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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 870-878
HEMATOPOIESIS
Characterization of hematopoietic lineage-specific gene
expression by ES cell in vitro differentiation induction system
Takumi Era,
Toshiaki Takagi,
Tomomi Takahashi,
Jean-Christophe Bories, and
Toru Nakano
From the Department of Molecular Cell Biology, Research Institute
for Microbial Diseases, Osaka University, Osaka, Japan; and Unité
462 Institut National de la Santé et de la Recherche
Médicale, Hôpital Saint-Louis, Paris, France.
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Abstract |
The continuous generation of mature blood cells from hematopoietic
progenitor cells requires a highly complex series of molecular events.
To examine lineage-specific gene expression during the differentiation
process, we developed a novel method combining LacZ reporter gene analysis with in vitro
hematopoietic differentiation induction from mouse embryonic stem
cells. For a model system using this method, we chose the erythroid and
megakaryocytic differentiation pathways. Although erythroid and
megakaryocytic cells possess distinct functional and morphologic
features, these 2 lineages originate from bipotential
erythro-megakaryocytic progenitors and share common lineage-restricted
transcription factors. A portion of the 5' flanking region of the
human glycoprotein IIb ( IIb) integrin gene extending from base
 598 to base +33 was examined in detail. As reported previously,
this region is sufficient for megakaryocyte-specific gene expression.
However, previous reports that used human erythro-megakaryocytic cell
lines suggested that one or more negative regulatory regions were
necessary for megakaryocyte-specific gene expression. Our data clearly
showed that an approximately 200-base enhancer region extending from
598 to 400 was sufficient for megakaryocyte-specific gene
expression. This experimental system has advantages over those using
erythro-megakaryocytic cell lines because it recapitulates normal
hematopoietic cell development and differentiation. Furthermore, this
system is more efficient than transgenic analysis and can easily
examine gene expression with null mutations of specific genes.
(Blood. 2000;95:870-878)
© 2000 by The American Society of Hematology.
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Introduction |
The process of cell differentiation is regulated
through the complex coordination of cell-type-specific gene activation
brought about by the interactions of cis- and
trans-acting regulatory elements. The hematopoietic system, one
of the stem cell systems, is a useful experimental model for analyzing
this coordination because more than 8 distinct blood cell lineages
arise from single multipotential hematopoietic stem cells within a
relatively short period.1 Recent gene-targeting experiments
have demonstrated that various lineage-specific or
differentiation-stage-specific transcription factors play crucial
roles during this process.2,3 However, our understanding of
how lineage commitment is orchestrated by many transcription factors
and how lineage-specific gene expression is tightly controlled within
individual hematopoietic lineages remains limited.4
In the hematopoietic system, the erythroid and megakaryocytic lineages
are considered closely related for 2 reasons. First, both erythrocytes
and megakaryocytes differentiate through common committed
erythro-megakaryocytic progenitors.5-7 Second, these 2 lineages share several features in common. For example, GATA-1, a
principal transcriptional regulator of erythropoiesis, also has an
important role in the production of platelets.8-10 Other important transcription factors and cofactors, such as NF-E2 and FOG,
which were first discovered in the erythroid lineage, play important
roles in platelet production.11-14 In addition, some leukemic cells possess both erythroid and megakaryocytic cell-surface markers.15-17 Although these findings illustrate
similarities in the control of the gene expression programs in these 2 lineages, the morphologic and functional features of these 2 lineages
are quite different. Therefore, these 2 lineages must possess discrete, specific gene expression programs for commitment to individual cell
lineages. Analyzing the regulation of differential gene expression within these 2 closely related lineages should provide clues to our
understanding of the mechanism of hematopoietic commitment.
The glycoprotein IIb (GPIIb) gene is an excellent lineage-restricted
marker for examining megakaryocyte-specific gene expression because the
gene is an early and specific marker of
megakaryocytes.18,19 Several groups have analyzed the
promoter region of the human GPIIb gene.10,16,17,20,21 All
of these studies used the transient expression of reporter genes in
human erythro-megakaryocytic leukemic cell lines such as HEL (human
erythroleukemia) and K562. These studies showed that 2 kinds of
transcription factor binding sites, GATA and Ets binding sites, play
important roles in the positive regulation of GPIIb promoter
activity.10,17,20 However, some evidence suggests that
these 2 sites are not sufficient for megakaryocyte-specific expression
and that negative cis-regulatory elements, such as those
located at 198/178 and 124/99
bp15 or 120/116 and 102/93
bp,21 are important for suppressing GPIIb expression in
cell lineages other than the megakaryocyte lineage.
The use of "erythro-megakaryocytic" leukemic cell lines may be
unsuitable for studying the mechanisms governing the discrimination between erythroid and megakaryocytic lineages because most
megakaryocytic and erythroid leukemic cell lines concomitantly express
erythroid and megakaryocytic markers, respectively. Most of the data on megakaryocyte-specific gene expression have been obtained by comparing gene expression in erythro-megakaryocytic cell lines with that in
irrelevant cell lines such as HeLa.20-22 In addition,
several groups using different erythro-megakaryocytic cell lines have reported conflicting results.21,23 To circumvent these
dubious situations, it seems preferable to use a different experimental system and to compare the results.
