested by the cell surface expression of Ig{kappa}, PU.1-/-Spi-B-/- cells increased their levels of {kappa}0 transcription more than wild-type pro-B cells after IL-7 withdrawal (Fig. 3B, upper panels). Real-time RT-PCR analysis of {kappa}0 transcripts confirmed that IL-7 withdrawal stimulated transcription at the Ig{kappa} locus to a greater degree in PU.1-/-Spi-B-/- cells (14.6 ± 2.0-fold, n = 2) than in wild-type cells (5.5 ± 2.1-fold, n = 2) (Fig. 3C). The Ig{lambda} locus contains three functional genes, termed {lambda}1, {lambda}2, and {lambda}3 (51). We analyzed germline transcription from the {lambda}2 gene (51) as well as transcription of the VJ-rearranged {lambda}1 gene (52). After IL-7 withdrawal, wild-type pro-B cells increased germline {lambda}2 as well as VJ-rearranged {lambda}1 transcription (Fig. 3B, second and third rows). In contrast, in PU.1-/-Spi-B-/- cells, VJ-rearranged {lambda}1 transcription was undetectable, and germline {lambda}2 transcription was greatly impaired. In summary, PU.1-/-Spi-B-/- cell lines actually increase their level of Ig{kappa} transcription and V{kappa}-J{kappa} rearrangement to greater than wild-type levels after IL-7 withdrawal. In contrast, Ig{lambda} transcription is absent or greatly impaired, demonstrating that PU.1 and/or Spi-B are important for Ig{lambda} transcription.

The PU.1-/-Spi-B-/-phenotype can be recapitulated by ectopic expression of an inactive form of PU.1

To confirm that the phenotype displayed by PU.1-/-Spi-B-/- pro-B cells is primarily the result of loss of expression of PU.1 and/or Spi-B, we considered that ectopic expression of a mutant form of PU.1 lacking the N-terminal 100 amino acids might interfere with endogenous PU.1 and/or Spi-B activity in wild-type pro-B cell lines. Deletion of the first 100 amino acids of PU.1 (PU.1{Delta}N100) removes both the acidic and the glutamine-rich activation domains of the protein, as measured on multimerized PU.1 binding sites (53). PU.1{Delta}N100 is also incapable of rescuing B cell or macrophage development from PU.1-/- progenitors (40). Therefore, we constructed a recombinant retroviral vector containing a cDNA encoding the {Delta}N100 mutant form of PU.1 (MIG-PU.1{Delta}N100). A stably infected MIG-PU.1{Delta}N100 packaging cell line was then used to infect wild-type pro-B cell lines by coculture. Infected pro-B cell lines that expressed high levels of GFP protein were sorted using flow cytometry and propagated in cultures containing IL-7 and stromal cells. As controls, wild-type pro-B cell lines were infected with control (MIGR1) retrovirus or retrovirus encoding wild-type PU.1 (MIG-PU.1). Infection of wild-type pro-B cell lines with control retrovirus had no effect on proliferation of the cells. In contrast, infection with MIG-PU.1 retrovirus blocked proliferation of wild-type pro-B cells, resulting in the rapid loss of sorted cell lines (data not shown). Strikingly, infection with PU.1{Delta}N100 resulted in cell lines with significantly increased proliferation over control-infected cell lines (doubling time of 19.3 ± 0.9 h, n = 8, compared with 22.0 ± 1.2 h, n = 8). High levels of PU.1{Delta}N100 expression relative to expression of the wild-type protein was confirmed by Western blot analysis using an Ab recognizing the C terminus of PU.1 (Fig. 4A).



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  		FIGURE 4. Phenotypic analysis of wild-type cells ectopically expressing an inactive form of PU.1. Wild-type pro-B cells were infected with a GFP-expressing retrovirus alone or one encoding PU.1 truncated by the 100 N-terminal amino acids (PU.1{Delta}N100). Positively infected cells were sorted and cultured in IL-7 medium. A, Western blot analysis of retrovirally infected pro-B cell lines. Control S17 stromal cells, wild-type pro-B cells expressing control GFP retrovirus, and wild-type pro-B cells expressing PU.1{Delta}N100 retrovirus were probed with C-terminal targeted anti-PU.1 Ab that detected both endogenous and truncated PU.1. B, RT-PCR analysis of total RNA extracted from retrovirally infected pro-B cell lines. Fold change is determined in H chain (Iµ) germline, {kappa}0 germline, RAG-1, and BLNK transcript expression in wild-type expressing PU.1{Delta}N100 relative to wild-type expressing control GFP retrovirus using the {Delta}{Delta}Ct method (49 ).

