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HEMATOPOIESIS
From the Department of Hematology/Oncology, Osaka
University Medical School, Osaka; Center for TARA and Institute of
Basic Medical Institute, University of Tsukuba, Tsukuba; and Department
of Immunology, Osaka City University Medical School, Osaka, Japan.
Lineage-specific transcription factors play crucial roles in
the development of hematopoietic cells. In a previous study, it was
demonstrated that Ras activation was involved in
thrombopoietin-induced megakaryocytic differentiation. In this study,
constitutive Ras activation by H-rasG12V evoked
megakaryocytic maturation of erythroleukemia cell lines F-36P and K562,
but not of myeloid cell line 32D cl3 that lacks GATA-1. However, the
introduction of GATA-1 led to reprogramming of 32D cl3 toward
erythrocytic/megakaryocytic lineage and enabled it to undergo
megakaryocytic differentiation in response to H-rasG12V. In
contrast, the overexpression of PU.1 and c-Myb changed the phenotype of
K562 from erythroid to myeloid/monocytic lineage and rendered K562 to
differentiate into granulocytes and macrophages in response to
H-rasG12V, respectively. In GATA-1-transfected 32D cl3,
the endogenous expression of PU.1 and c-Myb was easily detectable, but
their activities were reduced severely. Endogenous GATA-1 activities were markedly suppressed in PU.1-transfected and c-myb-transfected K562. As for the mechanisms of these reciprocal inhibitions,
GATA-1 and PU.1 were found to associate through their DNA-binding
domains and to inhibit the respective DNA-binding activities of
each other. In addition, c-Myb bound to GATA-1 and inhibited
its DNA-binding activities. Mutant GATA-1 and PU.1 that retained their
own transcriptional activities but could not inhibit the reciprocal
partner were less effective in changing the lineage phenotype of 32D
cl3 and K562. These results suggested that GATA-1 activities may be
crucial for Ras-mediated megakaryocytic differentiation and that its
activities may be regulated by the direct interaction with other
lineage-specific transcription factors such as PU.1 and c-Myb.
(Blood. 2000;96:2440-2450) Transcription factors play a key role during the
development of hematopoietic cells through a series of gene
transcriptions necessary for their growth, differentiation, and
survival. It has previously been shown that targeted disruption of a
certain transcription factor often causes a complete or selective
defect of hematopoietic cells. For example, AML-1 The GATA family is composed of 6 members and is indispensable for
the development and subsequent growth and differentiation of diverse
cell types.3 GATA family proteins possess 2 highly conserved zinc finger domains, both of which include the configuration Cys-X2-Cys-X17-Cys-X2-Cys. The carboxyl (C) finger is absolutely required for DNA binding, whereas the amino (N) finger stabilizes the
DNA binding and confers full specificity.4,5 The first cloned member of this family, GATA-1, was identified as an erythroid nuclear protein that binds to consensus GATA motifs, (A/T)GATA(A/G), in
globin gene promoters, enhancers, and locus control regions. It has
been shown that GATA-1 is expressed at high levels in erythroid cells,
megakaryocytes, and mast cells and at lower levels in hematopoietic progenitor cells and Sertoli cells of testis.3 In
contrast, GATA-2 is ubiquitously expressed, and GATA-3 is exclusively
expressed, in T lymphocytes.3 In chimeric mice generated
from mutant embryonic stem (ES) cells lacking GATA-1, the mutant cells
did not contribute to erythropoiesis.6 In addition,
GATA-1-null ES cells were unable to differentiate into mature
erythroid cells in vitro.7,8 Thus, GATA-1 is considered to
be essential for the terminal differentiation of erythroid progenitor
cells. Recently, Shivdasani et al9 reported that
lineage-selective GATA-1 knock-out mice exhibited striking
thrombocytopenia and severe anemia, which were accompanied by the
increased proliferation and impaired cytoplasmic maturation of
megakaryocytes.9 Furthermore, Takahashi et
al10 demonstrated that heterozygous mutant mice chimeric
for the GATA-1 gene displayed marked splenomegaly, anemia, and
thrombocytopenia. These results further suggest that GATA-1 plays
essential roles not only in erythropoiesis but also in megakaryopoiesis
and thrombopoiesis.
