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Blood, 15 September 2005, Vol. 106, No. 6, pp. 1995-2001. Prepublished online as a Blood First Edition Paper on May 26, 2005; DOI 10.1182/blood-2004-12-4744.
IMMUNOBIOLOGY Inhibition of EBF function by active Notch signaling reveals a novel regulatory pathway in early B-cell developmentFrom the Department for Stem Cell Biology, Lund University, Lund, Sweden; Department of Molecular Pharmacology, AstraZeneca R&D Mölndal, Mölndal, Sweden; Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA; and Department of Laboratory Medicine, Division of Molecular Medicine, Lund University, Malmö, Sweden.
The Notch signaling pathway is involved in several lineage commitment and differentiation events. One of these is fate determination of the common lymphoid progenitor, promoting T-cell development at the expense of B-cell differentiation. It has been suggested that this process relies on Notch's ability to inhibit E proteins, which are crucial for early B-cell development. Here, we report that Notch signaling also modulates the function of the transcription factor, early B-cell factor (EBF). Transient transfection of intracellular Notch1 (Notch1-IC) into a pre-B cell line resulted in the down-regulation of EBF-regulated promoters and diminished the capacity of EBF to activate these promoters in an epithelial cell line. This correlated with a reduction in the ability of EBF to bind DNA. Ligand-induced stimulation of endogenous Notch receptors with Delta4 mimicked the activity of Notch1-IC toward EBF. These data suggest that Notch signaling may affect B-versus T-lineage commitment by the targeting of both EBF and E2A.
The development of B- and T-lymphoid cells from hematopoietic stem cells involves a complex multistep differentiation pathway proceeding through common lymphoid progenitors (CLPs).1 The CLPs receive instructing signals from the surrounding cells that appear to promote a developmental branch point, where the choice of lineage, B cells versus T cells, takes place.2 One such signaling pathway with a crucial role in this process is mediated through the Notch family receptors.3,4 Transgenic mice expressing a constitutively active form of intracellular Notch1 (Notch1-IC) within the hematopoietic compartment, display gross abnormalities in lymphoid development.5 These are manifested as an induction of T-cell development apparently at the cost of B-lymphoid differentiation.5 Targeted disruption of the Notch1 gene has the opposite effect, resulting in a dramatic reduction of T-cell number and the development of B-lymphoid cells in the thymus.6,7 The signal transduction cascade, initiated by the interaction of a Notch receptor with any of its ligands, Jagged1, Jagged2, Delta1, Delta3, or Delta4, involves a  -secretase-mediated cleavage of the Notch protein.3,4 This releases the Notch intracellular domain (Notch-IC), which enters the nucleus, forms a complex with CSL (CBF1/RBP-Jk), and activates transcription.3,4 The potential of Notch signaling to impair B-cell development has been attributed to a functional inhibition of the E2A protein E47.8 E2A proteins are crucial in the development of B lymphocytes in a dose-dependent manner,9,10 supporting this notion. However, several lines of evidence are indicating that the function of E2A proteins in B-cell development is linked to the function of another transcription factor, early B-cell factor (EBF).11,12 This protein is of crucial importance for B-cell development because mice carrying a homologous disruption of the gene also display a block in early B-cell differentiation.13 EBF and E2A share several target genes, such as  5, VpreB,12 and Cd79a,14 and have also been shown to act in a coordinated manner to support B-cell development in vivo.11 Furthermore, it has been shown that B-cell development in E2A-deficient mice can be rescued by ectopic expression of EBF in hematopoietic progenitors, suggesting a partially redundant function of the E2A gene products in B-cell development.15 A direct role for EBF in B-versus T-lineage commitment per se has been suggested from studies in which the protein was ectopically expressed by retroviral transduction in hematopoietic progenitor cells.16 This resulted in an impairment of T lymphopoiesis, suggesting a key role for EBF in the initiation and commitment into the B-lymphoid pathway. The crucial function of both Notch signaling and EBF in early B-cell development prompted us to ask if EBF is affected by Notch signaling. We here present evidence that suggests that Notch signaling inhibits the activity of EBF as well as the coordinated activity of EBF and E2A. Thus, our results provide a novel insight into the orchestration of gene expression and lineage commitment in early lymphopoiesis.