 
To further characterize the wild-type-PU.1{Delta}N100 cell lines, these cells were analyzed for cell surface marker expression by flow cytometry and for gene expression using real-time RT-PCR analysis. We found that Fc{gamma}RIIb expression was reduced on WT-PU.1{Delta}N100 cells (MF = 101 ± 4.9, n = 2) relative to wild-type cells (MF = 205 ± 12.1, n = 2), indicating that PU.1 and/or Spi-B activity was reduced in these cells. We also observed that WT-PU.1{Delta}N100 cells expressed lower levels of c-Kit and IL-7R{alpha} than did the control infected wild-type cells. c-Kit levels on the WT-PU.1{Delta}N100 cells had a MF of 125 ± 4.3 (n = 3) while the wild-type levels were 130 ± 2.8 (n = 3). IL-7R{alpha} levels on the WT-PU.1{Delta}N100 cells had a MF of 32.6 ± 5.5 (n = 3) while the wild-type levels were 46.0 ± 5.7 (n = 3). CD19 and CD43 expression levels were equivalent on wild-type and WT-PU.1{Delta}N100 cells. Taken together, the phenotype exhibited by the WT-PU.1{Delta}N100 cells follows the trend of the PU.1-/-Spi-B-/- cells, although the differences in MF were smaller.

PU.1{Delta}N100 cell lines showed some similar patterns of gene expression to the PU.1-/-Spi-B-/- cells (Fig. 4B). Although smaller than in PU.1-/-Spi-B-/- cells, IgH germline transcripts were increased (1.4 ± 0.3-fold, n = 2) in WT-PU.1{Delta}N100 cell lines relative to wild-type cell lines. Transcription of Ig{kappa} was similarly reduced in WT-PU.1{Delta}N100 cell lines relative to control cells (4.9 ± 1.6-fold, n = 3). Finally, transcription of RAG-1 and BLNK were also reduced in WT-PU.1{Delta}N100 cell lines relative to control cells (1.58 ± 0.5-fold, n = 2; 1.6 ± 0.5-fold, n = 4). These changes followed the trend seen in wild-type vs PU.1-/-Spi-B-/- cells, as shown in Fig. 2, and therefore suggest that PU.1 and/or Spi-B are important for transcription of Ig{kappa}, RAG-1, and BLNK genes in pro-B cells. The milder phenotype suggests that ectopic expression of PU.1{Delta}N100 does not result in a complete block of PU.1 and/or Spi-B activity.

IL-7 withdrawal assays were used to induce Ig{kappa} expression in wild-type and WT-PU.1{Delta}N100 cells. Resembling the phenomenon seen in PU.1-/-Spi-B-/- cells, neither control-infected nor WT-PU.1{Delta}N100-infected cell lines contained significant numbers of Ig{kappa}-expressing cells when cultured in IL-7 (0.3 ± 0.2%, n = 2 and 0.5 ± 0.4%, n = 4, respectively) (Fig. 5A, upper row). However, a higher frequency of WT-PU.1{Delta}N100 cells expressed Ig{kappa} on their surface after 72-h withdrawal of IL-7 than control-infected wild-type cells (9.3 ± 2.0%, n = 4; compared with 1.4 ± 0.9%, n = 2) (Fig. 5A, middle row). IL-7 withdrawal from control-infected wild-type pro-B cells resulted in an increase in Ig{kappa} germline transcription (4.9 ± 1.2-fold, n = 2), based on real-time analysis. IL-7 withdrawal of WT-PU.1{Delta}N100-infected cells induced greater expression of Ig{kappa} germline transcripts than did IL-7 withdrawal of control infected wild-type pro-B cells (8.9 ± 1.3-fold, n = 2) (Fig. 5B). In contrast, germline transcription of the {lambda}2 gene was increased after IL-7 withdrawal of WT-PU.1{Delta}N100-infected pro-B cells (38.0 ± 7.0-fold, n = 2), but not to the same degree as in control cells (50.3 ± 3.8-fold, n = 2) (Fig. 5C). We note that in experiments performed with one particular wild-type cell line we were able to observe similar changes in Ig{kappa} and Ig{lambda} germline transcription in WT-PU.1{Delta}N100-infected cells after IL-7 withdrawal, but were unable to detect increased cell surface expression of Ig{kappa} using flow cytometry. Others have reported similar findings and suggest that this might be due to the particular cell line having a tendency to undergo unproductive Ig V{kappa}-J{kappa} rearrangements (54). In summary, the observation that the PU.1-/-Spi-B-/- phenotype can be reproduced in wild-type pro-B cells through expression of an activation-deficient form of PU.1 confirms that the phenotype seen in PU.1-/-Spi-B-/- pro-B cells is directly due to loss of function of PU.1 and/or Spi-B.



