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IMMUNOBIOLOGY
From the Immunology Group, Cell and Molecular
Biology, Lund University; and the Stem Cell Laboratory, Lund
University Hospital, Lund, Sweden.
Early B-cell factor (EBF) is a helix-loop-helix transcription
factor suggested to be essential for B-cell development in the mouse.
Several genetic targets for EBF have been identified in mice, among
these the surrogate light chain The development of a multipotent hematopoietic stem
cell into a highly differentiated immunoglobulin (Ig)-producing plasma cell can be divided into several stages based on expression of certain
surface markers and the recombination status of the Ig genes.1,2 The earliest human B-cell precursors express the surface molecule CD34 and carry their Ig genes in germline
configuration. Subsequent differentiation results in expression of
components in the pre-B-cell receptor3,4 and
signal-modulating molecules such as CD19 as well as the
recombination-activating genes RAG-1 and -2,
allowing for recombination of the Ig heavy chain genes (IgH). The
pre-B-cell receptor is composed of the signal transduction molecules
Ig Completion of this developmental pathway is dependent on stage-specific
expression of certain genes, demanding a system of transcriptional
activators that interact with regulatory elements controlling the
expression of these genes.18,19 The importance of
distinct transcription factors in the promotion of B-cell
differentiation in the mouse has been shown in a number of transgenic
animals.18,20,21 Early B-cell factor (EBF),22
B-cell-specific activator protein (BSAP),23 and
E4724,25 have all been shown to be essential for early
B-cell development by targeted disruptions of the coding genes.26-31 B-cell development in humans largely resembles
that in mice,1 and homologues of most of the proteins
shown to be important for B-cell development in mice have also been
cloned from humans. In line with this, human homologues of
BSAP23 and E4725 have been characterized and
suggested to play roles similar to their mouse
counterparts.25,32 However, no human homologue to
mouse22 and rat33 EBF/Olf-1 has
yet been described. The existence of such a protein located on human
chromosome 5 has been suggested from fluorescence in situ hybridization
(FISH) analysis using the mouse cDNA as probe34 and from
the finding that an EBF-like factor appears to interact with the human
CD19 promoter.35 EBF interacts with the DNA core CCCNNGGG
as a homodimer via a zinc-containing DNA-binding
domain.36,37 The dimerization is mediated by 2 helices
with homology to helix 2 of the bh-l-h protein.22,37 EBF
has been suggested to interact with and regulate the promoters of
several genes in the mouse pre-B cell, ie, the mb-1,38,39 B29,40
We here report cloning of the human homologue of mouse EBF (hEBF) and
the identification of potential target genes for this factor in human
B-cell development. We show binding to and activation of the human
mb-1, B29, and 14.1 promoters by hEBF
and thus suggest that hEBF plays an important role as a transcriptional
activator of a number of genes defining the early stages of the B
lineage in humans.