Tissue culture conditions and cell purification HeLa and 230-238 cells lines were grown in RPMI medium supplemented with 10% fetal calf serum (FCS), 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 2 mM pyruvate, 50 µM 2-mercaptoethanol, and 50 µg gentamicin per milliliter (complete RPMI media). BaF/3 and the EBF-expressing BaF/3-EBF17 cell lines were kept in complete RPMI supplemented with 10% interleukin 3 (IL-3) containing WEHI3-conditioned media. 293T cells were grown in Dulbecco modified Eagle medium (DMEM) with 10% FCS and 50 µM 2-mercaptoethanol added (all purchased from Gibco Invitrogen, Paisley, United Kingdom). To block Notch signaling, growth medium was supplemented with 10 µM N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT; Merck Biosciences, Darmstadt, Germany). CD19+ cells from murine bone marrow were enriched by positive selection by using magnetic-activated cell sorting (MACS) CD19 microbeads (Miltenyi Biotec, Auburn, CA), and kept in Optimem containing 10% FCS, 50 µM 2-mercaptoethanol, and 50 µg/mL gentamicin. All cells were cultured at 37°C and 5% CO2. Transient transfections and luciferase assays
On transfection, approximately 2.5 x 106 230-238 lymphoid cells were incubated for 30 minutes at 20°C in 1 mL diethylaminoethyl (DEAE)-dextran (0.8 mg/mL Tris-buffered saline [TBS] containing 140 mM NaCl, 5 mM KCl, 25 mM Tris [tris(hydroxymethyl)aminomethane], pH 7.4, 0.6 mM Na2HPO4, 0.7 mM CaCl2); Pharmacia, Uppsala, Sweden) containing 200 µg cytomegalovirus (CMV)-controlled Renilla luciferase reporter gene (Promega, Madison, WI), used as internal control, 2 µg reporter construct, controlled by the
HeLa cells, seeded into a 24-well tissue culture plate, were transfected according to the Invitrogen transfection protocol: "HeLa cells with LIPOFECTIN reagent and PLUS reagent." Briefly, DNA expression plasmids were mixed in 75 µL serum-free medium Optimem (Gibco, Invitrogen), 7 µL PLUS reagent and 0.75 µL LIPOFECTIN reagent (Invitrogen Life Technologies, Carlsbad, CA). After replacement of cell growth medium with 300 µL of serum-free medium, the DNA-PLUS-LIPOFECTIN mixture was added to the cells. Plates were then incubated at 37°C in 5% CO2 for 3 hours, after which each well was supplemented with 1 mL growth medium. Cells were harvested 48 hours after transfection and protein extracts were prepared by adding 80 µL cell lysis buffer to each well. In each transfection, 40 ng of the
To generate a soluble form of a Notch1 ligand, 293T cells grown in a 10-cm Petri dish were transfected in the same way as described for HeLa cells, with either 5 µg Fc TRAIL-R4 or 5 µg Fc-Delta4 expression plasmid.19 After 48 hours, the growth medium was collected and filtered through a 0.45-µm syringe filter. Fc-TRAIL-R4 or Fc-Delta4 fusion protein was immobilized by incubating nontissue culture plates overnight in 4°C with 10 µg/mL rabbit anti-human IgG Fc
BaF/3 cells were transfected with DEAE, as described for 230-238 cells. Then, 0.5 µg The luciferase assays for all transfections were performed with a Dual-Luciferase Reporter Assay System (Promega) using 20 µL of the total protein extract. All samples were made in triplicate.
The mouse RT-PCR and real-time quantitative PCR
RNA was prepared from cells using RNeasy Mini Kit (QIAGEN, VWR International, Stockholm, Sweden), and cDNA was generated by annealing 1 µg total RNA to 0.5 µg random hexamers in 10 µL diethyl pyrocarbonate-treated water. Reverse transcription (RT) reactions were performed with 200 U SuperScript II (Invitrogen Life Technologies, Carlsbad, CA) in accordance with the manufacturer's recommendations. One-twentieth of the RT reaction was used in the polymerase chain reaction (PCR) assays. PCR was performed with 1 U Taq polymerase (Invitrogen Life Technologies) in the manufacturer's buffer supplemented with 0.2 mM deoxynucleotide triphosphate (dNTP), in a total volume of 25 µL. Each quantitative (Q)-PCR (ABI PRISM 7700 Sequence Detector; PE Applied Biosystems, Foster City, CA) involved cDNA extracted from approximately 500 cells. Reactions were conducted by mixing 2 x TaqMan Universal PCR Master Mix, 20 x Gene Expression Assay Mix from Assays-on-Demand or Assays-by-Design service, RNase-free H2O and 5 µL cDNA to a final volume of 25 µL (all reagents purchased from Applied Biosystems). The reaction was initiated by a hold for 10 minutes at 95°C followed by 45 cycles 15 seconds at 95°C and 1 minute at 60°C. The following Assays-on-Demand probes were used: HPRT: Mm00446968_m1; EBF: Mm22395519_m1; CD79a: Mm00432423_m1; CD79b: Mm00434143_m1; CD19: Mm00515420_m1; and Pax5: Mm00435501_m1.