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  		FIGURE 5. Induction of Ig{kappa} in PU.1{Delta}N100-expressing wild-type pro-B cells after IL-7 withdrawal. A, Flow cytometric analysis of surface Ig{kappa} expression in retrovirally infected pro-B cells. Retrovirally infected pro-B cells were analyzed for surface {kappa} expression before and after culture in the presence or absence of IL-7 for 72 h; GFP expression is shown in the bottom panel as a control. B, Real-time RT-PCR analysis of {kappa}0 transcripts in retrovirally infected pro-B cells. Retrovirally infected pro-B cells were cultured in the presence or absence of IL-7 for 72 h and expression of {kappa}0 germline transcripts are shown as fold change in retrovirally infected pro-B cells after IL-7 withdrawal relative to transcript levels in cells cultured in the presence of IL-7 using the{Delta}{Delta}Ct method (49 ). C, Real-time RT-PCR analysis of {lambda}2 germline transcripts in retrovirally infected pro-B cells. Retrovirally infected pro-B cells were cultured in the presence or absence of IL-7 for 72 h and expression of {lambda}2 germline transcripts are shown as fold change in retrovirally infected pro-B cells after IL-7 withdrawal relative to transcript levels in cells cultured in the presence of IL-7 using the {Delta}{Delta}Ct method (49 ).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have analyzed IL-7-dependent, PU.1-/-Spi-B-/- cell lines for their phenotypes and patterns of gene expression relative to wild-type cell lines. PU.1 has been implicated in transcriptional regulation of the IgH, Ig{kappa}, and Ig{lambda} loci. Functional PU.1 binding sites have been extensively characterized in the IgH intronic enhancer (27); in Ig{kappa} V region promoters (24); in the Ig{kappa} 3' enhancer (24, 25); and in the Ig{lambda} 2–4 enhancer (26). Our most striking finding is that instead of being impaired, germline transcription of the IgH locus is significantly increased in the absence of PU.1 and Spi-B. The IgH transcripts that are increased were detected by multiple probe sets on the Affymetrix GeneChip, representing transcripts from the V region, the J region, and the intronic enhancer (Iµ). We confirmed, using relative quantitation real-time PCR, that levels of Iµ transcripts are up-regulated at least 10-fold in PU.1-/-Spi-B-/- cell lines. Therefore, these results suggest that PU.1 and/or Spi-B are not required for IgH transcription.

In contrast to the finding with IgH transcription, Ig{kappa} transcription was reproducibly decreased 2- to 3-fold in PU.1-/-Spi-B-/- pro-B cells relative to wild-type pro-B cells. When PU.1-/-Spi-B-/- cell lines were subjected to IL-7 withdrawal they exhibited increases in Ig{kappa} germline transcription and in VKJK recombination relative to wild-type cell lines, as demonstrated by increased numbers of cells expressing surface Ig{kappa} protein. These results imply that PU.1 and/or Spi-B are dispensable for Ig{kappa} transcription and rearrangement in the developing B cell. Sakano and colleagues (55) have previously suggested that PU.1 might have negative regulatory function in the Ig{kappa} 3' enhancer. They found that mutation of the PU.1 binding site in transgenic Ig{kappa} 3' enhancer constructs resulted in increased Ig{kappa} transcription and VKJK DNA rearrangement in pro-B cells, instead of restriction to the pre-B cell stage when it is normally initiated (55). These results, and our observations, suggest that PU.1 is not required to activate IgH and Ig{kappa} transcription and VDJ recombination, and that PU.1 may actually inhibit these functions in pro-B cells.

We found that Ig{lambda} transcription was severely reduced in PU.1-/-Spi-B-/- cell lines relative to wild-type cell lines after IL-7 withdrawal. The PU.1 binding sites in either the Ig{lambda} 2–4 or the Ig{lambda} 3–1 enhancers have been shown to be essential for the function of these enhancers when tested in transient transfection assays (26). Also, ectopic expression of a PU.1-IFN regulatory factor (IRF)-4 forced dimer lacking activation domains has been shown to silence Ig{lambda} transcription in a B cell line (56). Therefore our results provide further evidence that PU.1 and/or Spi-B are important for Ig{lambda} transcription and V{lambda}J{lambda} rearrangement. We speculate that the reason for the differential requirement of PU.1 and/or Spi-B for the Ig{lambda} vs the Ig{kappa} and IgH genes might be the nature of the enhancers present in these loci. The Ig{lambda} locus contains two enhancers that are nearly identical, and are believed to have arisen by gene duplication (57). Both of these enhancers contain PU.1-IRF-4 composite binding sites and therefore the cumulative effect of loss of PU.1 and/or Spi-B activity may be significant impairment of enhancer function (26). In the Ig{kappa} locus PU.1 interacts with the 3' enhancer but not the intronic enhancer (25), and therefore the intronic enhancer may compensate for the loss of activity in the 3' enhancer. In the IgH locus PU.1 interacts with the intronic enhancer but not the 3' enhancer region (27), and therefore the 3' enhancer may likewise be able to compensate for loss of activity of the intronic enhancer. Additional experiments must be performed to test this hypothesis.