Tissue culture conditions
Library construction and screening
Isolation and purification of bone marrow progenitors and mature peripheral blood cells Bone marrow (BM) cells were obtained from the posterior iliac crest of healthy volunteers after informed consent, as were peripheral blood (PB) samples. The BM and PB mononuclear cells (MNCs) were isolated by Ficoll-Hypaque (Nycomed, Oslo, Norway) gradient centrifugation. Positive selection of BM CD34+ cells was performed using a magnetically activated cell sorting CD34 isolation kit (Miltenyi Biotec, Bergish Gladbach, Germany), as described previously.46 The purity of CD34+ enriched cells was reproducibly greater than 80%. CD34-enriched or CD34-depleted BM cells were incubated with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or allophycocyanin-conjugated monoclonal antibodies (MoAbs) against CD34, CD19, CD38, CD33, or isotype-matched irrelevant control antibodies (all from Becton Dickinson, San Jose, CA) for 15 to 20 minutes at 6 to 12°C. CD34+CD19+ (proB), CD34+CD33+ (myeloid progenitor), CD34 CD19+CD38+ (preB), and
CD34CD19+CD38 (immature B) BM
cells were sorted on a FACSVantage Cell Sorter (Becton Dickinson)
equipped with a 488-nm argon-ion laser (Coherent Enterprise II, Santa
Clara, CA) and a 633-nm He-Ne laser (Model 127; Spectra-Physics,
Mountain View, CA). PB CD15+ (mature myeloid),
CD3+ (T), and CD19+IgM+ (mature B)
cell populations were sorted after incubation of MNCs with MoAbs
against CD3 (FITC; Becton Dickinson), CD15 (FITC; Pharmingen, San
Diego, CA), CD19 (PE; Becton Dickinson), and IgM (FITC; Pharmingen) or
control antibody for 15 minutes on ice. The purity of all sorted cell
populations was reproducibly greater than 90% to 95%. To obtain
activated PB B cells, we incubated the sorted
CD19+IgM+ cells in 50 ng/mL of phorbol
myristate acetate (PMA; Sigma, St. Louis, MO) and 500 ng/mL
Ionomycin (Sigma) at 37°C for 4 hours.
Transient transfections and luciferase assays A total of 500 000 cells were washed once with serum-free medium (OPTIMEM; Life Technologies), and 800 µL of the medium was added for transfection. Five microliters of Lipofectin (Life Technologies) was diluted in 100 µL of serum-free medium, incubated for 45 minutes at room temperature, and mixed with the DNA diluted in 100 µL of medium. The mixture was incubated for 25 minutes, and the combined volume of 200 µL was added to the cells. The cells were then incubated in a CO2 incubator at 37°C for 12 hours, after which the transfection medium was removed and replaced by RPMI supplemented with 10% fetal calf serum. The cells were harvested after 40 hours, and protein extracts were prepared directly in the 24-well plates by adding 80 µL of cell lysis buffer (Promega, SDS AB, Falkenberg, Sweden). This procedure results in protein extracts of even quality as judged by Western blots and repeated transfections of cytomegalovirus (CMV)-controlled reporter constructs, reducing the need to normalize for the protein content in the extracts from each transfection by co-transfection of a -gal reporter (M.S.,
unpublished data). The luciferase assay was conducted with 20 µL of
the obtained extracts and 200 µL of luciferase assay reagent (Promega).
Protein extracts and electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared according to Schreiber et al.47 DNA probes were labeled with [32P]-adenosine triphosphate by incubation with T4
polynucleotide kinase (Boehringer), annealed with the complementary
strand, and purified on a 5% polyacrylamide TBE gel. A total
of 5 to 10 µg of nuclear extract or 0.5 to 2 µL of in
vitro-transcribed/translated protein was incubated with labeled probe
(20 000 cpm, 3 fmol) for 30 minutes at room temperature in binding
buffer (10 mmol/L HEPES, pH 7.9; 70 mmol/L KCl; 1 mmol/L dithiothreitol
[DTT]; 1 mmol/L EDTA; 2.5 mmol/L MgCl2; 0.05% NP40) with
0.75 µg Poly dI/dC (Pharmacia). DNA competitors were added 10 minutes
before addition of the DNA probe. The samples were separated on a 6%
acrylamide TBE gel, which was dried and subjected to autoradiography.
Competitors based on synthetic oligonucleotides were added at molar
excesses indicated in the respective figures. Full-length mb-1,
B29, and 14.1 promoters were generated by polymerase
chain reaction (PCR; see below) and were added at molar excesses
indicated in the respective figures.
