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HEMATOPOIESIS
From the Institute of Molecular Genetics, Academy of
Sciences of the Czech Republic, Prague, and the Max-Delbrück
Center for Molecular Medicine, Berlin, Germany.
The development of blood cells proceeds from pluripotent stem cells
through multipotent progenitors into mature elements belonging to at
least 8 different lineages. The lineage choice process during which
stem cells and progenitors commit to a particular lineage is regulated
by a coordinated action of extracellular signals and transcription
factors. Molecular mechanisms controlling commitment are largely
unknown. Here, the transcription factor v-Myb and its leucine zipper
region (LZR) are identified as regulators of the commitment of
a common myeloid progenitor and progenitors restricted to the myeloid
lineage. It is demonstrated that wild-type v-Myb with the
intact LZR directs development of progenitors into the macrophage
lineage. Mutations in this region compromise commitment toward myeloid
cells and cause v-Myb to also support the development of erythroid
cells, thrombocytes, and granulocytes, similar to the c-Myb protein. In
agreement with that, the wild-type v-Myb induces high expression of
myeloid factors C/EBP Differentiation of hematopoietic stem cells and
progenitors into various lineages is controlled by a complex array of
extrinsic and intrinsic factors.1-4 Myeloid and erythroid
blood cells develop from a common myeloid progenitor, which
differentiates into either megakaryocytes and erythrocytes, or
granulocytes and macrophages. Several experimental strategies including
gene targeting, expression pattern analysis, antisense, and
overexpression studies led to the identification of transcription
factors that are required for formation, survival, and proliferation of
multilineage progenitors and that direct the differentiation and
maturation of individual lineages. Specifically, factors like
SCL, Rbtn2, GATA-2, and c-Myb were found to be essential for
multipotent cells.5 In more mature cells, the expression
of SCL, GATA-1, c-Myb, Rbtn2, FOG, and EKLF is a prerequisite for
proper development of the erythroid lineage. On the other hand, factors
of the C/EBP family, PU.1, Egr-1, c-Myb, and AML-1 play an
important role in the myeloid lineage.6-9
It has been suggested that in the common myeloid progenitor the
concentrations of intracellular lineage-determining regulators are to
be low and balanced.10,11 During the commitment process this balance is disturbed either in a stochastic or instructed manner,
resulting in the prevalence of a particular set of factors and the
subsequent development of the respective lineage. Several factors were
identified that instruct progenitor cells to develop along a specific
lineage. Using the model of primary chicken
myb-ets-transformed multipotent progenitors
(MEP),12 it was demonstrated that PU.1 and C/EBP The c-myb proto-oncogene is essential for early definitive
myeloid and erythroid cells as documented by gene targeting
experiments.16 It is required for the expansion of
immature cells of the myeloid, erythroid, and lymphoid lineages and is
down-regulated during their terminal differentiations.17
The v-myb gene transduced by avian myeloblastosis virus
(AMV), as well as its truncated homologue, transduced as the
myb-ets fusion by E26 leukemia virus, are oncogenes that
specifically affect the developmental programs of avian hematopoietic
cells. AMV v-Myb interferes exclusively with the development of the
macrophage lineage of both primitive and definitive hematopoietic cells
by blocking terminal differentiation of macrophage precursors and activating their self-renewal capacities in vitro. It causes fatal acute monoblastic leukemia in chicks. E26 v-Myb-Ets fusion
protein transforms multipotent progenitors of primitive hematopoietic cells (blastoderm), and committed erythroid and myeloid cells of bone
marrow. It induces erythroid leukemia in infected
chicks.18-22 Thus, the E26 v-Myb-Ets protein affects a
broader spectrum and more immature cells than the AMV v-Myb. In this
respect the biologic activities of E26 v-Myb-Ets resemble those of
c-Myb, which influence the development of many immature hematopoietic
cells. The very specific biologic activities of AMV v-Myb are thought
to result from the loss of some c-Myb functions due to N- and
C-terminal deletions and point mutations. Therefore, it was speculated
that the mechanisms by which AMV v-Myb deregulates development of
hematopoietic cells might also differ significantly from the mechanisms
of action of E26 v-Myb-Ets and c-Myb.23
In this paper we demonstrate that the long-observed restriction of AMV
v-Myb transforming potential to macrophage precursors is mainly due to
the activity of its leucine zipper region (LZR), which programs
development of hematopoietic progenitors into the macrophage lineage.
Mutations in the LZR rescue the Myb activity to affect uncommitted
progenitors as well as cells of erythroid and granulocytic lineages.
