SEARCH

SEARCH BY CITATION

Keywords:

  • Pax5;
  • STAT5;
  • IL-7;
  • Early B cell factor;
  • Transcriptional regulation

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Pax5 is an essential transcription factor for B cell development, and it is reported that Pax5 expression was reduced in the IL-7 receptor (IL-7R) knockout mouse. To investigate whether signals from the IL-7R regulate Pax5 transcription, we searched the consensus sequence of signal transducers and activators of transcription (STAT) in the Pax5 promoter region, since STAT is one of the components of cytokine signal transduction. A STAT-binding motif, termed SBM, was identified at 1,118 bp upstream of the transcriptional start site, and SBM completely overlapped with the binding sitefor early B cell factor (EBF). STAT5 was phosphorylated in the presence of IL-7 in the IL-7-dependent preB cell line, PreBR1, and phosphorylated-STAT5 as well as EBF was found to bind to the SBM. Moreover, we also revealed STAT5 binding to SBM in PreBR1 cells by chromatin immunoprecipitation assay. Transient co-transfection of reporter genes together with expression vectors of a constitutive active form of STAT5 and EBF into NIH3T3 cells demonstrated that STAT5 enhanced EBF-regulating transcription. Our results suggest that STAT5 phosphorylated by IL-7 can directly up-regulate Pax5 transcription in early B cells.

Abbreviations:
EBF:

Early B cell factor

EMSA:

Electrophoretic mobility shift assay

IL-7R:

IL-7 receptor

STAT:

Signal transducers and activators of transcription

SBM:

STAT-binding motif

ChIP:

Chromatin immunoprecipitation

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The transcription factor Pax5 is essential for B cell development 1. Although Pax5 is expressed during all stages of B lymphopoiesis, with the exception of terminally differentiated plasma cells 24, VH to DHJH recombination at the immunoglobulin heavy chain gene locus is impaired in Pax5 knockout (Pax5/–) mice, indicating that Pax5 plays an important role in the development from preB-I (DHJH rearranged) cells to preB-II (VHDHJH rearranged) cells 1. Pax5 expression is very strictly regulated during B lymphopoiesis, resulting in monoallelic transcription of Pax5 in preB cells and biallelic transcription in immature B cells 5.

During early B cell differentiation in vivo, pre-BI cells are proliferated by interleukin-7 (IL-7) secreted from stromal cells 6, thus these cells can be expanded in vitro using a recombinant IL-7 in combination with stromal cell feeders. IL-7 receptor α knockout (IL-7Rα–/–) mice show a defect in early B cell development, which is phenotypically similar to that of Pax5–/– mice, although the B cell defect in IL-7Rα–/– is incomplete 7. However, the phenotypic similarity of these two knockout mice raised the question whether IL-7 signaling regulates Pax5 transcription. In IL-7Rα–/– mice, the Pax5 transcription level was reduced in bone marrow cells compared to cells from heterozygous control mice 7. Moreover, when human fetal bone marrow cells were cultured in the presence of IL-7, CD19 expression, which is a target of Pax5 1, was increased three- to fourfold 8. These results indicate that IL-7 signaling could regulate Pax5 transcription. Recently, the mouse Pax5 promoter region was investigated, and it wasshown that the transcription factor EBF (early B cell factor) bound directly to the promoter region, thereby inducing Pax5 transcription 9.

Signal transducers and activators of transcription (STAT) are indispensable for intracellular signaling after stimulation with cytokines. In fact, the IL-7/IL-7R association mainly leads to the phosphorylation, dimerization and translocation of STAT5 to the nucleus, resulting in the regulation of target genes after binding to the consensus sequence, TTCNNNGAA 10. It isreported that the STAT5 deficient mice exhibited a reduction of preB and proB cells in the bone marrow 11.