Oligonucleotides used for the EMSA were as follows: mb-1 EBF sense, 5'-AGCCACCTCTCAGGGGAATTGTGG; mb-1 EBF antisense, 5'-CCACAATTCCCCTGAGAGGTGGCT; mutated mb-1 EBF sense, 5'-AGCCACCTCTCAGCCGTTTTGTGG; mutated mb-1 EBF antisense, 5'-CCACAAAACGGCTGAGAGGTGGCT; CD19 PyG sense, 5'-CGCCTTCCTCTCTGGGGGGACTGCCTG; CD19 PyG antisense, 5'-CAGGCAGTCCCCCCAGAGAGGAAGGCG; Oct binding-site sense, 5'-CATCTCAAGTGATTTGCATCGCATGAGACG; Oct binding-site antisense, 5'-CGTCTCATGCGATGCAAATCACTTGAGATC; 14.1-1 sense, 5'-GCTCAAGCCCTGGGGGACTCCTGC; 14.1-1 antisense, 5'-GCAGGAGTCCCCCAGGGCTTGAGC; 14.1-2 sense, 5'-TCTCTGGACCCCAGTGAGATGCTC; 14.1-2 antisense, 5'-GAGCATCTCACTGGGGTCCAGAGA; 14.1-3 sense, 5'-CCAGGGCGCCCTCGGGGAAGTGGG; 14.1-3 antisense, 5'-CCCACTTCCCCGAGGGCGCCCTGG; 14.1-4 sense, 5'-GGACCAGCCCCGCGGGGACTCAAG; 14.1-4 antisense, 5'-CTTGAGTCCCCGCGGGGCTGGTCC; 14.1-5 sense, 5'-AGGAAAGCCCCAAGGGAGGGTCTT; 14.1-5 antisense, 5'-AAGACCCTCCCTTGGGGCTTTCCT; 14.1-6 sense, 5'-CCCAGGGCCCCAGGGGCAAGGCCA; 14.1-6 antisense, 5'-TGGCCTTGCCCCTGGGGCCCTGGG; 14.1-7 sense, 5'-GGACAGAACCCCAGGGGTAACGGT; and 14.1-7 antisense, 5'-ACCGTTACCCCTGGGGTTCTGTCC. Plasmids and constructs Full-length hEBF cDNA was obtained by 22 cycles of high-fidelity PCR (LaRoche) with an oligonucleotide containing the predicted translation initiation codon and a T7 promoter primer using an hEBF cDNA in the viral vector as template (hEBF 5': 5'-AGACATATGTTTGGGATCCAGGAAAGCATCCAACGGAGTGG). The EBF expression plasmid was based on the eukaryotic expression vector pcDNA3 (Invitrogen, BV, NV Leek, The Netherlands), which places the inserted hEBF cDNA under the control of a CMV promoter. The human mb-1 ( 284 to translation start 2), B29 (146
to +54), and 14.1 (335 to +239) promoters were amplified
by PCR with promoter-specific sense and antisense primers with genomic
HeLa-cell DNA as template. The resulting PCR products were cloned in
the SmaI site of the luciferase reporter plasmid pGL3 basic
(Promega). All constructs were verified by sequencing.