One of the best experimental systems to analyze promoter activity
during development and cell differentiation is the transgenic mouse,
and this has been validated by the numerous studies of human  -globin
locus gene expression.24 However, the analysis of many
promoters and their mutants requires many transgenic lines and is
tremendously time and labor intensive. We have developed an alternative
strategy, which combines promoter-reporter gene analysis and in vitro
hematopoietic differentiation from mouse embryonic stem cells (ES
cells).25,26 ES cells can give rise to hematopoietic cells,
including erythroid and megakaryocyte lineages, when cultured with the
macrophage colony-stimulating factor (M-CSF)-deficient OP9 stromal cell
line (OP9 system).27-29 The process of hematopoiesis in
this system is considered to reflect accurately the early phase of in
vivo hematopoietic development.
Here, we show that a 200-bp enhancer element containing a GATA site and
an adjacent Ets binding site is necessary and sufficient for
megakaryocyte lineage-specific expression. The experimental system
developed in this study is a useful, rapid, and reliable method to
examine gene-regulatory mechanisms during hematopoiesis.
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Materials and methods |
Construction of reporter plasmid
The pBS-LacZ vector was constructed by inserting a 4.4-kb
HindIII-ApaI fragment of the bacterial LacZ
gene with a nuclear localization signal (kindly provided by Dr
Takeuchi30) into the HindIII and ApaI sites
of pBluescript (StrataGene, La Jolla, CA). The human GPIIb
promoter from bases 1121 to +516 (transcriptional initiation
site is designated as +1) was amplified by polymerase chain reaction
(PCR) using primers 5'-TTCCGCTTACCGAGAGAAAA-3' and
5'-GGGTGGAGTGGTTCATACAA-3', as described
previously.31 The PCR product was cloned into pBluescript
and the sequence was confirmed. The sequences of all the PCR products
produced in this study were confirmed by DNA sequencing using the
Thermosequenase kit (Amersham, Uppsala, Sweden) and a
LI-CORauto sequencer (Lincoln, NE).32 The
GPIIb fragment was digested with HindIII and MboII to
generate a HindIII-MboII segment containing the genomic
sequence from nucleotide 598 to +33. The pBS-GPIIb-LacZ parental
vector was constructed by inserting this GPIIb
HindIII-MboII fragment into the HindIII site of
the pBS-LacZ vector. Vector pMC1NEO EF-1 LacZ was constructed by
inserting the LacZ gene into the XbaI site of vector
pMC1NEO EF-1 .
Various mutants containing deletions or point mutations were produced
by PCR.33 To produce a mutant of the region from 118 to 93, all the A's in this region were replaced by C's, the
G's by T's, and vice versa. The GATA at site 463 was converted
from 5'-TTTATCGG-3' (the GATA site is underlined) to
5'-TTTAGAGG-3' using the oligonucleotide
5'-TACAGAAGCCTCAGGTTTTAGAGGGGGCAGCAGCTTCCTTCT-3'. The Ets
at site 515 was converted from 5'-AAGGAGG-3' to
5'-AAGGATT-3' using the oligonucleotide
5'-CGCCCAAGGTCCTAGAAGGATTAAGTGGGTAAATGCCATATC-3'. The
vector pBS-TK-LacZ was constructed by ligating the LacZ gene and the minimal TK promoter, which was obtained by PCR from the pTK
vector (Nippon Gene, Toyama, Japan) using primers
5'-CCCGGATCCGGCCCCGCCCAGCGTC-3' and
5'-CCCAAGCTTAGATCTGCGGCACGCTGTT-3', into the ApaI
and BamHI site of pBluescript, respectively. Four tandem
repeats of oligonucleotides including the GATA site (463)
flanked with 13 bp on each end and 4 base pairs of
XhoI-cohesive ends
(5'-CGAGAAGCCTCAGGTTTTATCGGGGGCAGCAGCT-3' and
5'-CTCGAGCTGCTGCCCCCGATAAAACCTGAGGCTT-3') were cloned
into the BamHI site of the pBS-TK-LacZ vector by blunt end
ligation. Two tandem repeats of oligonucleotides
containing 2 tandem repeats of the distal Ets binding site flanked with
13 bp on both ends and overhanging BamHI-cohesive ends
(5'-GATCCAAGGTCCTAGAAGGAGGAAGTGGGTAAATGAAGGTCCTAGAAGGAGGAAGTGGGTAAATGG-3' and
5'-GATCCCATTTACCCACTTCCTCCTTCTAGGACCTTCATTTACCCACTTCCTCCTTCTAGGACCTTG-3') were cloned into the BamHI site of the pBS-TK-LacZ vector.