PU.1 is a member of the Ets family transcription factors that
specifically bind to the GGAA/T motif in the target DNA.11 Structural and functional analyses of PU.1 revealed that DNA binding is
executed through the C-terminal Ets homology region and that the
transactivating domain is located in the N-terminal
region.12 In addition, there is a central region, called a
PEST domain, rich in proline, glutamic acid, serine, and threonine
residues.12 Expression of PU.1 is restrictedly detected in
hematopoietic tissues, especially with high levels in monocytic,
granulocytic, and B-lymphoid lineages.11 Several
presumptive target genes of PU.1 have been identified, some of which
are essential for the growth and survival of the cells in these
lineages.13 In agreement with these findings, PU.1-targeted mice showed defects in the development of multiple hematopoietic lineages, including B and T lymphocytes, monocytes, and
granulocytes.14,15 In addition, PU.1 was shown to have the
potential to impose myeloid lineage commitment on multipotent hematopoietic progenitors,16 suggesting that PU.1 is a
master regulator of myeloid lineage commitment.
The growth and differentiation of hematopoietic stem/progenitor cells
are regulated by a number of growth factors. Among them, thrombopoietin
(TPO) is a fundamental regulator of megakaryopoiesis and
thrombopoiesis. In previous studies,17-19 others and we
have shown that Ras/MAPK (mitogen-activated protein kinase) activation is involved in TPO-induced megakaryocytic differentiation. However, Ras
activation is not necessarily coupled with megakaryocytic differentiation in other cell types. For example, Ras activation was
shown to promote terminal differentiation toward macrophages in a
monocytic cell line, U937.20 In addition, activated Ras was reported to induce cell growth but not to affect the stage of
maturation in a murine myeloid cell line, 32D cl3, and a murine pro B
cell line, Ba/F3.21,22 Therefore, it remains largely unknown which instinctive cell properties are prerequisite for Ras-mediated megakaryocytic differentiation. Because instinctive characters of hematopoietic cells are primarily controlled by lineage-specific transcription factors such as GATA-1 and PU.1, in the
current study we investigated the roles of these transcription factors
in Ras-mediated megakaryocytic differentiation. We here found that
GATA-1 enabled the myeloid cell line 32D cl3 to undergo megakaryocytic
differentiation in response to constitutive Ras activation by
H-rasG12V. In addition, though H-rasG12V
induced megakaryocytic differentiation of an erythroid cell line K562,
the introduction of PU.1-and c-myb into
H-rasG12V-expressing K562 cells led to granulocytic and
macrophage maturation, respectively. In these cells, the
transcriptional activity of GATA-1 was suppressed by PU.1 or c-Myb and
vice versa. Thus, we here provide unique evidence that GATA-1
activities are required for Ras-mediated megakaryocytic differentiation
and that the cell lineage and differentiation may be determined by the
combined effects of lineage-specific transcription factors, including
GATA-1, PU.1, and c-Myb.
Reagents and antibodies
Cell lines and cultures
Plasmid constructions and cDNA Full-length human GATA-1 and murine PU.1 cDNA were subcloned into pcDNA3 (Invitrogen, De Schelp, Netherlands) to generate each expression vector. To construct NZF of GATA-1 and AD of PU.1,
cDNA coding amino acids 194 to 250 and 33 to 101 were eliminated by
polymerase chain reaction, respectively. This cDNA was subcloned into
pcDNA3-HA or pcDNA3-Flag to generate N-terminus epitope-tagged expression vectors. An expression vector of c-myb, pact-c-myb, was
kindly provided from Dr S. Ishii (Tsukuba Life Science Center; RIKEN,
Ibaraki, Japan). Probes for platelet factor 4 (PF4) and GPIIb were
kindly provided from Dr Y. Furukawa (Jichi Medical School, Tochigi,
Japan) and Dr Y. Tomiyama (Osaka University, Osaka, Japan), respectively.