Oligonucleotides used for promoter constructs were as follows: mb-1 PCR sense, 5'-GTGACGAGCCAGCCCTTGAACCA; mb-1 PCR antisense, 5'-TCTCCCAGTGAGTCGGTTAGTTTG; B29 PCR sense, 5'-CCCAGCTGACAAAAGCCTGC; B29 PCR antisense, 5'-GGTCACTGCTCTGTCCCCGACC; 14.1 PCR sense, 5'-GAGCTCAAGCCCTGGGGGACTCCT; and 14.1 PCR antisense, 5'-CGGCCCTGACCCTCAGAGGTCCTT. The basal fos promoter construct has been described earlier.42 In vitro transcription and translation Recombinant protein was generated by coupled in vitro transcription/translation with a reticulocyte lysate kit (Promega) in the presence or absence of [35S]methionine. Two microliters of a 15-µL reaction mix was loaded on SDS-polyacrylamide gel electrophoresis (PAGE), and 0.5 to 2 µL was used for EMSA.Reverse transcriptase (RT) and PCR RNA was prepared from cells with Trizol (Life Technologies), and cDNA was generated by annealing 1 µg of total RNA to 0.5 µg of random hexamers in 10 µL DEPC-treated water. RT reactions were performed with 200 U of SuperScript Reverse Transcriptase (Life Technologies) in the manufacturer's buffer supplemented with 0.5 mmol/L dNTP, 10 mmol/L DTT, and 20 U RNase inhibitor (Boehringer Mannheim, Bromma, Sweden) in a total volume of 20 µL at 37°C for 1 hour. One twentieth of the RT reaction was used in the PCR assays. PCR reactions were performed with 1 U of Taq-polymerase (Life Technologies) in the manufacturer's buffer supplemented with 0.2 mmol/L dNTP in a total volume of 25 µL. Reduced glyceraldehyde phosphate dehydrogenase (GAPDH) was amplified by 25 cycles (94°C, 30 seconds, 55°C, 30 seconds, and 72°C, 30 seconds), whereas 30 cycles were used to amplify EBF cDNA (94°C, 30 seconds, 61°C, 30 seconds, 72°C, 30 seconds). Primers were added to a final concentration of 1 mmol/L. The PCR products were blotted onto Hybond N+ nylon membranes (Amersham) using capillary blotting with 0.4 mol/L NaOH. Membranes were prehybridized in 5× Denharts, 6× SSC, 0.1% SDS, and 50 µg/mL salmon sperm DNA at 57°C for 90 minutes; they were hybridized with[32P]-labeled oligonucleotide for 12 hours at 57°C in the same solution. The membranes were washed at room
temperature 2 times in 2× SSC supplemented with 0.1% SDS for
15 minutes.
Oligonucleotides used for RT-PCR were as follows: GAPDH sense, 5'-CCACCCATGGCAAATTCCATGGCA; GAPDH antisense, 5'-TCTAGACGGCAGGTCAGGTCCACC; GADPH hybridization, 5'-AAGATCATCAGCAATGCCTCCTGC; hEBF sense, 5'-AGACATATGTTTGGGATCCAGGAAAGCATCCAACGGAGTGG; hEBF antisense, 5'-TGAGCAAGACTCGGCACATTTCTG; and hEBF hybridization, 5'-GCCAACAGCGAAAAGACCAATAAC.
Human pre-B cells and B cells express a homologue of mouse EBF Studies of the human CD19 promoter35 as well as FISH analysis of human chromosomes with a mouse EBF probe34 have suggested the existence of a human homologue of mouse EBF. To clone this factor, we made a GT11 cDNA
library from the human pre-B-cell line Nalm6. Low-stringency screening
of 500 000 plaques with a 32P-labeled random-primed mouse
EBF cDNA resulted in 12 clones that also hybridized to the cDNA in a
Southern blot of phage DNA. Sequence analysis revealed a 93% overall
as well as coding-region cDNA homology (GenBank accession number
AF208502) and a 98.8% amino acid homology (Figure
1) to mouse EBF.22 The
longest clone we obtained started at base pair 60 of the defined mouse
cDNA, so to obtain the translation start site, we made a GenBank query using the hEBF sequence. This resulted in the identification of an EST
fragment from a human germinal center library (GenBank accession number
AA504812) spanning the 5' end of hEBF, including the
translation initiation ATG. Using the partial hEBF cDNA as a template,
we extended our cDNA by PCR with a primer containing the missing amino
acids to obtain the full reading frame of hEBF. To investigate whether
the cloned cDNA could be translated into a protein with biochemical
properties similar to those of the mouse EBF, we made in vitro
translations of hEBF. The
[35S]-methionine-labeled proteins were then
detected by SDS-PAGE (Figure 2A). The
empty vector did not result in any translation product, whereas both
the mouse EBF and the hEBF-encoding plasmids generated proteins with an
apparent molecular weight of approximately 55 to 60 kd, which
correlates fairly well with the predicted molecular weight of 64 kd. To
investigate the ability of hEBF to interact with the suggested
EBF-binding site in the human mb-1 promoter,48 we performed the EMSA with either 0.5 or 2 µL of reticulocyte lysate
programmed with empty expression vector or hEBF-encoding vector (Figure
2B). This resulted in one prominent band with the EBF-programmed lysate
that could not be detected with the lysate programmed with empty cDNA3
vector. To confirm the binding specificity between hEBF and the
mb-1 promoter, we performed the EMSA with in
vitro-translated recombinant hEBF and with competition for complex
formation by the addition of duplex oligonucleotides spanning the
mb-1 EBF site, a point-mutated mb-1 EBF site, or
the EBF-binding PyG box from the human CD19
promoter.35,49 Whereas the wild-type mb-1 EBF
site as well as the CD19 PyG box competed for complex formation, the mutated EBF site did not. Hence, we conclude that the
cloned cDNA encodes a protein with the ability to interact specifically
with EBF-binding sites from the human mb-1 and
CD19 promoters.