The LZR of Myb therefore appears to be a part of a mechanism that
regulates a lineage choice in hematopoietic tissues.
Cells and cell culture
Differentiation assays
Cytochemical assays To evaluate cell morphology and hemoglobin content, cytospin preparations were stained with neutral benzidine and counterstained with Diff-Quik (Baxter, Deerfield, IL).26,30 May-Grünwald-Giemsa staining followed standard procedures. To detect heterophilic granules, cells were stained with Astra blue.31 The peroxidase activity in eosinophilic granules was determined as described.32 In each experiment, 300 to 500 cells were evaluated and the proportion of individual cell types was determined. Microphotographs were taken from representative fields.Plasmids The following AMV-v-myb mutants were used: C
contains a deletion of 27 C-terminal amino acid codons (362-388);
LZ1, LZ2, P1, P2, and P3 represent internal in-frame
deletions of codons 325-362, 311-325, 301-311, 294-300, and 279-300, respectively. The numbering of codons is based on the v-myb
cDNA sequence.33 All deletion mutants were generated by
polymerase chain reaction (PCR) and verified by DNA sequencing. The
L3,4A mutant was described previously.25 Mutant genes were
inserted into the pNeoAMV vector34 and transfected into
CEFs or QT6 cells along with MAV-1 helper virus DNA by calcium
phosphate.25
Antibodies, flow cytometry, and immunofluorescence The following antibodies were used: MC51/2, MC47/83, MC22/3,35 and JS436 (kindly provided by Dr H. Beug); 11C3 monoclonal antibody37 (generous gift of Dr M. Corbel); MEP26, MEP21, MEP17 antibodies38 (generously provided by Dr T. Graf); anti-GATA-1,39 anti-Myb monoclonal antibody 2.32,40 and polyclonal anti-C/EBP antibody41 (kind gift of Dr K.-H. Klempnauer).
Flow cytometry was performed as described25 using the EPICS Elite ESP Flow Cytometer (Coulter). For immunofluorescence analysis, cells were attached to coated slides (Bio-Rad, Hercules, CA) and processed according to the manufacturer's protocol with the following modifications. Briefly, the cells were recovered, resuspended in phosphate-buffered saline (PBS) and loaded onto adhesion slide reaction fields. The cells were fixed with 3% paraformaldehyde for 5 minutes, washed with PBS and permeabilized with 0.4% NP-40 in PBS for 2 minutes. After washing in PBS the reaction fields were blocked with 2% BSA in PBS for 1 hour. Then, the cells were reacted with primary monoclonal antibodies (1 hour), washed with PBS, 0.5% BSA, and stained with fluorescein isothiocyanate (FITC)-conjugated and/or rhodamin-conjugated secondary antibody (1 hour). All reactions were done at room temperature. Finally, the cells were mounted into Mowiol (Hoechst, Strasbourg, France) and images were taken with a Leica DMIRB microscope (Leica, Wetzlar, Germany) and processed with Leica Qwin and Adobe Photoshop software. Northern blot analysis and RNase protection assay Total RNA was prepared42 and 15 µg were separated on 1% agarose/formaldehyde gels, transferred to nylon membrane (GeneScreen, NEN, Boston, MA), and hybridized with specific probes. The following probes were used: 0.8-kb EcoRI fragment of chicken PU.1 cDNA43 (kind gift of Dr J. Ghysdael); 305-base pair (bp) BglII-NheI fragment of chicken c-myb cDNA44; chicken D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA45; and full-length cDNA of chicken GATA-146 (kindly provided by Dr J. D. Engel). The chicken egr-1-, C/EBP-, and SCL-specific probes (589 bp,
610 bp, and 968 bp, respectively) were generated by reverse
transcriptase (RT)-PCR (nucleotides: 1-589, 493-1102, and 208-1175, respectively; GenBank accession nos. AF026082, Z21646, and X63371).
Probes were labeled by nick-translation and hybridized overnight in
ULTRAhyb (Ambion, Austin, TX) at 42°C. Filters were washed twice in
2 × standard saline citrate (SSC)/0.1% sodium dodecyl
sulfate (SDS) and then in 0.1 × SSC/ 0.1% SDS at 42°C, and exposed
to BioMax MS film (Kodak, Rochester, NY) with intensifying screen.