The Protein extracts and electrophoretic mobility shift assay
Nuclear extracts were prepared according to Schreiber et al.22 DNA probes were labeled with
Oligonucleotides used for electrophoretic mobility shift assays (EMSAs) were as follows: Mb-1 EBF sense: 5'-GAGAGAGACTCAAGGGAATTGTGG; Mb-1 EBF antisense: 5'-CCACAATTCCCTTGAGTCTCTCTC; Oct sense: 5'-TTCATTGATTTGCATCGCATGAGACGCTAACATCGTACGTTC; Oct antisense: 5'-GAACGTACGATGTTAGCGTCTCATGCGATGCAAATCAATGAA; Western blotting For Western blotting, the HeLa cell nuclear extracts used for EMSA and the BaF/3 extracts used for luciferase assay were separated on a 7.5% PAGEr Gold Precast gel (Cambrex, In vitro Sweden AB, Stockholm, Sweden) and blotted onto a nylon membrane (anti-myc; Amersham Pharmacia Biotech) or immobilon polyvinylidene difluoride (PVDF; Millipore, Billerica, MA; anti-HES-1) by semidry electroblotting. For EBF detection, a primary mouse monoclonal anti-c-myc antibody (9E10) diluted 1:1000 in phosphate-buffered saline (PBS; Gibco Invitrogen) supplemented with 0.5% Tween 20 (PBS-T: Amersham Life Science, Ohio), and a 1:2000 dilution of a secondary horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used. The primary anti-HES-1 antibody was diluted 1:8000 (kindly provided by Dr Tetsuo Sudo, Kamakura, Japan) in PBS-T, and HRP-conjugated donkey anti-rabbit antibody was here used as secondary antibody. Detection of the secondary antibody was obtained using an enhanced chemoluminescence (ECL) system (Amersham Pharmacia Biotech). All the steps were performed at room temperature.
Ectopic expression of Notch1-IC reduces the functional activity of B-lineage promoters in a pre-B cell line
Even though it has been shown that active Notch signaling alters the expression of B cell-restricted genes,23 it is unclear if this effect is due to a general influence on lymphoid progenitors or a direct activity on pre-B cell-restricted promoters. The study of these phenomena in primary cells is complicated by the fact that functional alterations can be related to a lineage switch rather than the direct action of Notch signaling on target promoters. Thus, to investigate if Notch signaling has a direct impact on the activity of promoters active in a pre-B cell, we transiently transfected the pre-B cell line 230-238 with luciferase reporter constructs controlled by either the B29, Cd79a, or
Ectopic expression of a constitutively active Notch1 reduces the functional activity of EBF
The
Whereas both the
We next transfected a set of luciferase reporters containing EBF-responsive promoters into HeLa cells together with expression plasmids encoding EBF and Notch1-IC (Figure 3). The Notch1-IC reduces the DNA-binding ability of EBF
Notch signaling has been shown to induce expression of hairy/enhancer of split homologues (HES) proteins,3,4,25 potential repressors of E-protein activity, but also to stimulate ubiquitination and degradation of E47.26,27 To investigate the influence of Notch1-IC on EBF protein levels, we transfected HeLa cells with a myctagged EBF protein and assayed the level of EBF in nuclear extracts by Western blot using anti-myc antibody (Figure 4A). The vector-transduced cells did not generate any band of the expected size, whereas myc-EBF-transduced cells expressed the expected protein of roughly 55 to 60 kDa. The same protein (comparable intensity and size) could be detected in nuclear extracts from cells transduced with myc-EBF and Notch1-IC. Thus, we were unable to detect any signs that EBF is modified or degraded as a consequence of Notch1 signaling. To investigate the ability of the protein to bind DNA, we performed an EMSA, using the same nuclear extracts and an Cd79a promoter EBF-binding site as probe (Figure 4B). Even though all the extracts contained comparable amounts of Oct1 DNA-binding activity as assayed by binding of protein to a consensus octamer site, DNA binding by EBF to the Cd79a promoter site was reduced in the presence of Notch1-IC. To verify that the activity of Notch1 on EBF was directed to the DNA-binding part of the protein, we transiently transfected HeLa cells with an EBF fusion protein (EBFVp16), where the transactivation domain of EBF (Figure 4C) was replaced with that of the Vp16.18 This fusion protein activated the