Expression of EBF in the hematopoietic system in mice is restricted to
pre-B cells and B cells.22,39 To examine whether hEBF is
expressed in a similar pattern, we performed RT-PCR analysis of total
RNA from a set of cell lines representing different cell types (Figure
3A). The presence of GADPH-encoding RNA
was used as a control for the integrity of the cDNA in all of the
samples. Although GADPH message could be detected in all tested cell
lines, EBF message could be found only in KM3 and Nalm6 pre-B-cell
lines, in Namalwa and Raji B-cell lines, and in human PB cells. No EBF message could be detected in epitheloid HeLa cells, Jurkat T cells, or
U937 plasmacytoid/myeloid cells or in THP-1 myeloid cells. To
investigate the presence of proteins with the ability to interact with
the mb-1 promoter EBF site in the same cell lines, we
performed the EMSA with nuclear extracts (Figure 3B). The integrity of
the extracts was confirmed by an EMSA with a 32P-labeled
Oct protein-binding site. Oct proteins could be detected in all the
tested cell lines, whereas only the pre-B cell and B-cell lines
contained high levels of a protein with the ability to interact with
the EBF-binding site. This suggests that human pre-B cell and B-cell
lines express a homologue of the mouse EBF.
To investigate the expression pattern of EBF in primary hematopoietic
cells, we sorted a set of cell populations from human BM and PB by flow
cytometry. RNA was extracted from the cells, and we analyzed the
expression of EBF-encoding message by RT-PCR analysis, as
described earlier (Figure 4).
CD34+CD19+CD10+ pro-B cells from
BM expressed EBF, although at a lower level than
CD34
EBF-binding sites in the mb-1 and B29 promoters are functionally conserved between man and mouse The high homology and overlapping expression patterns of human and mouse EBF suggest that the biochemical properties and the lineage specificity of EBF within the hematopoietic system are conserved between man and mouse. However, to evaluate whether EBF plays similar roles in B-cell development in these 2 species, we wanted to investigate whether the factor was able to interact functionally with the promoters of human homologues of defined genetic targets for EBF in mice. To this end, we compared the promoter structures of mouse and human mb-138,48 and B2950,51 promoters with regard to the presence of potential EBF-binding sites (Figure 5A). Site-selection experiments have suggested that EBF binds to variants of the palindrome sequence ATTCCCNNGGGAAT.36 The mouse mb-1 promoter contains an EBF-binding site with 12 matching base pairs out of 14.38,39 The human promoter contains a slightly different variant of this site because even though the same number of matching bases are found, 12 of 14 of the 5' part as well as the central NN core differ (Figure 5A). The mouse B29 promoter contains 3 independent EBF-binding sites40 with 7, 8, and 10 base pairs homologous to the consensus site, whereas the corresponding EBF sites in the human promoter have 8, 9, and 9 base pairs matching the defined consensus site. This finding suggests that even though differences could be detected within the EBF-binding sites, these would probably not interfere with EBF binding. However, naturally occurring binding sites appear to be hard to predict because some sites with high homology to the consensus sequence do not interact well with EBF, whereas others with apparently lower homology bind EBF with high affinity40,42,52 (M.S., unpublished data). Thus, the ability of EBF to interact functionally with a target promoter cannot be determined solely on the basis of sequence homology, but must be addressed experimentally. To do