For RNase protection assays, a 306-bp cDNA fragment of chicken GBX2
(nucleotide number 905-1211; GenBank accession no. AF022151) was
subcloned into pGEM3Z. Similarly, a 182-bp cDNA fragment of chicken
C/EBP Western blot analysis Protein lysates were separated by 10% SDS-PAGE, blotted onto nitrocellulose membranes (BA85, Schleicher and Schuell, Dässel, Germany) and processed for western blot analysis as described.25 The 2.32 anti-Myb monoclonal antibody served as primary antibody.Transient transactivation assay CEFs (6 × 105 cells) were seeded onto 60-mm dishes and incubated in DMEM medium (D5546, Sigma) supplemented with 8% FCS and 2% ChS serum, 580 mg/L L-glutamine, and 100 units/mL penicillin/streptomycin (Gibco-BRL). The cells were transfected and processed as described earlier.25 The cells transfected with empty pNeo vector were used as a control. The chloramphenicol acetyl transferase (CAT) in transfected cells was determined by the CAT enzyme-linked immunosorbent assay (ELISA) (Boehringer Mannheim, Mannheim, Germany). The CAT value of wild-type v-Myb was assigned a value of 100%. Transcriptional activities of v-Myb mutants were calculated in percents of the wild-type v-Myb activity.
Mutations in the LZR activate the transforming potential of Myb oncoprotein in erythroid cells Previous work on myeloid cells demonstrated that v-Myb LZR is required for proliferation, growth factor independence, and leukemic properties of v-Myb-transformed monoblasts.25,49 In order to obtain more information about the biologic activity of v-Myb LZR, a series of deletion mutants were constructed that span the C-terminus of v-Myb protein (Figure 1). The point mutant v-MybL3,4A, where Leu3 and Leu4 of LZR are replaced by Ala25 was also used. Mutations affecting the LZR, namelyLZ1, LZ2, P1, and L3,4A are in the following text referred to
as LZR mutations. Since C-terminal mutations could negatively influence
the basic property of v-Myb, that is, its transcriptional activity, CAT assays in transiently transfected fibroblasts were performed. It was
found that LZR mutations did not impair but rather slightly enhanced
(1.5 fold-2.5 fold) the transcriptional activity of v-Myb (Figure 1).
The activity of P2 and P3 mutants was reduced to 70% and 45%,
respectively, of wild-type Myb.
Next, the transforming potential of the Myb mutants was analyzed.
Retroviruses carrying mutant myb genes were used to infect blastoderm cells from 20- to 28-hour-old chicken embryos. The
These results define LZR as a functional domain and demonstrate that its inactivation by mutation enables v-Myb to also affect erythroid cells in addition to monoblastlike cells. Importantly, mutations of LZR have no deleterious effect on transcriptional properties of v-Myb. The C and P2 mutants, containing an intact LZR, induced a monoblastic phenotype identical to that of wild-type v-Myb.
The P3 mutant displayed a rather weak transforming activity (data
not shown). Among LZR mutants, P1 induced in infected cells (a
mixture of myeloid and erythroid cells) a proliferation rate, life
span, and colony-formation efficiency that was comparable to wild-type
v-Myb, whereas myeloerythroid cultures formed by LZ1, LZ2, and
L3,4A mutants grew more slowly and the cells showed a somewhat reduced
lifespan and colony-formation efficiency. Since the intracellular
stability and subcellular distribution of P1 and wild-type v-Myb
proteins were also very similar,49 the P1 mutant was
selected for further studies and analyzed along with wild-type
v-Myb.
The v-Myb LZR influences expression of lineage-specific genes Blastoderm cells transformed by wild-type andP1 v-Myb were
first characterized for their growth potential, morphology, and surface
marker expression, and second for expression of genes important for the
development of the myeloid and erythroid lineages. Figure 2A,B shows
that within the first 7 days after infection, wild-type v-Myb cultures
were composed of monoblastlike cells and some erythroid cells, whereas
from day 7 on only rapidly proliferating monoblasts were observed. In
contrast, in P1 cultures there was an initial outgrowth of primarily
erythroid cells within the first 7 days after infection. Then, a high
proportion of cells underwent spontaneous differentiation into
erythrocytes, and resulting cultures contained erythroid cells of
different maturation stages and monoblasts (Figure 2A,B). At later time
points, cells belonging to additional hematopoietic
lineages thrombocytes, eosinophils, and heterophilic granulocytes
(equivalent to neutrophils in mammals)were also observed (see below).
Protein analysis in cultured cells demonstrated essentially identical
amounts of wild-type and P1 v-Myb proteins, comparable with v-Myb
expression in the myeloid cell line BM2 (Figure 2C). Thus, accumulation
of different cells in wild-type and P1 v-Myb cultures was not due to
different expression levels of Myb proteins.