In further support of the validity of our results, we interfered with PU.1 and/or Spi-B function in wild-type pro-B cell lines by ectopic expression of an inactive mutant form of PU.1 (PU.1{Delta}N100). We expect that PU.1{Delta}N100 protein functions by displacement of PU.1 and/or Spi-B at DNA binding sites. Alternatively, ectopic expression of PU.1{Delta}N100 protein may result in formation of aberrant protein complexes due to interactions with other proteins. Therefore, a caveat of this approach is that PU.1{Delta}N100 protein may modulate the function of factors other than PU.1 and Spi-B. We found that ectopic expression of PU.1{Delta}N100 protein promoted proliferation of wild-type pro-B cell lines, whereas wild-type PU.1 protein inhibited their proliferation. As shown in Figs. 4 and 5, wild-type pro-B cells that ectopically express PU.1{Delta}N100 acquire a phenotype that is similar to that possessed by PU.1-/-Spi-B-/- cell lines. In addition to increased proliferation, these cell lines have decreased germline Ig{kappa} and RAG-1 transcription. Withdrawal of IL-7 from wild-type-PU.1{Delta}N100 cell lines results in an increase in Ig{kappa} transcription relative to wild-type cell lines, and increased VKJK rearrangement as demonstrated by surface expression of Ig{kappa}. The phenotype of these cells is milder than that of PU.1-/-Spi-B-/- cells, probably due to an incomplete block of PU.1 and/or Spi-B activity. In summary, interference with PU.1 and/or Spi-B activity does not inhibit but rather promotes Ig transcription and VDJ rearrangement.

PU.1-/-Spi-B-/- and WT-{Delta}N100 (low/mutant) pro-B cell lines have altered phenotypic characteristics relative to wild-type pro-B cell lines. A hypothesis that integrates most of our observations is that the PU.1/Spi-B low/mutant cell lines are advanced in their state of differentiation relative to wild-type pro-B cell lines, for the following reasons (see Fig. 6). In vivo, large pre-B cells proliferate more rapidly than pro-B cells, as a result of signaling through the combination of the IL-7R and pre-BCR (6). This is consistent with the observation that PU.1/Spi-B low/mutant cell lines proliferate more rapidly than wild-type cells. Second, IL-7R{alpha} and c-Kit expression are normally down-regulated during the pro-B to pre-B cell transition, consistent with our observations that they are down-regulated in PU.1/Spi-B low/mutant cell lines (Fig. 1). Third, CD25 expression is increased on PU.1-/-Spi-B-/- cell lines, consistent with the observation that CD25 expression is initiated at the pre-B cell stage (45). Fourth, VH-DHJH recombination is normally completed during the pro-B to pre-B cell transition, followed by deposition of pre-BCR on the cell surface, consistent with our observation that the PU.1/Spi-B low/mutant cell lines have acquired large amounts of cytoplasmic H chain protein. Because the PU.1/Spi-B low/mutant cell lines have not yet acquired significant levels of surface IgH expression, this is consistent with the idea that they are not yet differentiated to pre-B cells. Fifth, expression of the RAG-1 and RAG-2 genes are normally down-regulated during the pro-B to pre-B cell transition, as VH-DHJH recombination is completed and these cells enter a proliferative burst phase. As shown in Table I and in Figs. 2 and 4, PU.1/Spi-B low/mutant cells also have lower levels of RAG-1 transcription than wild-type cell lines. Finally, the expression of CD43 on PU.1 low/mutant cells (Fig. 1 and data not shown) indicates that these cells have not differentiated to the small pre-B cell stage. Therefore, although PU.1/Spi-B low/mutant cell lines have acquired many of the characteristics of large pre-B cells, they remain arrested in a stage of development just before the pre-B cell stage (Fig. 6). We suggest that lowering the level of PU.1 and/or Spi-B activity in late pro-B cells promotes differentiation to this later stage.



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  		FIGURE 6. Model of PU.1 function in early B cell development.

 
There are several genes whose level of expression in wild-type vs PU.1/Spi-B low/mutant cell lines is not predicted according to the hypothesis described above. BLNK (SLP-65) is an adaptor protein required for signaling through the BCR and for normal B cell development (58, 59). There is no suggestion in the literature that BLNK transcription should be down-regulated during the pro-B to pre-B cell transition, but we found that BLNK transcription was reduced in PU.1/Spi-B low/mutant cells by an average of 3-fold. Therefore BLNK may represent a novel target of PU.1 and/or Spi-B during B cell development. Secondly, Btk is a B cell-restricted protein tyrosine kinase required for normal BCR signaling and for normal B cell development (60, 61). The btk gene is known to contain PU.1 binding sites in its promoter region (32, 62). Because btk is also not expected to be down-regulated during the pro-B to pre-B transition, our finding that btk transcription is decreased in PU.1/Spi-B low/mutant cells suggests that it is indeed a target gene. Further biochemical experiments will have to be performed to confirm that BLNK and btk, as well as other genes shown in Table I, are targets of PU.1 and/or Spi-B in the B cell lineage.