Morphologic analysis The morphology of the cultured cells was analyzed by staining the cytospin preparations with May-Grünwald-Giemsa.Benzidine staining Cultured cells were suspended in staining solution, which is a 14:1 mixture of benzidine solution (3% benzidine in acetic acid) and 31% H2O2, for 5 minutes, and subjected to cytospin centrifugation. The black-stained cells were counted as benzidine-positive cells by microscopy.Flow cytometry Surface phenotypes of cells were examined by indirect immunofluorescence with FACSort (Beckon Dickinson, Oxnard, CA). DNA content of cultured cells was quantitated by propidium iodide staining.23Northern blot analysis The isolation of total cellular RNA and the method used for Northern blot analysis were described previously.23Immunoprecipitation and immunoblotting Preparation of cell lysates, immunoprecipitation, gel electrophoresis, and immunoblotting were performed as described previously.23 Immunoreactive proteins were visualized with the enhanced chemiluminescence detection system (Dupont NEN, Boston, MA).Lac-inducible system and plasmids To express a target cDNA, we used a LacSwitch II (Stratagene, La Jolla, CA) inducible expression system in which the expression of the target cDNA is induced by isopropyl- -D-thiogalactopyranoside (IPTG) treatment. In short, F-36P, K562, and 32D cl3 cells were initially transfected with an expression vector of Lac-repressor (Lac-R), pCMV-LacI, by electroporation. After selection with hygromycin (0.5 mg/mL), one clone, in which Lac-R was most intensely expressed, was further transfected with a Lac-inducible expression vector of
H-rasG12V, pOPRSVI-H-rasG12V.17
After selection with G418 (1.0 mg/mL), the induction levels of
H-rasG12V were examined before and after IPTG treatment
(0.5 mmol/L) in each clone by Western blot analysis. To further
transfect an expression vector of PU.1, c-myb, or GATA-1 into
hygromycin- and G418-resistant cells, an expression vector of
blasticidin S deaminase pSV2bsr (Kaken Pharma, Tokyo, Japan) was
cotransfected with an appropriate expression vector and cultured with
30 µg/mL blastcidin S hydrochloride (Funakoshi, Tokyo, Japan).
Luciferase assay At first, the murine minimal JunB promoter ( 42 to +136) was
subcloned into the luciferase plasmid to construct JunB-MP-Luc. To
generate functional and nonfunctional reporter genes for GATA-1, PU.1,
and c-Myb, concatamerized double-stranded oligonucleotides were
subcloned just upstream of minimal JunB promoter in JunB-MP-Luc, and
their sequences were as follows: a wild-type (WT) and a mutated (MT)
reporter gene for GATA-1 (3× WT-M -Luc,
5'-GGGCAACTGATAAGGATTCC-3'; 3× MT-M-Luc, MT,
5'-GGGCAACTGGTCAGGATTCC-3'; recognition sites are
underlined); reporter genes for PU.1 (3×
WT-MHC-Luc,5'-AAAGAGGAACTTGG-3'; 3×
MT-MHC-Luc,5'-AAAGAGCTACTTGG-3'); and reporter genes for
c-Myb (4× WT-MBS-Luc,5'-CTCTACACCCTAACTGACACACATTCT-3';
4× MT-MBS-Luc,5'-CTCTACACCATCACAAACACACATTCT-3'). Luciferase assay was performed with the Dual-Luciferase Reporter System
(Promega, Madison, WI) as previously described.24 Briefly, the cultured cells were transfected with an appropriate reporter gene
together with pRL-CMV-Rluc, an expression vector of renilla luciferase, by electroporation. In addition, luciferase assays were
performed in NIH3T3 cells by the calcium phosphate coprecipitation method.24 Relative firefly luciferase activities were
calculated by normalizing transfection efficiency according to the
renilla luciferase activities. Experiments were performed in
triplicate, and the similar results were obtained from at least 3 independent experiments.
Transient transfection into 293T cells 293T Cells were transfected with expression vectors of GATA-1, PU.1, and c-myb alone or in combination with pcDNA3-GFP (an expression vector of green fluorescence protein) by the calcium phosphate coprecipitation method. After 48 hours, total cellular lysates or nuclear extracts were isolated. Transfection efficiencies were monitored by the intensities of green fluorescence protein by flow cytometric analyses.Electrophoretic mobility shift assay Nuclear extracts were prepared from 293T cells transfected with expression vectors of GATA-1, PU.1, and c-myb alone or in combination. Sequences of probes and competitors are as follows: probe for GATA-1 (M), 5'-GATCTCCGGCAACTGATAAGGATTCCCTG-3'; mutated GATA-1
competitor, 5'-GATCTCCGGCAACTCAGAAGGATTCCCTG-3'; probe for
PU.1 (MHC, major histocompatibility complex),
5'-TCGAAAAGAGGAACTTGGGTA-3'; mutated PU.1 competitor,
5'-TCGAAAAGACGTACTTGGGTA-3'. The binding reaction and
electrophoresis were performed as previously described.24 In competition assays, nuclear extracts were preincubated with a
200-fold molar excess of unlabeled competitor before the binding reaction. In a supershift assay, nuclear proteins were preincubated with 1 µg anti-GATA-1 antibody or anti-PU.1 antibody for 30 minutes at 4°C, followed by the binding reaction.