These observations led us to investigate whether STAT5 is activated by IL-7, and whether it binds to the Pax5 promoter region thereby controlling transcription. In this study, we report that the sequence for STAT binding in the Pax5 promoter was found by database program searches. EMSA revealed that phosphorylated-STAT5 (p-STAT5) bound to this sequence in the presence of IL-7. Chromatin immunoprecipitation (ChIP) assay also demonstrated STAT5 binding to SBM in PreBR1 cells. Furthermore, a constitutive active form of STAT5 up-regulated EBF-regulating transcription in reporter gene assays. From these results, we demonstrate that STAT5 can directly regulate the transcription of Pax5.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 A STAT-binding motif is located within the Pax5 promoter region and overlaps with an EBF-binding site

To investigate whether STAT transcription factors bind to the mouse Pax5 promoter region, we first searched the consensus sequence for STAT binding (GenBank no. 148961) using the TFSEARCH program (http://pdap1.trc.rwcp.or.jp/research/db/TF-SEARCH.html). A potential STAT-binding motif, termed SBM, was identified at 1,118 bp upstream of exon1A (Fig. 1), although the sequence was not completely identical to the consensus sequence (TTCNNNGAA) 10. Interestingly, the SBM overlapped entirely with the binding site for EBF described by O'Riordan et al. 9.

thumbnail image

Figure 1. Analysis of the STAT binding motif in the Pax5 promoter region. A STAT-binding motif (SBM) was searched in the sequence of 1.8 kb upstream of Pax5 exon1A (GenBank no. AF148961) using the TFSEARCH database. The SBM was identified at –1,118 bp from exon 1A (boxed), which was found to entirely overlap with a consensus binding site for EBF. The sequence indicated in bold letters was used for EMSA.

Download figure to PowerPoint

2.2 Phosphorylation of STAT5 upon IL-7 stimulation of PreBR1 cells

IL-7-induced phosphorylation of STAT5 has been reported in the T cell line, CT6 12, as well as in human peripheral blood T lymphoblasts 13. Therefore, we were prompted to examine STAT5 phosphorylation in a preB cell line, PreBR1, the mouse bone marrow-derived IL-7-dependent cell line displaying a phenotype of large pre-BII cells 14. PreBR1 cells were treated with recombinant IL-7 (1,000 U/ml) for 5, 15 and 30 min, and after starvation of IL-7, nuclear extracts were prepared. Western blot analysis revealed that STAT5 was phosphorylated already after 5 min of IL-7 treatment, whereas it was not detected in untreated cells (0 min; Fig. 2 upper panel). The amount of p-STAT5 in nuclei increased gradually after IL-7 treatment, indicative of the nuclear translocation of p-STAT5. Western blot analysis using antibodies specific for phosphorylated STAT5 revealed a strong upper band and a weaker lower band (upper panel in Fig. 2). In contrast, anti-STAT5a specific antibody detected only one immunoreactive band. However, the antibody recognizing both STAT5a and STAT5b again detected two immunoreactive bands, representing STAT5a and STAT5b. These results showed that both STAT5a and STAT5b were phosphorylated in PreBR1 cells in the presence of IL-7.

thumbnail image

Figure 2. Phosphorylation of STAT5 in PreBR1 cells upon stimulation of IL-7. IL-7 was removed from PreBR1 cell cultures of 1×107–2×107 cells for 2–4 h, and rIL-7 (1,000 U/ml) was then added to the cultures for the indicated times. Then, the nuclear extracts were prepared and Western blot analysis was performed with anti-phospho-STAT5 (top), anti-STAT5a (middle) or the antibody that recognizes both STAT5a and b (bottom).

Download figure to PowerPoint

2.3 Binding of proteins to SBM in extracts from PreBR1 cells upon IL-7 stimulation

To identify proteins binding to the SBM site, EMSA was performed using a 21 bp synthetic target sequence containing the SBM (the sequence shown in bold in Fig. 1), and nuclear extracts were prepared from PreBR1 cells treated with or without IL-7. EMSA revealed an additional strong band (Fig. 3, lane 3, arrow) that was detected with slower electrophoretic mobility in extracts from cells treated with IL-7.

thumbnail image

Figure 3. Analysis of proteins binding to the SBM upon IL-7 stimulation. PreBR1 cells were treated with recombinant IL-7 and nuclear extracts were prepared. The nuclear extracts and the 32P-labeled SBM probe were subjected to EMSA as described in Sect. 4.

Download figure to PowerPoint

2.4 STAT5 and EBF binding to the SBM

To identify the proteins contained in the gel-retarded complex, we performed EMSA using antibodies specific for STAT5a/b and EBF, respectively. The band signal induced by IL-7 was completely diminished by addition of anti-STAT5a/b antibody (Fig. 4, lane 1). Furthermore, when anti-EBF antibody was added, we observed that the signal was almost disappeared, and the super-shifted band was observed (lane 3, SS). These results demonstrated that the observed band in EMSA, appeared with IL-7 treatment, contained p-STAT5 as well as EBF.

thumbnail image

Figure 4. Binding analysis of STAT5 by EMSA. The nuclear extracts from PreBR1 cells were prepared as described in Fig. 2. For EMSA, the extracts were preincubated with anti-STAT5 (lane2) or anti-EBF (lane3) for 1.5 h on ice prior to the addition of the labeled SBM probe. SS: supershift.