What are the possible mechanisms by which interference with PU.1 and/or Spi-B activity promotes Ig transcription? First, PU.1 and/or Spi-B occupancy of Ig promoters and enhancers may directly inhibit transcription and VDJ rearrangement. This seems unlikely because most studies have demonstrated that these proteins function as transcriptional activators. Therefore it seems more likely that in the absence of PU.1 and/or Spi-B, other transcription factors might be increased in expression or activity such that they can compensate for the reduced PU.1 and/or Spi-B activity. We note that in the Affymetrix GeneChip analyses, the expression of several transcription factors, including IRF-4 and Pax-5, are significantly up-regulated in PU.1-/-Spi-B-/- cell lines (supplemental Table II). IRF-4 requires an interaction partner, such as PU.1, to efficiently bind DNA and activate transcription (63). It has recently been shown that E47 can bind to DNA cooperatively with IRF-4 to synergistically activate transcription of Ig germline transcription (64). Therefore it is possible that these types of transcription factor interactions could compensate for the loss of PU.1 and/or Spi-B activity. It is also possible that other uncharacterized Ets-family transcription factors might occupy PU.1 and/or Spi-B binding sites in their absence and bypass the normal requirement for these factors. Additional experiments will be required to clarify which mechanisms explain the increased rates of Ig transcription.

Based on our suggestion that interference with PU.1 and/or Spi-B activity in pro-B cells promotes differentiation to a stage intermediate between late pro-B and large pre-B cells, we speculate that modulation of the activity of these factors may normally occur during the late pro-B to large pre-B cell transition in B cell development (Fig. 6). Intriguingly, the transcription factor Spi-C (Prf), which is closely related to PU.1 and Spi-B, was initially cloned in a screen for factors which could occupy PU.1 DNA binding sites (17). Spi-C has extremely weak transcription-promoting activity, and unlike PU.1 and Spi-B, cannot interact with IRF-4 to synergistically activate gene expression on Ets/IRF composite elements (17, 65). Spi-C has an extremely restricted pattern of expression: its transcription is initiated at the pre-B cell stage of B cell development, it is expressed in mature B cells, and its transcription is extinguished in plasma cells (16, 17). Taken together, these results suggest that the normal function of Spi-C might be as a negative regulator of PU.1/Spi-B activity (17, 65). We speculate that Spi-C may normally interfere with PU.1/Spi-B activity starting at the large pre-B cell stage of B cell development, and that this negative regulation might be required for normal B cell development.

In summary, we have demonstrated that contrary to what has been predicted, PU.1 and/or Spi-B do not seem to be required for transcription and VDJ rearrangement of the IgH and Ig{kappa} loci in pro-B cell lines. However, our evidence demonstrates that PU.1 and Spi-B are required for transcription of the Ig{lambda} locus. Taken together with other phenotypic changes in PU.1-/-Spi-B-/- and wild-type-PU.1{Delta}N100 cell lines relative to control cell lines, our observations suggest that reduction of PU.1 activity in pro-B cell lines promotes the differentiation of these cells to a stage intermediate between late pro-B cells and large pre-B cells (Fig. 6). We speculate that a reduction in PU.1 and/or Spi-B activity starting before the large pre-B cell stage might be a normal feature of B cell development. Additional experiments will have to be performed to test this hypothesis and distinguish between various mechanisms by which PU.1 activity might be regulated.


   Acknowledgments
 
We thank Lee-Ann Garret-Sinha (State University of New York, Buffalo, NY) and Celeste Simon (University of Pennsylvania, Philadelphia, PA) for giving us PU.1-/-Spi-B-/- (PUB) mice and Antonius Rolink (University of Basel, Basel, Switzerland) for generously providing the J558-IL-7 cell line. We thank Harinder Singh and Jagan Pongubala (University of Chicago, Chicago, IL) for helpful comments and discussion, and Isaac Houston (University of Cincinnati, Cincinnati, OH) and Pierre-Yves Berclaz (Cincinnati Children’s Hospital, Cincinnati, OH) for critically reading the manuscript.


   Footnotes
 
1 This work was supported in part by a Functional Genomics Seed Grant (to B.L.S.) from the University of Cincinnati, and by Grant Number 1R01AI052175-01A1 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Rodney P. DeKoter, Department of Molecular Genetics, University of Cincinnati College of Medicine, Medical Sciences Building 3006, 231 Albert Sabin Way, Cincinnati, OH 45267-0524. E-mail address: dekoterp{at}ucmail.uc.edu Back

3 Abbreviations used in this paper: pro-B, progenitor B; BCR, B cell receptor; d.p.c., days postcoitum; Btk, Bruton’s tyrosine kinase; GFP, green fluorescent protein; Ct, threshold cycle; MF, mean fluorescence; BLNK, B cell linker protein; IRF, IFN regulatory factor. Back