H-rasG12V induces megakaryocytic differentiation of K562 and F-36P but not of 32D cl3 To examine which types of hematopoietic cells are able to undergo megakaryocytic differentiation in response to H-rasG12V, we expressed H-rasG12V in human erythroleukemia cell lines K562 and F-36P and also in a murine myeloid cell line, 32D cl3, by using a Lac-inducible system in which expression of the target protein was induced by IPTG treatment. The clones were designated K562/ras, F-36P/ras, and 32D/ras, respectively. In all clones, IPTG treatment led to the efficient induction of H-rasG12V protein in Western blot analysis with anti-Ras antibody (Figure 1A), whereas actin, a loading control, was found to be expressed at similar levels in each lane (data not shown). As shown in Figure 1, panel B, K562/ras, F-36P/ras, and 32D/ras were absolutely composed of small, undifferentiated blast cells before IPTG treatment. After 5-day culture with IPTG, a substantial fraction (60%-70%) of F-36P/ras and K562/ras showed morphologic alterations indicative of megakaryocytic maturation, whereas most of 32D/ras still revealed blastoid features.
Ectopic overexpression of GATA-1 reprograms 32D/ras into erythrocytic/megakaryocytic lineage Because GATA-1 has been shown to change the lineage phenotype of hematopoietic cells,25-27 we introduced GATA-1 into 32D/ras. As shown in Figure 2A, GATA-1 protein was hardly detected in a parental clone (32D/ras) and a control clone transfected with an empty vector (32D/ras/Mock), but it was expressed in GATA-1 transfectants 32D/ras/GATA-1 cl1 and cl2 at high levels almost similar to those in a murine erythroleukemia cell line (MEL). We initially explored the expression of-globin, a marker of mature erythroid cells, in GATA-1 transfectants by benzidine staining. Although 32D/ras and 32D/ras/Mock were essentially negative on benzidine staining, approximately 30% of 32D/ras/GATA-1 became positive. As shown in Figure 2, panel C, a megakaryocytic antigen CD61
(GP IIIa) was scarcely expressed on 32D/ras/Mock, whereas a weak but
easily detectable level of CD61 expression was observed on GATA-1
transfectants. These results suggested that the ectopic overexpression
of GATA-1 reprogrammed 32D/ras toward the
erythrocytic/megakaryocytic lineage.
GATA-1-transfected 32D/ras undergoes megakaryocytic differentiation in response to H-rasG12V Next, we investigated whether biologic responses of 32D cl3 to H-rasG12V were modulated by GATA-1. Like parental 32D/ras (Figure 1B), an apparent morphologic change was not induced by IPTG in 32D/ras/Mock (Figure 3A, upper panel), whereas 32D/ras/GATA-1 underwent morphologic changes indicating megakaryocytic maturation after a 5-day IPTG treatment (Figure 3A, lower panel). DNA content analysis showed that IPTG treatment resulted in polyploid formation (2N, 32%; 4N, 38%; 8N, 24%; 16N, 6%) in 32D/ras/GATA-1 but not in 32D/ras/Mock (Figure 3B, left panel). In addition, CD61 expression was augmented after IPTG treatment in 32D/ras/GATA-1, whereas its expression was hardly detected in 32D/ras/Mock before and after IPTG treatment (Figure 3B, right panel). In addition, the expression of GPIIb mRNA was up-regulated and that of PF4 mRNA was induced after IPTG treatment in 32D/ras/GATA-1 (Figure 3C), yet these expressions were scarcely detected in 32D/ras/Mock before and after IPTG treatment (data not shown). These results suggested that ectopically introduced GATA-1 enabled 32D/ras to differentiate into mature megakaryocytes in response to H-rasG12V.
Granulocyte and macrophage differentiation of PU.1- and c-myb-transfected K562 after induction of H-rasG12V We next investigated whether H-rasG12V-mediated megakaryocytic differentiation was influenced by other transcription factors. PU.1 and c-myb were introduced into K562/ras, and the clones were designated K562/ras/PU.1 and K562/ras/c-myb, respectively. As shown in Figure 4A, PU.1 protein was efficiently expressed in K562/ras/PU.1, but endogenous PU.1 was detected only faintly in a control clone K562/ras/Mock transfected with an empty expression vector. Although c-Myb was expressed in K562/ras/Mock endogenously, the more increased level of c-Myb was expressed in K562/ras/c-myb (Figure 4B). Without IPTG treatment, K562/ras/PU.1, K562/ras/c-myb, and K562/ras/Mock were primarily composed of blastoid cells, and no significant difference was observed between the clones (Figure 4C, left panel). After a 5-day IPTG treatment, megakaryocytic maturation was observed in K562/ras/Mock just as parental K562/ras (Figure 4C, right panel). In contrast, a significant proportion of K562/ras/PU.1 revealed morphologic changes suggesting granulocytic maturation. Furthermore, a noticeable proportion of K562/ras/PU.1 underwent apoptosis after IPTG treatment, which was characterized by shrunken cell size, nuclear fragmentation, or both. Moreover, K562/ras/c-myb revealed macrophage-like features characterized by enlarged cell size and vacuoles in the cytoplasm after IPTG treatment. In agreement with morphologic changes, DNA content analysis showed that 5-day culture with IPTG induced polyploid formation up to 16N in K562/ras/Mock (Figure 5A). In K562/ras/PU.1, the proportion of cells in S or G2/M phase was reduced from 40% to 7% by IPTG treatment. In addition, IPTG treatment yielded apoptotic cells in approximately 45% of cultured cells, which was detected as a subdiploid fraction, and H-rasG12V induced growth suppression in K562/ras/c-myb (proportion of cells in S or G2/M phase: IPTG42% vs IPTG +5%; Figure 5A). This severe growth inhibition in
K562/ras/PU.1 and K562/ras/c-myb after IPTG treatment was coupled with
terminal differentiation of these clones.