EBF function is modulated by receptor-mediated Notch signaling in hematopoietic progenitor cells
Although constitutively active Notch proteins (Notch-IC) are commonly used to study Notch activity, they may not fully mimic the Notch signaling pathway. To verify that EBF function is modulated by the interaction of the endogenous Notch1 receptor with its ligand Delta4, we transfected a Notch1-expressing hematopoietic progenitor cell line, BaF/3 (Figure 5A), with EBF and the
Active Notch1 signaling reduces EBF DNA-binding capacity in pre-B cells
To investigate if EBF DNA-binding ability could be modulated by Notch signaling mediated through the endogenous receptors, we incubated 230-238 pre-B cells (which express Notch1 as judged by RT-PCR; Figure 5A) on plates coated either with the control Fc-TRAIL-R4 or the Fc-Delta4 fusion protein. In contrast to the BaF/3 cells, the 230-238 cells contained a background expression of HES-1, as judged by both real-time PCR and Western blot, but incubation on Fc-Delta4 still resulted in an about a 2-fold increase in HES-1 expression (data not shown). EMSA revealed that even though nuclear extracts from 230-238 cells grown on Fc-Delta4 or Fc-TRAIL-R4 gave rise to the same amounts of Oct1 DNA binding, there was a reduction in EBF binding to the Cd79a promoter in cells grown on Fc-Delta4 (Figure 6A). This effect was most dramatic after 48 hours of ligand exposure, whereas both earlier and later time points gave less definitive results (data not shown). Similar results were obtained when using an EBF site from the
To investigate if Notch signaling could modulate EBF DNA binding in a normal B-lymphoid cell, we purified CD19+ cells from mouse BM and incubated these on plates coated with Fc-TRAIL-R4 or Fc-Delta4. This resulted in a down-regulation of DNA-binding activity after 3 hours (Figure 7A) and 24 hours (Figure 7B) of incubation without any dramatic effect on EBF or Pax-5 expression at these time points (Figure 7C-D). Although we may have expected a more widespread effect on gene expression, we conclude that bona fide Notch signaling reduces EBF DNA-binding activity in B-lineage cells.
We provide data suggesting that the capacity of EBF to activate several target promoters is reduced by active Notch signaling and that this is associated with an impaired ability of EBF to bind DNA. This has important implications in the understanding of the mechanisms underlying the role of Notch signaling in B-cell versus T-cell commitment. The potential of Notch to inhibit both EBF and E47 function, while possibly enhancing the function of Pax-5, may provide an extended insight into definitive B-cell commitment. It has been shown that despite the fact that both EBF and E47, as well as several of their target genes, are expressed at high levels in B-cell progenitors from Pax-5-deficient mice,31,32 these cells are able to differentiate into other lineages, including T cells, in vivo.33 In addition, Pax-5-deficient cells are able to develop along the T-lineage pathway when cultured on Delta-ligand-expressing stromal cells.23 This strongly suggests that the definitive choice to proceed into the B-lymphoid pathway is incomplete even in the presence of EBF and E47 in the progenitor cell and proposes an absolute requirement for Pax-5 expression in the establishment of B lymphopoiesis. One mechanism that has been suggested to be of significant importance in the Pax-5-mediated lineage decision is the ability to suppress expression of Notch-1 in the progenitor cells.34 Thus, our current findings, in combination with previous data showing a functional repression of E47 by Notch signaling,8 provide a possible molecular explanation for the inability of EBF and E47 to lock the cells into the B-lymphoid developmental pathway in the absence of Pax-5 expression. Expression of Notch-1 in the absence of Pax-5 makes the cells susceptible to Delta or Jagged stimulation, and the corresponding drop in the functional activity of EBF and E47. Thus, the apparent role of EBF and E47 to induce but not lock B-cell development could be a result of susceptibility to the action of Notch signaling. Moreover, EBF is noted to be involved in the regulation of Pax-5 expression, suggesting an intricate interplay between transcription factors and Notch signaling in lymphoid lineage commitment.