Received for publication May 29, 2003. Accepted for publication October 27, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rolink, A. G., E. ten Boekel, T. Yamagami, R. Ceredig, J. Andersson, F. Melchers. 1999. B cell development in the mouse from early progenitors to mature B cells. Immunol. Lett. 68:89.[Medline]
  2. Hardy, R. R., K. Hayakawa. 2001. B cell development pathways. Annu. Rev. Immunol. 19:595.[Medline]
  3. Lu, L., D. G. Osmond. 2000. Apoptosis and its modulation during B lymphopoiesis in mouse bone marrow. Immunol. Rev. 175:158.[Medline]
  4. Hirose, J., T. Kouro, H. Igarashi, T. Yokota, N. Sakaguchi, P. W. Kincade. 2002. A developing picture of lymphopoiesis in bone marrow. Immunol. Rev. 189:28.[Medline]
  5. Igarashi, H., S. C. Gregory, T. Yokota, N. Sakaguchi, P. W. Kincade. 2002. Transcription from the RAG1 locus marks the earliest lymphocyte progenitors in bone marrow. Immunity 17:117.[Medline]
  6. Fleming, H. E., C. J. Paige. 2002. Cooperation between IL-7 and the pre-B cell receptor: a key to B cell selection. Semin. Immunol. 14:423.[Medline]
  7. Chowdhury, D., R. Sen. 2003. Transient IL-7/IL-7R signaling provides a mechanism for feedback inhibition of immunoglobulin heavy chain gene rearrangements. Immunity 18:229.[Medline]
  8. Kee, B. L., C. Murre. 2001. Transcription factor regulation of B lineage commitment. Curr. Opin. Immunol. 13:180.[Medline]
  9. Hardy, R. R.. 2003. B-cell commitment: deciding on the players. Curr. Opin. Immunol. 15:158.[Medline]
  10. 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]
  11. Scott, E. W., R. C. Fisher, M. C. Olson, E. W. Kehrli, M. C. Simon, H. Singh. 1997. PU.1 functions in a cell-autonomous manner to control the differentiation of multipotential lymphoid-myeloid progenitors. Immunity 6:437.[Medline]
  12. DeKoter, R. P., H.-J. Lee, H. Singh. 2002. PU.1 regulates expression of the interleukin-7 receptor in lymphoid progenitors. Immunity 16:297.[Medline]
  13. McKercher, S. R., B. E. Torbett, K. L. Anderson, G. W. Henkel, D. G. Vestal, H. Baribault, M. Klemsz, A. J. Feeney, G. E. Wu, C. J. Paige, R. A. Maki. 1996. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 15:5647.[Medline]
  14. Anderson, K. L., K. A. Smith, K. Conners, S. R. McKercher, R. A. Maki, B. E. Torbett. 1998. Myeloid development is selectively disrupted in PU.1 null mice. Blood 91:3702.[Abstract/Free Full Text]
  15. Ray, D., R. Bosselat, J. Ghysdael, M. G. Mattei, A. Tavitian, F. Moreau-Gachelin. 1992. Characterization of Spi-B, a transcription factor related to the putative oncoprotein Spi-1/PU.1. Mol. Cell. Biol. 12:4297.[Abstract/Free Full Text]
  16. Bemark, M., A. Martensson, D. Liberg, T. Leanderson. 1999. Spi-C, a novel Ets protein that is temporally regulated during B lymphocyte development. J. Biol. Chem. 274:10259.[Abstract/Free Full Text]
  17. Hashimoto, S., H. Nishizumi, R. Hayashi, A. Tsuboi, F. Nagawa, T. Takemori, H. Sakano. 1999. Prf, a novel Ets family protein that binds to the PU.1 binding motif, is specifically expressed in restricted stages of B cell development. Int. Immunol. 11:1423.[Abstract/Free Full Text]
  18. 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]
  19. 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]
  20. Su, G. H., H. S. Ip, B. S. Cobb, M. M. Lu, H. M. Chen, M. C. Simon. 1996. The Ets protein Spi-B is expressed exclusively in B cells and T cells during development. J. Exp. Med. 184:203.[Abstract/Free Full Text]
  21. Su, G. H., H. M. Chen, N. Muthusamy, L. A. Garrett-Sinha, D. Baunoch, D. G. Tenen, M. C. Simon. 1997. Defective B cell receptor-mediated responses in mice lacking the Ets protein, Spi-B. EMBO J. 16:7118.[Medline]
  22. Garrett-Sinha, L. A., G. H. Su, S. Rao, S. Kabak, Z. Hao, M. R. Clark, M. C. Simon. 1999. PU.1 and Spi-B are required for normal B cell receptor-mediated signal transduction. Immunity 10:399.[Medline]
  23. Rao, S., L. A. Garrett-Sinha, J. Yoon, M. C. Simon. 1999. The Ets factors PU.1 and Spi-B regulate the transcription in vivo of P2Y10, a lymphoid restricted heptahelical receptor. J. Biol. Chem. 274:34245.[Abstract/Free Full Text]
  24. Schwarzenbach, H., J. W. Newell, P. Matthias. 1995. Involvement of the Ets family factor PU.1 in the activation of immunoglobulin promoters. J. Biol. Chem. 270:898.[Abstract/Free Full Text]