Next, we examined the surface expression of lineage-specific antigens
such as an erythroid marker glycophorin A (GPA), a megakaryocytic marker GPIIb/IIIa, and the myeloid/macrophage markers CD11b, CD14, and
CD33 (Figure 5B). Without IPTG treatment, GPA was expressed intensely
on K562/ras/Mock, whereas its expression decreased distinctly on
K562/ras/PU.1 and K562/ras/c-myb. In contrast, expression levels of
CD14 and CD33 were up-regulated slightly in K562/ras/PU.1 and K562/ras/c-myb. In addition, CD11b expression emerged on
K562/ras/c-myb. Consistent with the findings on morphologic and DNA
content analyses, K562/ras/Mock showed phenotypic changes indicative of
megakaryocytic maturation in response to H-rasG12V Sustained expression of PU.1 and c-myb in 32D/ras/GATA-1 and that of GATA-1 in K562/ras/PU.1 and K562/ras/c-myb during IPTG treatment Because 32D cl3 is a myeloid cell line and expresses PU.1 and c-myb originally, we investigated changes of these expressions in 32D/ras/GATA-1 during H-rasG12V-induced megakaryocytic differentiation by Northern blot analysis. As shown in Figure 6, panel A, expression levels of PU.1 and c-myb did not reveal an apparent difference between 32D/ras/Mock and 32D/ras/GATA-1 before IPTG treatment (Figure 6A, left 2 lanes). These expressions did not show significant change during H-rasG12V-induced megakaryocytic differentiation of 32D/ras/GATA-1 for up to 120 hours. We next examined the expression of endogenous GATA-1 during H-rasG12V-induced granulocytic differentiation of K562/ras/PU.1 and of macrophage differentiation of K562/ras/c-myb. Before IPTG treatment, GATA-1 expression was slightly reduced in K562/ras/PU.1 and K562/ras/c-myb compared with that in K562/ras/Mock (Figure 6B, left 2 lanes). After IPTG treatment, GATA-1 expression was down-regulated modestly in K562/ras/PU.1 and K562/ras/c-myb (Figure 6B). However, GATA-1 expression was still detectable even after 120 hours of IPTG treatment, at which time granulocytic or macrophage differentiation was already obvious with morphologic and surface phenotypic analyses (Figures 4C, 5B). Coincident with the findings on Northern blot analysis, each protein was expressed at comparable levels on Western blot analysis (data not shown).