These findings further strengthen the idea that the functions of EBF and E47 are closely linked in the earliest stages of B-cell development. These proteins have been shown to cooperate in the direct activation of pre-B cell-restricted target promoters such as Cd79a,14 It has been suggested that the ability of Notch signaling to modulate E47 activity results from increased protein degradation26,27 and from direct inhibition of E2A function by HES proteins.25 We have not observed any apparent Notch1-induced EBF degradation, and the modest induction of HES-1 in 230-238 cells (Figure 6A and data not shown) was in contrast to the dramatic effect on EBF DNA binding, arguing against a direct influence of HES-1 on EBF DNA binding. To further investigate this we cotransfected EBF and HES-1 into HeLa cells and analyzed protein expression by Western blotting and DNA binding by EMSA. We were unable to observe any alternations in either EBF protein expression or DNA binding (data not shown), providing further support for the idea that the inability of EBF to bind DNA is not a direct effect of HES-1 action. These findings suggest that Notch signaling reduces the function of EBF by modulation of the DNA-binding activity of the protein in the context of a pre-B cell. Thus, we have not yet been able to unravel the mechanisms by which Notch inhibits EBF DNA binding, demanding additional biochemical analyses of the interaction between EBF and the Notch signaling pathway. In addition to its important role in lymphoid development, Notch signal modulation of EBF activity may also be of critical importance in differentiation systems outside of the developing immune system. EBF stimulates neurogenesis in Xenopus38,39 as well as adipogenesis in mice,40 2 processes that are modulated by active Notch signaling.30,38,39 Key roles for the Notch/EBF link may also be revealed from further studies of these model systems for cellular differentiation.
We are grateful to Drs Warren Pear, Björn Olde, and Meinrad Busslinger for the kind gifts of reagents; Gerd Sten and Lillian Wittman for technical assistance; and Sten Eirik Jacobsen and Jalal Taneera for stimulating discussions.
Submitted December 14, 2004; accepted May 11, 2005.
Prepublished online as Blood First Edition Paper, May 26, 2005; DOI 10.1182/blood-2004-12-4744.
Supported by the Swedish Cancer Society (Cancerfonden), The Swedish Research Council (VR) [Vetenskaprådet], the Barn Cancer, Kocks, Österlunds, and Crafoord foundations, and the Medical Faculty at Lund University. The Lund Strategic Center for Stem Cell Biology and Cell Therapy is sponsored by a center grant from Stiftelsen för Strategisk Forskning (SSF).
E.M.K.S. conducted the large majority of the experiments with some help from P.Å., H.A., and M.S. T.K. provided crucial reagents as well as important ideas about experimental design. All authors contributed to the writing of the manuscript.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Correspondence: Mikael Sigvardsson, Department for Stem Cell Biology, Lund University, BMC B12, S-221 84 Lund, Sweden; e-mail: mikael.sigvardsson{at}med.lu.se.
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Abstract
Introduction
-promoter TATA-box. The SV40 reporter plasmid was obtained from Promega. 
-actin was amplified by 25 cycles, and 40 cycles were used to amplify Notch1 cDNA (94°C, 30 seconds, 60°C, 30 seconds, and 72°C, 30 seconds). The following primers were added to a final concentration of 1 mM: 
) or Fc-Delta4 (
). The data are based on 3 transfections from a single representative of 3 experiments normalized to the internal control CMV Renilla. The reporter activities obtained with empty expression plasmid were set to 1 and error bars indicate SD. (E) Display of an EMSA where the DNA binding of ectopically expressed EBF to the Cd79a promoter EBF sites were assayed following incubation of the stably transduced cells on the control Fc-TRAIL-R4 or the Fc-Delta4 protein for 40 hours. (F) Diagrams show the expression of the endogenous 