  25. Pongubala, J. M., S. Nagulapalli, M. J. Klemsz, S. R. McKercher, R. A. Maki, M. L. Atchison. 1992. PU.1 recruits a second nuclear factor to a site important for immunoglobulin {kappa} 3' enhancer activity. Mol. Cell. Biol. 12:368.[Abstract/Free Full Text]
  26. Eisenbeis, C. F., H. Singh, U. Storb. 1993. PU.1 is a component of a multiprotein complex which binds an essential site in the murine immunoglobulin {lambda}-2-4 enhancer. Mol. Cell. Biol. 13:6452.[Abstract/Free Full Text]
  27. Nelsen, B., G. Tian, B. Erman, J. Gregoire, R. Maki, B. Graves, R. Sen. 1993. Regulation of lymphoid-specific immunoglobulin µ heavy chain gene enhancer by ETS-domain proteins. Science 261:82.[Abstract/Free Full Text]
  28. 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]
  29. Hu, M., D. Krause, M. Greaves, S. Sharkis, M. Dexter, C. Heyworth, T. Enver. 1997. Multilineage gene expression precedes commitment in the hemopoietic system. Genes Dev. 11:774.[Abstract/Free Full Text]
  30. Himmelmann, A., A. Riva, G. L. Wilson, B. P. Lucas, C. Thevenin, J. H. Kehrl. 1997. PU.1/Pip and basic helix loop helix zipper transcription factors interact with binding sites in the CD20 promoter to help confer lineage- and stage-specific expression of CD20 in B lymphocytes. Blood 90:3984.[Abstract/Free Full Text]
  31. Hagman, J., R. Grosschedl. 1992. An inhibitory carboxyl-terminal domain in Ets-1 and Ets-2 mediates differential binding of ETS family factors to promoter sequences of the mb-1 gene. Proc. Natl. Acad. Sci. USA 89:8889.[Abstract/Free Full Text]
  32. Muller, S., P. Sideras, C. I. Smith, K. G. Xanthopoulos. 1996. Cell specific expression of human Bruton’s agammaglobulinemia tyrosine kinase gene (Btk) is regulated by Sp1- and Spi-1/PU.1-family members. Oncogene 13:1955.[Medline]
  33. Ma, E. L.-G., K. Maeda, H. Nomiyama, M. Nakao, K. Kuwahara, N. Sakaguchi. 2001. PU.1 is involved in the regulation of B lineage-associated and developmental stage-dependent expression of the germinal center-associated DNA primase GANP. J. Biol. Chem. 276:48000.[Abstract/Free Full Text]
  34. 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]
  35. Kinsella, T. M., G. P. Nolan. 1996. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum. Gene Ther. 7:1405.[Medline]
  36. Markowitz, D., S. Goff, A. Bank. 1988. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J. Virol. 62:1120.[Abstract/Free Full Text]
  37. Collins, L. S., K. Dorshkind. 1987. A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis. J. Immunol. 138:1082.[Abstract/Free Full Text]
  38. Winkler, T. H., F. Melchers, A. G. Rolink. 1995. Interleukin-3 and interleukin-7 are alternative growth factors for the same B-cell precursors in the mouse. Blood 85:2045.[Abstract/Free Full Text]
  39. Pear, W. S., J. P. Miller, L. Xu, J. C. Pui, B. Soffer, R. C. Quackenbush, A. M. Pendergast, R. Bronson, J. C. Aster, M. L. Scott, D. Baltimore. 1998. Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood 92:3780.[Abstract/Free Full Text]
  40. DeKoter, R. P., H. Singh. 2000. Regulation of B lymphocyte and macrophage development by graded expression of PU.1. Science 288:1439.[Abstract/Free Full Text]
  41. Pfaffl, M. W.. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29:e45.[Abstract/Free Full Text]
  42. Engel, P., L. J. Zhou, D. C. Ord, S. Sato, B. Koller, T. F. Tedder. 1995. Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule. Immunity 3:39.[Medline]
  43. Berclaz, P. Y., Y. Shibata, J. A. Whitsett, B. C. Trapnell. 2002. GM-CSF, via PU.1, regulates alveolar macrophage Fc{gamma}R-mediated phagocytosis and the IL-18/IFN-{gamma}-mediated molecular connection between innate and adaptive immunity in the lung. Blood 100:4193.[Abstract/Free Full Text]
  44. Hardy, R. R., C. E. Carmack, S. A. Shinton, J. D. Kemp, K. Hayakawa. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173:1213.[Abstract/Free Full Text]
  45. Rolink, A., U. Grawunder, T. H. Winkler, H. Karasuyama, F. Melchers. 1994. IL-2 receptor {alpha} chain (CD25, TAC) expression defines a crucial stage in pre-B cell development. Int. Immunol. 6:1257.[Abstract/Free Full Text]
  46. Schlissel, M. S., L. M. Corcoran, D. Baltimore. 1991. Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription. J. Exp. Med. 173:711.[Abstract/Free Full Text]