GATA-1 inhibits PU.1 and c-myb activities reciprocally Next, we performed luciferase assays with functional and nonfunctional reporter genes for GATA-1 (3× WT-M-Luc and 3×
MT-M-Luc), PU.1 (3× WT-MHC-Luc and 3× MT-MHC-Luc), and c-Myb (4×
WT-MBS-Luc and 4× MT-MBS-Luc) in 32D/ras/Mock, 32D/ras/GATA-1,
K562/ras/Mock, K562/ras/PU.1, and K562/ras/c-myb (Figure
7A). In 32D/ras/Mock, reporter genes for
PU.1 and c-Myb were activated 6.8-fold and 2.8-fold, respectively, with
reference to the activities of the mutant reporter genes, whereas those
for GATA-1 were scarcely activated. In contrast, PU.1 and c-Myb
activities were inhibited drastically in 32D/ras/GATA-1 instead of
GATA-1 activities (relative luciferase activities: PU.1, 1.4-fold;
c-Myb, 1.1-fold; GATA-1, 3.8-fold). In K562/ras/Mock, endogenous GATA-1
activated 3× WT-M-Luc by 4.1-fold, yet PU.1 and c-Myb activities
were hardly detectable (PU.1, 1.1-fold; c-Myb, 1.1-fold). However,
GATA-1 activities were reduced to 1.2-fold in both K562/ras/PU.1 and
K562/ras/c-myb, but ectopically introduced PU.1 and c-Myb stimulated
3× WT-MHC-Luc by 5.8-fold and 4× WT-MBS-Luc by 4.0-fold,
respectively. These results raised the possibility that GATA-1 may
antagonize PU.1 and c-Myb and, conversely, that PU.1 and c-Myb may
antagonize GATA-1. To examine this possibility, we performed luciferase
assays in NIH3T3 cells. As shown in Figure 7, panel B, both
PU.1-induced 3× WT-MHC-Luc activities and c-Myb-induced 4×
WT-MBS-Luc activities were repressed by cotransfected GATA-1 in a
dose-dependent manner. GATA-1-stimulated 3× WT-M-Luc
activities were reduced by cotransfected PU.1 or c-myb, depending on
their doses. In these experiments, neither expression vector
affected the luciferase activities of mutant reporter
genes for GATA-1, PU.1, or c-Myb (data not shown). Furthermore,
negative cross-talk was observed among other members of GATA and Ets
families, because PU.1-, Ets-1, or Ets-2-induced luciferase
activities were dose-dependently inhibited by GATA-1, GATA-2, or GATA-3
in NIH3T3 cells (data not shown).
Mechanisms of reciprocal inhibition of GATA-1-PU.1 and GATA-1-c-Myb We examined the mechanisms of the reciprocal inhibition of GATA-1-PU.1 and GATA-1-c-Myb. At first, 293T cells were transfected with expression vectors of HA-tagged GATA-1, Flag-tagged PU.1, or both, and the formation of GATA-1-PU.1 complex was examined by a coimmunoprecipitation method. In agreement with recent findings,28-30 GATA-1 and PU.1 were found to associate through the 2 zinc finger domains of GATA-1 and the Ets domain of PU.1 (data not shown). Similarly, we found that c-Myb was coimmunoprecipitated with GATA-1 and vice versa by the coimmunoprecipitation method (Figure 8A).
Next, we examined whether the interaction between GATA-1 and PU.1
affects the respective DNA-binding activities. Nuclear extracts were
isolated from 293T cells transfected with GATA-1 and PU.1 expression
vectors. Although the nuclear extract prepared from mock (an empty
expression vector)-transfected 293T cells did not show any binding
activity to the probe for GATA-1 (M Characterization of NZF-GATA-1, which lacks N-zinc finger
(NZF) of GATA-1 (Figure 9A), had little
suppressive effect on PU.1 activities, whereas its transcriptional
activity on 3× WT-M-Luc was almost similar to that of full-length
(FL)-GATA-1 (data not shown). In addition, AD-PU.1, which
lacks a part of the activating domain (AD) of PU.1 (Figure 9A), hardly
affected GATA-1 activities but showed transcriptional activity on 3×
WT-MHC-Luc approximately 50% that of FL-PU.1 (data not shown). To
characterize these mutants, the formation of the GATA-1-PU.1 complex
was examined by coimmunoprecipitation. We transfected FL-PU.1 together
with HA-tagged FL-GATA-1 or HA-tagged NZF-GATA-1 into 293T cells. Although FL-GATA-1 and NZF-GATA-1 were expressed at similar levels on Western blot analysis of the anti-HA-immunoprecipitated proteins with anti-HA antibody (Figure 9B, left, lower panel), FL-GATA-1 but not
NZF-GATA-1 was coimmunoprecipitated with PU.1 (Figure 9B, left,
upper panel). We transfected FL-GATA-1 in combination with Flag-tagged
FL-PU.1 or Flag-tagged AD-PU.1 into 293T cells and found that both
FL-PU.1 and AD-PU.1 were coimmunoprecipitated with GATA-1 with
similar efficiency (Figure 9B, right, upper panel). Next, we prepared
nuclear extracts from 293T cells transfected with these expression
vectors. Although FL-GATA-1 significantly inhibited the DNA binding of
PU.1, NZF-GATA-1 could scarcely affect the DNA binding (Figure 9C,
upper panel). In contrast to FL-PU.1, AD-PU.1 scarcely inhibited the
DNA binding of GATA-1 (Figure 9C, lower panel), though AD-PU.1 could
bind to GATA-1 as efficiently as FL-PU.1 (Figure 9B, right upper
panel). These results suggested that NZF of GATA-1 is necessary for its
binding to PU.1 and its inhibition of the DNA-binding activities of
PU.1 and that the AD of PU.1 is required for the inhibition of the DNA-binding activities of GATA-1 but not for the interaction between GATA-1 and PU.1.