To further extend these observations, cells were analyzed for
lineage-specific surface markers using monoclonal antibodies MC51/2 and
JS4, which recognize myeloid and erythroid surface molecules,
respectively. The Next, wild-type and Expression of the homeobox transcription factor GBX2 was also analyzed,
as GBX2 was suggested to activate expression of chicken myelomonocytic
growth factor (cMGF) and to generate an autocrine loop important for
the development and proliferation of AMV v-Myb-transformed myeloid
cells.50 In cultures of primary blastoderm cells, however, only very low levels of GBX2-specific transcripts were detected in
v-Myb cooperates with growth factors to block differentiation of multipotent progenitors In early cultures ofP1 v-myb-infected and to a
lesser extent also of wild-type v-myb-infected blastoderm
hematopoietic cells, some cells expressed surface protein markers of
multipotent progenitors recognized by MEP17, MEP21, and MEP26
monoclonal antibodies.12,38 We therefore reasoned that the
different v-Myb cells described so far in this paper might represent
the progeny of multipotent progenitors, which were not completely
blocked in differentiation and hence spontaneously developed into
myeloid or erythroid cells. To enhance the self-renewing capacity
and/or delay spontaneous differentiation of putative Myb progenitors,
different growth factors and combinations thereof were tested. TGF ,
bFGF, and SCF, when applied simultaneously, were effective in
increasing growth rates of both wild-type and P1 v-Myb cells
positive for MEP antigens. Maximal expression of MEP antigens was
achieved on approximately days 14 to 21 of culture (Figure
3A). At this point all cells also
expressed the erythroid marker JS4. No myeloid cells were present, as
documented by the absence of the myeloid marker MC51/2 (Figure
4A). Thus, v-Myb and specific growth
factors cause accumulation of early undifferentiated cells. These cells are progenitors since they can develop into different types of hematopoietic cells as will be documented in following experiments. Importantly, these progenitors accumulated in cultures only when growth
factors were added at the time of infection with myb
constructs. Later addition of factors yielded only higher numbers of
erythroid cells in P1 cultures and had no effect on the strictly
myeloid character of wild-type myb-infected cells. This
observation ruled out the possibility that v-Myb monoblasts and
erythroid cells were reprogrammed into progenitors by growth
factors.
v-Myb LZR influences expression of lineage-specifying factors in progenitors As stated above, the highest proportion of progenitors was obtained in 14- to 21-day-old cultures. Their prolonged cultivation in the presence of TGF, bFGF, and SCF (for about 30 days) caused both
wild-type and P1 v-Myb progenitors to down-regulate MEP markers and
to commit to the erythroid lineage, as evidenced by their ability
to differentiate into hemoglobinized erythrocytes (P.B. et al,
manuscript submitted, June 2001). To find out whether Myb LZR
influences levels of C/EBP, PU.1, Egr-1, c-Myb, SCL, and GATA-1
mRNAs in progenitors and early erythroid cells, RNA from cells grown
for 21 and 30 days in growth factor mixture was analyzed. Figure 3B
shows that both wild-type and P1 v-Myb cells abundantly expressed
SCL and GATA-1 mRNAs at both time points. In contrast, c-Myb mRNA was
lower in wild-type v-Myb cells than in P1 cells on day 21, whereas
its levels were essentially the same on day 30 when both cultures were
erythroid. The mRNAs of myeloid factors C/EBP and PU.1 were more
abundant in wild-type v-Myb cells than in P1 cells on day 21 and
barely detectable in both cultures on day 30. Virtually no difference
was seen for Egr-1 mRNA levels at any time.
The data show that in v-Myb progenitors (day 21), wild-type LZR
causes elevated levels of C/EBP Wild-type v-Myb progenitors differentiate predominantly into the
macrophage lineage; , bFGF, and SCF, and
then their differentiation was induced by withdrawal of growth factors.
As the phorbol ester TPA was found to be a potent inducer of
differentiation of Myb progenitors,12 factor removal and
TPA treatment were combined in some experiments. Resulting cells were
first analyzed for the presence of lineage-specific surface proteins
(Figure 4A). Immediately prior to differentiation induction both
wild-type and P1 v-Myb cells expressed the erythroid JS4 marker and
no myeloid MC51/2 (Figure 4A, +GF). From additional myeloid markers the
MC47/83 was weakly detectable in both cultures whereas the MC22/3 was
present only on P1 cells (Figure 4A, +GF). Several days after
differentiation induction by growth factor removal and TPA treatment,
wild-type v-Myb cells lost the erythroid marker JS4 and upregulated the
myelomonocytic markers MC51/2, MC47/83, and MC22/3 (Figure 4A, +TPA).