Download figure to PowerPoint

2.5 STAT5 binds to SBM in PreBR1 cells

To detect whether STAT5 binds to the SBM in PreBR1 cells, we used the ChIP technique. PreBR1 cells were stimulated with rIL-7 in the same manner, and cross-linked chromatin was immunoprecipitated with anti-STAT5 antibody, which was previously used in EMSA. Precipitated DNA was purified and was amplified with PCR. The DNA fragment containing the SBM was observed in the presence of the antibody (Fig. 5, lane 2), while it was undetectable in the absence of the antibody (Fig. 5, lane 1). Taken together, p-STAT5 binds to the SBM in PreBR1 cells.

thumbnail image

Figure 5. Chromatin immunoprecipitation assay for SATA5 in PreBR1 cells. Chromatin immunoprecipitation assay of PreBR1 cells stimulated with rIL-7. PreBR1 cells were stimulated with rIL-7, fixed, lysed and precipitated with or without anti-STAT5 antibody twice. PCR and Southern blotting were performed to detect the SBM site among the precipitated chromatin. Similar results were obtained in two independent experiments.

Download figure to PowerPoint

2.6 STAT5 has a synergistic effect on EBF-induced transcription

To investigate the regulation of transcription by p-STAT5 and EBF, a luciferase reporter construct (pGL3-stat) harboring five tandem repeated SBM sequences upstream of a luciferase gene (Fig. 6A) was analyzed in a reporter gene assay. Expression vectors for a constitutive active mutant of STAT5 (m-STAT5) 15 as well as for EBF were co-transfected with pGL3-stat into NIH3T3 cells. The results of the reporter gene assay demonstrated that the expression of EBF alone was active in the luciferase activity, consistently with a previous report 9, while the expression of m-STAT5 alone also slightly activated the reporter gene. When EBF and m-STAT5 expression vectors were co-transfected to the cells, the synergistic activation was observed; the activity of m-SATA5 plus EBF was about threefold or fivefold relative to EBF alone or m-STAT5 alone (Fig. 6B).

For further investigation, the 1.8-kb promoter fragment of Pax5 gene (Pax5p-pGL3) 9 was examined in a luciferase reporter gene assay employing same conditions of transfection. As shown in Table 1, the synergistic effect of m-STAT5 and EBF was also observed when 1.5 μg of the m-STAT5 expression plasmid together with 0.5 μg of the EBF expression vector were transfected into the NIH3T3 cells. These results imply that IL-7 signaling can enhance the Pax5 transcription by the synergistic effect of p-STAT5 and EBF.

thumbnail image

Figure 6. The luciferase reporter assay with STAT5 and EBF constructs. (A) Schematic diagram of the luciferase reporter construct (pGL3-stat). The DNA fragment containing five tandem repeats of SBM was inserted into pGL3-basic (Promega). (B) Luciferase activity in transient transfection of NIH3T3 cells. Amounts of the transfected expression construct for the constitutively active form of STAT5 (m-STAT5) 22 are indicated, as well as amounts for the EBF expression vector. Constructs were transiently co-transfected with 3 μg of β-galactosidase expression vector into NIH3T3 cells as described in Sect. 4. After 48-h incubation, cells were lysed and luciferase activities were measured. Relative activities were normalized with the β-galactosidase activity of the reporter gene control. Data represent means of triplicate experiments and an error bar represents SEM. Data representative of two independent experiments are shown.

Download figure to PowerPoint

Table 1. Luciferase reporter gene assay of 1.8-kb promoter fragment of Pax5 genea)
EBF (μg)m-STAT5 (μg)Relative activity
  1. a) The expression vectors for EBF and m-STAT5, Pax5p-pGL3 and pKM18-β-galactosidase plasmids were co-transfected to NIH3T3 cells as in Fig. 5. Relative luciferase activities compared with negative control were calculated. Data represent means of triplicate experiments. Representative of three independent experiments are shown.