so, we amplified the human mb-1 and B29 promoters by PCR from genomic HeLa-cell DNA and used these fragments as competitors for hEBF-mb-1 EBF-site complex formation (Figure 5B). The human mb-1 and B29 promoters competed efficiently (Figure 5B), whereas no efficient competition for complex formation could be seen after the addition of a CD19 promoter with a mutated EBF-binding site (data not shown). To test the function of these interactions, we cloned the promoters in front of a luciferase reporter gene and transfected these plasmids into HeLa cells together with either empty or hEBF-encoding expression plasmid (Figure 5C). This resulted in an 8-fold induction of mb-1 promoter activity and a 5-fold increase of B29 promoter activity in the presence of EBF expression plasmid, but no significant effect on the activity of a fos promoter reporter construct.42 This indicates that the human homologue of EBF shares target genes with its mouse counterpart, suggesting conserved functions for EBF in human and mouse B-cell development.
The surrogate light chain gene 14.1 is a genetic target for hEBF Another target gene for EBF activation in the mouse is the surrogate light chain5. The promoter of this gene has
been suggested to be a direct target for synergistic activation by EBF
and the E2A-encoded bh-l-h protein E47.42 To
investigate a potential role for EBF in the regulation of the human
functional homologue 14.1, we compared the sequences of the 5' regions
from the 57 and
14.153 genes (Figure
6A). This showed that even though
conserved regions could be observed, the overall homology was only
about 40%. Furthermore, only 2 of the defined EBF-binding sites in the 5 promoter42 appeared to be conserved,
whereas several other potential binding sites were present only in the
14.1 promoter. To investigate whether the 14.1 promoter was capable of interacting with EBF, we performed an EMSA in
which competition existed for complex formation between recombinant in
vitro-translated EBF and the mb-1 promoter EBF site by the
addition of PCR-amplified 14.1 promoter (335 to
+239) (Figure 6B). Inclusion of unlabeled 14.1 promoter in the EMSA competed efficiently for complex formation, suggesting that EBF indeed has the ability to interact with this control element. To approximate the number of functional EBF-binding sites in the 14.1 promoter, we synthesized oligonucleotides
spanning 7 potential sites and used these as competitors in EMSA
experiments (Figure 6B). Oligonucleotide 14.1-1 contains a
potential EBF-binding site with 10 of 14 matching base pairs and
competed for binding of recombinant EBF, albeit with a low efficiency.
Oligonucleotides 14.1-2 and 14.1-3, which could
be considered conserved between the 5 and 14.1 promoter, both have 9 base pairs matching the EBF consensus site but
appeared unable to interact efficiently with EBF. Both
14.1-4 and 14.1-5 displayed a 10-base pair match to the consensus site and competed efficiently for EBF binding; 14.1-6 and 14.1-7 contained 9- and 10-base pair
homology with the consensus EBF site, respectively, and both competed
with a low efficiency. The wild-type mb-1 promoter EBF site
competed efficiently, whereas no efficient competition could be
detected using the mutated mb-1 promoter EBF site (Figure
2C). This suggests that the 14.1 promoter has the ability to
interact with EBF through at least 5 independent sites with
varying affinities.
To investigate the ability of EBF to induce transcription from
the 14.1 promoter, we cloned the PCR-amplified
14.1 promoter in a luciferase reporter vector and made
transient transfections of this construct and hEBF-encoding expression
plasmid in HeLa cells (Figure 7A).