  47. Van Ness, B. G., M. Weigert, C. Coleclough, E. L. Mather, D. E. Kelley, R. P. Perry. 1981. Transcription of the unrearranged mouse C {kappa} locus: sequence of the initiation region and comparison of activity with a rearranged V{kappa}-C{kappa} gene. Cell 27:593.[Medline]
  48. Schlissel, M. S., D. Baltimore. 1989. Activation of immunoglobulin {kappa} gene rearrangement correlates with induction of germline {kappa} gene transcription. Cell 58:1001.[Medline]
  49. Rolink, A., A. Kudo, F. Melchers. 1991. Long-term proliferating early pre-B-cell lines and clones with the potential to develop to surface immunoglobulin-positive, mitogen-reactive B-cells in vitro and in vivo. Biochem. Soc. Trans. 19:275.[Medline]
  50. Melamed, D., J. A. Kench, K. Grabstein, A. Rolink, D. Nemazee. 1997. A functional B cell receptor transgene allows efficient IL-7-independent maturation of B cell precursors. J. Immunol. 159:1233.[Abstract]
  51. Engel, H., A. Rolink, S. Weiss. 1999. B cells are programmed to activate {kappa} and {lambda} for rearrangement at consecutive developmental stages. Eur. J. Immunol. 29:2167.[Medline]
  52. Sun, T., U. Storb. 2001. Insertion of phosphoglycerine kinase (PGK)-neo 5' of J{lambda}1 dramatically enhances VJ{lambda}1 rearrangement. J. Exp. Med. 193:699.[Abstract/Free Full Text]
  53. Klemsz, M. J., R. A. Maki. 1996. Activation of transcription by PU.1 requires both acidic and glutamine domains. Mol. Cell. Biol. 16:390.[Abstract]
  54. Ray, R. J., A. Stoddart, J. L. Pennycook, H. O. Huner, C. Furlonger, G. E. Wu, C. J. Paige. 1998. Stromal cell-independent maturation of IL-7-responsive pro-B cells. J. Immunol. 160:5886.[Abstract/Free Full Text]
  55. Hayashi, R., T. Takemori, M. Kodama, M. Suzuki, A. Tsuboi, F. Nagawa, H. Sakano. 1997. The PU.1 binding site is a cis-element that regulates pro-B/pre-B specificity of V{kappa}-J{kappa} joining. J. Immunol. 159:4145.[Abstract]
  56. Brass, A. L., A. Q. Zhu, H. Singh. 1999. Assembly requirements of PU.1-Pip (IRF-4) activator complexes: inhibiting function in vivo using fused dimers. EMBO J. 18:977.[Medline]
  57. Hagman, J., C. M. Rudin, D. Haasch, D. Chaplin, U. Storb. 1990. A novel enhancer in the immunoglobulin {lambda} locus is duplicated and functionally independent of NF {kappa}B. Genes Dev. 4:978.[Abstract/Free Full Text]
  58. Jumaa, H., B. Wollscheid, M. Mitterer, J. Wienands, M. Reth, P. J. Nielsen. 1999. Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65. Immunity 11:547.[Medline]
  59. Pappu, R., A. M. Cheng, B. Li, Q. Gong, C. Chiu, N. Griffin, M. White, B. P. Sleckman, A. C. Chan. 1999. Requirement for B cell linker protein (BLNK) in B cell development. Science 286:1949.[Abstract/Free Full Text]
  60. Kerner, J. D., M. W. Appleby, R. N. Mohr, S. Chien, D. J. Rawlings, C. R. Maliszewski, O. N. Witte, R. M. Perlmutter. 1995. Impaired expansion of mouse B cell progenitors lacking Btk. Immunity 3:301.[Medline]
  61. Satterthwaite, A. B., H. Cheroutre, W. N. Khan, P. Sideras, O. N. Witte. 1997. Btk dosage determines sensitivity to B cell antigen receptor cross-linking. Proc. Natl. Acad. Sci. USA 94:13152.[Abstract/Free Full Text]
  62. Muller, S., A. Maas, T. C. Islam, P. Sideras, G. Suske, S. Philipsen, K. G. Xanthopoulos, R. W. Hendriks, C. I. Smith. 1999. Synergistic activation of the human Btk promoter by transcription factors Sp1/3 and PU.1. Biochem. Biophys. Res. Commun. 259:364.[Medline]
  63. Brass, A. L., E. Kehrli, C. F. Eisenbeis, U. Storb, H. Singh. 1996. Pip, a lymphoid-restricted IRF, contains an autoinhibitory domain that is important for ternary complex formation with the Ets factor, PU.1. Genes Dev. 10:2335.[Abstract/Free Full Text]
  64. Nagulapalli, S., A. Goheer, L. Pitt, L. P. McIntosh, M. L. Atchison. 2002. Mechanism of e47-Pip interaction on DNA resulting in transcriptional synergy and activation of immunoglobulin germ line sterile transcripts. Mol. Cell. Biol. 22:7337.[Abstract/Free Full Text]
  65. Carlsson, R., C. Persson, T. Leanderson. 2003. SPI-C, a PU-box binding ETS protein expressed temporarily during B-cell development and in macrophages, contains an acidic transactivation domain located to the N-terminus. Mol. Immunol. 39:1035.[Medline]



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