 1  
0.533.8
1.5 4.7
0.51.558.1

3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

In this study, we reported the identification of a STAT5 binding sequence in the Pax5 promoter, which completely overlaps with a binding site for EBF. IL-7 induced the phosphorylation of STAT5, and p-STAT5 bound to the SBM which EBF bound to. Previously, it has been reported that other transcription factors shared the same binding site with STAT5; in the IL-2rE, the STAT5 binding site was shared with ETS family proteins, ETS-1 and ETS-2 16, and these factors made a complex that was capable of transactivation 17. Thus, it is possible that SATA5 associates with EBF. However, we have failed to detect such a physical association of these two transcription factors in immunoprecipitation (data not shown). The p-STAT5 together with EBF is able to activate transcription of the Pax5 promoter. To our knowledge, this finding is the first indication that implicates a direct link between IL-7R signaling and the transcriptional regulation of Pax5. Our data support the notion that IL-7 is a positive regulator of Pax5 transcription, which was demonstrated in the analysis of IL-7 receptor α knockout (IL-7Rα–/–) mice 7. In the early stage of B lymphopoiesis, cells greatly expand by IL-7. Wakatsuki et al. 18 demonstrated that Pax5 regulated proliferation of mature B cells. Although physiological meaning of Pax5 up-regulation by IL-7 have to be investigated in detail in vivo studies, we speculate that the increased amount of Pax5 by IL-7R signaling may be used to regulate proliferation of early B cells.

There are many transcription factors that cooperatively regulate B cell development. Among them, Pax5 plays a key role and coordinates the expression of many target genes required for the early B cell development, and STAT5 may indeed be involved in modulating the function of Pax5 as a transcription factor in some stages in early B cell differentiation.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Cell lines and antibodies

PreBR1 cells were cultured in SF-O3 medium (Sanko Jyunyaku, Kyoto, Japan) containing 5×10–5 M of 2-mercaptoethanol (Sigma, St. Louis, MI), 1× nonessential amino acids (Gibco BRL, Gaithersburg), 0.03% primatone (Quest International, Naarden), 2% FCS and 100 U/ml of rIL-7 14. The following antibodies were purchased: anti-STAT5a (sc-1081, Santa Cruz Biotechnology, Santa Cruz, CA), anti-STAT5 (sc-835, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-phospho-STAT5 (PYS5, Zymed, South San Francisco, CA). An anti-EBF antiserum was kindly provided by Dr. R. Grosschedl (Ludwig-Maximilians-Universität, München, Germany). The recombinant murine IL-7 was purchased from Genzyme (Cambridge, MA).

4.2 Nuclear extraction and Western blotting

Preparation of nuclear extract was described previously 19. In brief, 1×107–2×107 cells were washed, resuspended in 400 μl of buffer A (10 mMHEPES (pH 7.5), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM PMSF, 2 μg/ml aprotinin, 2.5 μg/ml leupeptin, 5 μg/ml pepstatin, 1 mM DTT and 400 μM Na3VO4) and chilled on ice for 15 min. After adding 25 μl of 10% NP-40, the suspension was mixed and centrifuged. The nuclear proteins were extracted with 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, 2.5 μg/ml leupeptin, 5 μg/ml pepstatin, 1 mM DTT and 400 μM Na3VO4) by shaking for 15 min at 4°C, and were stored at –80°C until use. The amount of extracts was measured using a Bio-Rad protein assay (Bio-Rad, Hercules, CA)

For the Western blotting, 5–10 μg of protein extracts were subjected to SDS-PAGE using 7.5% gels and transferred to nitrocellulose membranes followed by blocking with 5% nonfat-dry milk inTTBS (10 mM Tris-HCl [pH7.5], 150 mM NaCl, 0.1 % Tween 20). The filters were incubated sequentially with a primary Ab followed by a secondary horseradish peroxidase-conjugated antibody (1:1,000 dilution in TTBS with 1% non-fat dry milk), and signals were detected using the chemiluminescence ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ).

4.3 Electrophoretic mobility shift assay (EMSA)

The reaction mixtures (20 μl) contained nuclear extracts (5–10 μg), 4 μl of 5 × binding buffer [100 mM HEPES, pH 7.6, 5 mM EDTA, 50 mM (NH4)2SO4, 5 mM DTT, 1% Tween 20 and 150 mM KCl], 1 μl of 1 mg/ml poly[d(I-C)] and 1 μl of 32P-labeled double-stranded oligonucleotides of the SBM site (15,000–20,000 cpm/μl). Following incubation for 15 min at room temperature, the reaction mixtures were separated by 5% polyacrylamide gel electrophoresis (150 V at 4°C). Gels were dried and analyzed by BAS2000 (Fuji film, Tokyo, Japan). Supershift experiments were performed by incubating 2 μg of anti-STAT5 Ab or 3 μl of anti-EBF antiserum with nuclear extracts on ice for 1.5 h before reaction.