NZF-GATA-1 into 32D/ras and AD-PU.1 into
K562/ras; these clones were named 32D/ras/NZF and K562/ras/AD, respectively. Western blot analysis of 32D/ras/NZF and
K562/ras/AD showed that NZF-GATA-1 and AD-PU.1 were expressed
in each clone at similar or increased levels compared with full-length
products (Figure 10A). GATA-1 and PU.1
activities were detected in 32D/ras/NZF (GATA-1 activities,
4.1-fold; PU.1 activities, 6.3-fold, with reference to the respective
nonfunctional reporter gene; Figure 10B), whereas PU.1 activities were
severely reduced in 32D/ras/GATA-1 (GATA-1 activities, 3.8 fold; PU.1
activities, 1.4 fold; Figure 7A). PU.1 and GATA-1 activities were
detected in K562/ras/AD (GATA-1 activities, 5.2-fold; PU.1
activities, 5.6 fold; Figure 10B), whereas GATA-1 activities were
intensely inhibited in K562/ras/PU.1 (GATA-1 activities, 1.2-fold; PU.1
activities, 5.8 fold; Figure 7A). Although the induced expression of
H-rasG12V for 5 days led to the development of fully
matured megakaryocytes from 32D/ras/GATA-1 (Figure 3A), it was less
effective in promoting maturation of 32D/ras/NZF (Figure 10C, upper
panel). In addition, IPTG treatment failed to induce PF4 mRNA in
32D/ras/NZF, whereas it was effectively induced in 32D/ras/GATA-1
(Figure 10D, left panel). CD61 induction and polyploid formation were
perturbed distinctly in 32D/ras/NZF (data not shown). In
K562/ras/AD morphology, 5-day treatment with IPTG still induced
megakaryocytic maturation (Figure 10C, lower panel), whereas
K562/ras/PU-1 underwent granulocytic maturation (Figure 4C). In
accordance with the morphologic findings, PF4 mRNA was induced by IPTG
treatment in K562/ras/AD but not in K562/ras/PU-1 (Figure 10D, right
panel). Together, these results suggested that NZF-GATA-1 and
AD-PU.1, which respectively had little inhibitory activity on PU.1
and GATA-1, were less effective in reprogramming the lineage phenotype
in spite of the retained transcriptional activities. Similar findings
on luciferase assays and responses to IPTG were also observed in other
stable transformants with NZF-GATA-1 and AD-PU.1 (data
not shown).
During the development of hematopoietic cells from pluripotent stem cells to terminally differentiated cells, lineage-specific transcription factors play crucial roles in lineage determination and subsequent maturation through the transcriptional regulation of lineage-specific genes. In a previous study,17 we found that TPO-induced Ras activation was necessary and sufficient for promoting megakaryocytic differentiation of a human erythroleukemia cell line. However, Ras activation is not specific for megakaryocytic differentiation; it is also observed in other cell types, and it evokes various effects in the cells.20-22 Therefore, it was speculated that there might be a key molecule that is prerequisite for Ras-mediated megakaryocytic differentiation. In previous studies, it has been shown that GATA-1 is unique in its ability to influence the lineage phenotype of hematopoietic cells. For example, enforced expression of GATA-1 reprogrammed transformed chicken myeloblasts into erythroblasts, thromboblasts, or eosinophils.25 Moreover, the overexpression of GATA-1 in the early myeloid 416B and M1 cells provoked megakaryocytic differentiation accompanied by a marked decrease in myeloid surface phenotype.26,27 These findings led us to speculate that GATA-1 might confer the characteristics that facilitate the cells to undergo megakaryocytic differentiation in response to H-rasG12V. In agreement with this hypothesis, ectopically introduced GATA-1 reprogrammed myeloid 32D cl3 into an erythroid/megakaryocytic lineage and caused 32D cl3 to undergo megakaryocytic differentiation in response to H-rasG12V. These results suggested that GATA-1 is one of the key molecules necessary for megakaryocytic differentiation, whereas the direct target gene(s) of GATA-1 remains unknown. In addition, because our preliminary experiments showed that H-rasG12V enhanced GATA-1 activities approximately 2.5-fold, it was suggested that H-rasG12V-evoked megakaryocytic differentiation might result from these augmented GATA-1 activities or from the cooperation of GATA-1 and H-rasG12V-induced unidentified product(s). PU.1 is known as a pivotal transcription factor involved in the commitment and terminal differentiation of myeloid cells.11,31 It has been reported that constitutive expression of PU.1 inhibits terminal erythroid differentiation of the murine cell line MEL.32-34 The enforced expression of c-Myb, in