Addition of 100 or 600 ng of hEBF-encoding expression vector resulted
in a 4-fold or 24.5-fold induction of the 14.1 promoter
activity, respectively, whereas no significant effect on the function
of a fos promoter could be detected. This suggests that hEBF has the
ability to interact functionally with the 14.1 promoter.
The function of the mouse
We here report the cloning of the human homologue of mouse EBF. Mouse and human EBFs have a high homology at the protein and RNA levels and also appear to share expression patterns in the hematopoietic system. We also show data supporting the idea that these proteins share a number of B-lineage-restricted target genes. Thus, we suggest that the role of EBF in human B-cell development may be highly analogous to that in mice, positioning EBF as a key transcriptional regulator in human B lymphopoiesis. In the mouse, it has been suggested that EBF is an activator of
several genes encoding components of the pre-B-cell and B-cell receptors.39,40,42 The data we present suggest not only
that EBF is conserved as a protein, but also that the functional role of EBF in B-cell development may be conserved between man and mouse.
This is based on the ability of hEBF to interact functionally with
promoters of target genes defined in mouse B-cell development. In the
case of the B2951 and
mb-148 genes, this seems to be a consequence of
conserved 5' regions. As a result, the structural features of the
promoter elements as well as the EBF-binding sites appear to be
conserved between man and mouse. It is notable that mutations have
occurred also within the EBF-binding sites, but that most of these are
positioned so that they should not impair binding of EBF (Figure 5A).
This may reflect a selection for functional ability to interact with
EBF rather than a precise base-pair composition of the promoters.
Functional conservation of EBF binding is even more striking when
comparing the promoter of 14.1 with that of the mouse
homologue When studying the role of transcription factors in an evolutionary
perspective, it may be interesting to compare functional and genetic
conservation. One striking example of functional conservation is the
role of NF The presence of a helix-loop-helix dimerization domain in EBF has led to the suggestion that EBF belongs to the helix-loop-helix family of transcription factors. This family includes transcription factors such as myc, Tal, and E47, all known to play important roles in the development of leukemia.66-68 EBF has not yet been implicated in the development of human cancer. However, abnormalities in the p34 region of human chromosome 5, where hEBF is located,34 have been associated with a number of myeloid leukemias.69 A role for EBF in the development of myeloid leukemia could occur because EBF appears to be a key factor in the determination of the B-cell as opposed to the myeloid lineage. Ectopic expression of EBF in macrophage-converted 70Z/3 pre-B cells results in a partial restoration of the B-cell phenotype.45 Such a function of EBF may be of interest in human malignancies in which the conversion of B lymphomas into mixed or myeloid-like cells is associated with lower remission rates and poor prognosis.70-72 The data we present suggest that hEBF is involved in the regulation of several genes defining the B lineage. However, our understanding of the precise role of this factor in normal and malignant B-cell development remains elusive and demands further studies of genetic targets and of direct involvement in the initiation or progression of human cancer.
We thank Y. Zhuang and R. Grosschedl for the kind gifts of plasmids, K. Riesbeck for the gift of human peripheral blood cell RNA, Ingbritt Åstrand-Grundström for help with cell sorting, and D. Liberg and R. Carlsson for helpful comments and critical reading of the manuscript.
Submitted December 2, 1999; accepted April 14, 2000.
Supported by the Swedish Medical Research Council, The Swedish Cancer Foundation, Magnus Bergwall-Åke Wibergs-Österlunds Foundation, The Crafoord Foundation, and The American Cancer Society.
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.
Reprints: Mikael Sigvardsson, Immunology Group, CMB, Lund University, Sölvegatan 21, S-223 62 Lund, Sweden; e-mail: mikael.sigvardsson{at}immuno.lu.se.
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Top
Abstract
Introduction
(mb-1) and Ig
14.1, 16.1, and 16.2
B homologues in stimulating inflammatory responses in both
mouse and Drosophila.