4.4 Chromatin immunoprecipitation (ChIP) assay

ChIP assay was performed as described 20, 21. Briefly, PreBR1 cells (5×106–10×106) stimulated with rIL-7 were fixed with formaldehyde for 5 min at room temperature, and immunoprecipitation was carried out with 4–6 μg of anti-STAT5 sc-835 antibody overnight. Precipitation was performed twice. Precipitated DNA was purifiedafter washing and reversely cross-linking the precipitates. Purified DNA was subjected to PCR for 30–40 cycles and to Southern blotting. Primers were designed as follows: forward, 5′-CAA AAC GCA AGA AAC AAA AGA-3´; reverse, 5´-GAG GAT AGG GGG AGG GGT CAG-3´. A probe for Southern blotting was a PCR product provided with the reaction using these primers and Pax5p-pGL3 as a template.

4.5 Reporter gene assay

For the luciferase reporter construct (pGL3-stat), the fragment containing five tandem repeats of the SBM was inserted upstream of the luciferase gene in pGL3-basic (Promega, Madison, WI). Thefive tandem repeats of the SBM were artificially constructed (Amersham Pharmacia Biotech, Piscataway, NJ). The expression vector of a constitutive active mutant form of STAT5 (m-STAT5) 15 was a kind gift of Dr. T. Kitamura (Institute of Medical Science, University of Tokyo, Tokyo, Japan), and the EBF expression vector and the Pax5p-pGL3 reporter plasmid were kindly suppliedby Dr. R. Grosschedl (Ludwig-Maximilians-Universität, München, Germany). NIH3T3, 5×105 cells were transiently transfected with 1.5–3.0 μg of m-STAT5 and/or EBF expression vectors and 1 μg of pGL3-stat together with 1.5 μg of pKM18-β-galactosidase. A total amount of plasmid was adjusted to 10 μg using the pMX only. Transfection was performed with SuperFect-Transfection-Reagent (QIAGEN, Hilden) according to the manufacture's instruction manual. After 48-h incubation, cells were lysed with 60 μl Reporter Lysis Buffer (Promega, Madison, WI) and 20 μl and 30 μl lysates were used for measurements of luciferase activity and β-galactosidase activity for normalization of transfection efficiency, respectively. In the second experiment, 1 μg of Pax5p-pGL3 plasmid was used for the reporter gene, and then 1.5 μg of pKM18-β-galactosidase and the indicated amount of m-STAT5 and/or EBF expression vectors were co-transfected into NIH3T3 cells in the same conditions as described above.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

We thank Dr. R. Grosschedl for EBF expression vector, Pax5p-pGL3 plasmid and anti-EBF antibody. We also thank Dr. T. Kitamura for the expression vector for the constitutive active form of STAT5. We thank Dr. U. Grawunder for critical reading of the manuscript. This work was supported in part by grants-in-aid from the Ministry of Education, Sports, Science and Technology.