addition to PU.1, was shown to block the erythroid differentiation of MEL cells.35,36 In this study, we examined whether these transcription factors could affect H-rasG12V-induced megakaryocytic differentiation of K562. As a result, the overexpression of PU.1 and c-Myb changed the phenotype of K562 to myeloid/monocyte lineage and caused K562 to differentiate into granulocytes and macrophages, respectively, in response to H-rasG12V. For this mechanism, the transcription activity of GATA-1 was antagonized by either PU.1 or c-Myb, and transcriptional activities of PU.1 or c-Myb were also suppressed by GATA-1. Although the expression patterns of lineage-specific transcription factors are known to be regulated reciprocally during the differentiation programs,37 it is also reported that conflicting lineage-specific transcription factors, such as GATA-1 and PU.1, coexist in multipotent hematopoietic progenitor cells.38 Therefore, it was suggested that relative levels of these transcription factors may have a great influence on progenitor cells to commit and differentiate toward myeloid or erythroid/megakaryocyte lineage. Our data also showed that, compared with wild types, Regarding the mechanisms of the reciprocal inhibition, GATA-1 and PU.1
were reported to associate in vivo,28-30 and we found similar results in the current study. In this association, the respective DNA-binding domains (zinc finger domains of GATA-1 and Ets
domain of PU.1) were to be used as a surface of protein-protein interaction. In previous studies,40-50 the zinc finger
domains of GATA-1 and the Ets domain of PU.1 were reported to interact with various types of nuclear proteins: FOG, CREB-binding protein, Sp1,
EKLF, U-shaped, and the estrogen receptor for GATA-1; C/EBP It has been shown that several megakaryocyte-specific genes, such as
c-mpl, GPIb In agreement with our result indicating the mutual negative regulation between GATA-1 and PU.1, Zhang et al28 have recently shown that both GATA-1 and GATA-2 interact with PU.1 through the C-terminal zinc finger, thereby resulting in reciprocal inhibition. Furthermore, they have shown that GATA-1 inhibits the binding of PU.1 to c-jun, a critical coactivator of PU.1. Rekhtman et al29 and Nerlov et al30 have recently demonstrated that GATA-1 and PU.1 directly interact through both zinc fingers of the GATA-1 and Ets domains of PU.1, which led to functional antagonism of these factors.29,30 In addition, Rekhtman et al29 have shown that PU.1 inhibits erythroid differentiation of a murine erythroid cell line, MEL, by repressing GATA-1 activities. These results, including ours, suggested that direct interaction of these lineage-specific transcription factors profoundly affects the activities of other lineage-specific transcription factors and influences the lineage commitment and subsequent differentiation of hematopoietic cells. Further studies concerning the interaction between GATA-1 and PU.1 using our system should provide more useful information on normal and abnormal hematopoiesis.
We thank Dr S. Ishii for providing us with an expression vector of c-myb.
Submitted December 21, 1999; accepted June 8, 2000.
Supported in part by grants from the Japanese Ministry of Education, Science, Sports and Culture, the Japanese Ministry of Health and Welfare, Senri Life Science Foundation, Uehara Memorial Foundation, Naito Foundation, and the Japan Medical Association.
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: Itaru Matsumura, Department of Hematology and Oncology, Osaka University Medical School, 2-2, Yamada-oka, Suita, Osaka 565, Japan; e-mail: matsumura{at}bldon.med.osaka-u.ac.jp.
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© 2000 by The American Society of Hematology.
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Top
Abstract
Introduction
/
for example, t(8;21)(q22;q22), AML-1-ETO; inv16(p13;q22),
CBF
, AML1, c-fos, c-jun, and Pip for PU.1.
In most cases, GATA-1 and PU.1 activities are reported to be positively
or negatively regulated through these interactions, though limited
information is available for the biologic significance of the
GATA-1-PU.1 complex in this protein assembly. Our data revealed that,
as a result of the interaction, DNA-binding activities of GATA-1 and PU.1 were inhibited reciprocally by electrophoretic mobility shift assay. However, GATA-1 was less effective than PU.1 in inhibiting DNA-binding activities of PU.1. More mechanisms might be involved in
the suppression of PU.1 activities by GATA-1. In addition,