These cells clearly represented macrophage precursors as also judged by
morphology (data not shown). In contrast, cells infected with P1
v-myb still retained high JS4 expression and were negative
for MC51/2 and MC22/3, and weakly positive for MC47/83 (Figure 4A,
+TPA). Additionally, these cells kept their immature phenotype
(not shown).
In similar experiments, differentiating cells were analyzed by
cytochemical staining for the presence of granulocytes and by
immunofluorescence for thrombocytes 20 days after differentiation induction (Figure 4B). In both wild-type and The treatment of blastoderm cells transformed with Myb LZR determines commitment of a common myeloid progenitor to the macrophage lineage Our experiments suggest that LZR represents a critical element for development of progenitors into either erythroid or myeloid cells. To demonstrate this at the clonal level, blastoderm cells infected with wild-type orP1 v-myb genes were seeded into semisolid media containing TGF, bFGF, and SCF. Colonies were isolated 12 days
later. In a representative experiment, 94 wild-type-myb and 155 P1-myb colonies were obtained (Figure
5A). More than 90% of both
wild-type-myb and P1-myb clones
could be expanded in liquid culture. Ten representative clones of each
type were finally studied in detail. At the time of isolation both
wild-type and P1 v-myb colonies contained cells
displaying an immature phenotype, rather weak JS4 staining, and no
MC51/2 staining.
Individual colonies were then grown for 10 to 14 days in the absence of
factors to allow their differentiation and analyzed again for the
presence of erythroid and myeloid markers. The analysis of one
representative wild-type and one These data demonstrate unequivocally that the colonies were composed of
both myeloid and erythroid cells, which must have originated from a
common myeloid progenitor. Additionally, wild-type v-Myb progenitors
developed predominantly into MC51/2+/C/EBP Several well-growing clones were later analyzed for provirus integration sites by southern blot hybridization. The majority of clones contained a single integration site, indicating that they originated from a single cell (data not shown).
In this paper we describe the capacity of the Myb oncoprotein and
its LZR to affect the lineage commitment of various hematopoietic progenitors. The lineage-determining activity of Myb LZR is
particularly obvious when self-renewal of Myb progenitors is enhanced
by specific growth factors (TGF
The hematopoietic progenitors transformed by v-Myb LZR mutants
described in this paper, and exemplified by The analysis of selected mRNAs and proteins with an impact on the
development of myeloid and erythroid lineages revealed higher amounts
of C/EBP Our data show that at least the elevated levels of C/EBP The data indicate that Myb can affect, through its LZR, expression of
factors controlling development of myeloid and erythroid lineages. The
exact mechanism of how this is achieved is not known at present. Recent
work demonstrates that Myb can directly activate the C/EBP Additionally, a common feature of regulatory protein complexes in
hematopoietic cells appears to be their association with the
multifunctional regulator CBP, which recruits many hematopoietic transcription factors, including c-Myb.59,60 It is
therefore tempting to speculate that Myb LZR (if present and
accessible) favors the formation of a protein complex composed of
CBP-Myb and myeloid factors (eg, C/EBP This model also implies that Myb LZR itself binds specific protein factors. None of the above-mentioned factors bind Myb LZR, and therefore some others are likely to be involved. It can be expected that the complex biologic activity of Myb LZR is caused by rather complex interactions. At the present state of knowledge it is hard to indicate any protein whose interaction with Myb LZR might have been decisive for the lineage choice. The only well-characterized partner of Myb leucine zipper, the p160 protein,61 cannot be readily implicated in the regulation of hematopoietic commitment. Perhaps the analysis of recently identified transcription factors that bind to Myb leucine zipper (V.C., unpublished data, June 2000) might bring more information about mechanisms of action of Myb in hematopoietic progenitors.
We thank Drs J. D. Engel and J. Ghysdael for recombinant plasmids; and Drs H. Beug, C. Corbel, T. Graf, and K.-H. Klempnauer for providing antibodies. We are indebted to Drs Z. Kozmik and P. Urbanek for critical reading of the manuscript and I. Gallagher and S. Takacova for the help with manuscript preparation.
Submitted April 10, 2001; accepted August 10, 2001.
Supported by grants 301/98/K042 and 204/00/0554 from the Grant Agency of the Czech Republic (M.D.), A5052805 from the Grant Agency of the Academy of Sciences of th |
áková,
í,
ermák,
n
k,
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Abstract
Introduction
leukosis-free chick embryos in Hamburger
and Hamilton stages 5 through 8.
and C/EBP