  • 1

    WILEY-VCH

  • 2

    WILEY-VCH

  • 3

    WILEY-VCH

  • 4

    WILEY-VCH

  • 5

    WILEY-VCH

  • 6

    WILEY-VCH

  • 1
    Nutt, S. L., Urbanek, P., Rolink, A. and Busslinger, M., Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus. Genes Dev .1997. 11: 476491.
  • 2
    Barberis, A., Widenhorn, K., Vitelli, L. and Busslinger, M., A novelB-cell lineage-specific transcription factor present at early but not late stages of differentiation. Genes Dev .1990. 4: 849859.
  • 3
    Adams, B., Dorfler, P., Aguzzi, A., Kozmik, Z., Urbanek, P., Maurer-Fogy, I. and Busslinger, M., Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes, the developing CNS, and adult testis. Genes Dev .1992. 6: 15891607.
  • 4
    Li, Y. S., Wasserman, R., Hayakawa, K. and Hardy, R. R., Identification of the earliest B lineage stage in mouse bone marrow. Immunity 1996. 5: 527535.
  • 5
    Nutt, S. L., Vambrie, S., Steinlein, P., Kozmik, Z., Rolink, A., Weith, A. and Busslinger, M., Independent regulation of the two Pax5 alleles during B-cell development. Nat. Genet .1999. 21: 390395.
  • 6
    Namen, A. E., Lupton, S., Hjerrild, K., Wignall, J., Mochizuki, D. Y., Schmierer, A., Mosley, B., May, C. J., Urdal, D. and Gillis, S., Stimulation of B-cell progenitors by cloned murine interleukin-7. Nature 1988. 333: 571573.
  • 7
    Corcoran, A. E., Riddell, A., Krooshoop, D. and Venkitaraman, A. R., Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor. Nature 1998. 391: 904907.
  • 8
    Billips, L. G., Nunez, C. A., Bertrand, F. E., 3rd, Stankovic, A. K., Gartland, G. L., Burrows, P. D. and Cooper, M. D., Immunoglobulin recombinase gene activity is modulated reciprocally by interleukin 7 and CD19 in B cell progenitors . J. Exp. Med. 1995. 182: 973982.
  • 9
    O'Riordan, M. and Grosschedl, R., Coordinate regulation of B cell differentiation by the transcription factors EBF and E2A. Immunity 1999. 11: 2131.
  • 10
    Leonard, W. J. and O'Shea, J. J., Jaks and STATs: biological implications. Annu. Rev. Immunol .1998. 16: 293322.
  • 11
    Sexl, V., Piekorz, R., Moriggl, R., Rohrer, J., Brown, M. P., Bunting, K. D., Rothammer, K., Roussel, M. F. and Ihle, J. N., Stat5a/b contribute to interleukin 7-induced B-cell precursor expansion, but abl- and bcr/abl-induced transformation are independent of stat5. Blood 2000. 96: 22772283.
  • 12
    Foxwell, B. M. J., Beadling, C., Guschin, D., Kerr, I. and Cantrell, D., Interleukin-7 can induce the activation of Jak1, Jak3 and STAT5 proteins in murine T cells. Eur. J. Immunol. 1995. 25: 30413046.
  • 13
    Rosenthal, L. A., Winestock, K. D. and Finbloom, D. S., IL-2 and IL-7 induce heterodimerizationof STAT5 isoforms in human peripheral blood T lymphoblasts. Cell. Immunol. 1997. 181: 172181.
  • 14
    Kato, I., Miyazaki, T., Nakamura, T. and Kudo, A., Inducible differentiation and apoptosis of the pre-B cell receptor-positive pre-B cell line .Int. Immunol 2000. 12: 325334.
  • 15
    Nosaka, T., Kawashima, T., Misawa, K., Ikuta, K., Mui, A. L. and Kitamura, T., STAT5 as a molecular regulator of proliferation, differentiation and apoptosis in hematopoietic cells. EMBO J .1999. 18: 47544765.
  • 16
    Lecine, P., Algarte, M., Rmeil, P., Beadling, C., Bucher, P., M., N. and Imbert, J., Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptoralpha gene. Mol. Cell. Biol. 1996. 16: 68296840.
  • 17
    Rameil, P., Lecine, P., Ghysdael, J., Gouilleux, F., Kahn-Perles, B. and Imbert, J., IL-2 and long-term T cell activation induce physical and functional interaction between STAT5 and ETS transcription factors in human T cells. Oncogene 2000. 19: 20862097.
  • 18
    Wakatsuki, Y., Neurath, M. F., Max, E. E. and Strober, W., The B cell-specific transcription factor BSAP regulates B cell proliferation. J. Exp. Med .1994. 179: 10991108.
  • 19
    Schreiber, E., Matthias, P., Muller, M. M. and Schaffner, W., Rapiddetection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucl. Acids Res .1989. 17: 6419.
  • 20
    Ye, S., Agata, Y., Lee, H.-C., Kurooka, H., Kitamura, T., Shimizu, A., Honjo, T. and Ikuta, K., The IL-7 receptor controls the accessibility of the TCRγ locus by stat5 and histone acetylation. Immunity 2001. 15: 813823.
  • 21
    Weinmann, A. S., Bartley, S. M., Zhang, T., Zhang, M. Q. and Farnham, P. J., Use of chromatin immunoprecipitation to clone novel E2F target promoters. Mol. Cell. Biol .2001. 21: 68206832.
  • 22
    Travis, A., Hagman, J., Hwang, L. and Grosschedl, R., Purification of early-B-cell factor and characterizeation of its DNA-binding specificity. Mol. Cell. Biol. .1993. 13: 33923400.