Cell culture and electroporation
D3 ES cells were maintained on embryonic fibroblasts using standard
procedures.34 OP9 cells were maintained in -minimal essential medium supplemented with 20% fetal calf serum and standard antibiotics.27 For differentiation induction, ES cells were seeded onto confluent OP9 cell layers on 6-well plates at a density of
104 cells per well. On day 5 of the differentiation
induction, these cells were treated with 0.25% trypsin and seeded onto
a fresh confluent OP9 cell layer in the presence of erythropoietin
(EPO), thrombopoietin (TPO), or M-CSF to support the differentiation and proliferation of erythroid cells, megakaryocytes, and macrophages, respectively. Erythroid cells and megakaryocytes were analyzed on days
8 and 12 of differentiation induction. Macrophages were analyzed on day
10 or 11.
Reporter plasmids and plasmid pMC1NEO EF-1 containing the neomycin
resistance gene were cotransfected at a 20:1 molar ratio into ES cells
by electroporation using a gene pulser (Bio-Rad, Hercules,
CA) set at 500 µF, 230 V. Transfected ES cells were seeded on a
neomycin-resistant layer of embryonic fibroblasts in 10-cm dishes or
6-well plates with G418. The puromycin resistance gene was similarly
used to examine gene expression in
c-ets-1 /
ES cells, which were established as described
previously.35 Dr Yagi (National Institute for
Physiological Science, Okazaki, Japan) kindly provided the
puromycin-resistant gene and puromycin-resistant STO
cells.36 In most experiments, ES cells were harvested from individual wells of 6-well plates when confluent, and the total cells
harvested from individual wells were induced to differentiate as
described earlier.
Staining and histologic analysis of hematopoietic cells
The hematopoietic cells generated from the transfected ES cells were
collected from the supernatants of the induction cultures, and
cytospins were produced. These cells were fixed in 1% formaldehyde for
8 minutes and washed 3 times with phosphate-buffered saline (PBS) at
room temperature. X-gal staining was carried out overnight at 37°C
in PBS containing 1 mg/mL X-gal, 5 mmol/L
K3Fe(CN)6, 5 mmol/L
K4Fe(CN)6, and 2 mmol/L MgCl2, pH
7.3. Stained cells were washed twice with deionized water. To verify
the lineages of the cells, we used acetylcholine esterase (AChE)
staining, which is specific for mouse megakaryocytes, and dianisidine
staining for hemoglobinized cells. After drying the slides completely,
we performed AChE staining at room temperature for 3 hours in 0.1 mol/L
PBS containing 0.05% acetylthiocholine iodide, 0.1 mol/L sodium
citrate, 30 mmol/L copper sulfate, and 5 mmol/L potassium ferricyanide (pH 6.0). Dianisidine staining for erythroid lineage cells was carried
out as described previously.37 Macrophages were not counterstained because more than 95% of the cells harvested from cultures containing M-CSF were macrophages. The percentages of LacZ-positive megakaryocytes, erythroid cells, and macrophages harvested from the cultures containing EPO, TPO, and M-CSF,
respectively, were counted under a microscope. We counted 250 AChE-positive cells, 1000 erythroid cells, and 1000 macrophages to
calculate the percentage of LacZ-positive cells. The
percentages of LacZ-positive cells were compared statistically
with the t test using StatView statistical analysis software.
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Results |
Selective proliferation of megakaryocytes, erythroid cells, and
macrophages by the OP9 differentiation induction system with the
addition of growth factors
Our previous studies demonstrated that multipotential hematopoietic
progenitors developed from ES cells in the OP9 system.27,38 TPO, EPO, or M-CSF was added from day 5 of differentiation induction for the efficient propagation of megakaryocytes, erythroid lineage cells, and macrophages, respectively (Figure
1). Preferential proliferation of
megakaryocytes was observed in the presence of TPO, and more than 90%
of the cells at day 8 expressed AChE, a cytologic marker of murine
megakaryocytes. As in our previous studies, primitive embryonic and
definitive adult erythroid cells appeared between days 6 and 8 and
between days 12 and 15 of the differentiation induction, respectively.
In the presence of EPO, more than 90% of the cells were
dianisidine-positive hemoglobinized cells at days 8 and 12 of
induction. On days 10 and 11, the vast majority (> 95%) of the
cells induced in the presence of M-CSF were macrophages, which was
confirmed by staining with anti-Mac-1 antibody.
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| Fig 1.
Differentiation induction into megakaryocytes, erythroid
cells, and macrophages and LacZ expression driven by the GPIIb
promoter and EF1- promoter in these cells.
Hematopoietic cells were generated from ES cells by the addition of TPO
(A, E, I), EPO (B, F, J for primitive embryonic erythrocytes and C, G,
K for definitive adult erythrocytes), and M-CSF (D, H, L) for
megakaryocytes, erythroid cells, and macrophages, respectively.
May-Grunwald Giemsa staining was used to detect morphologic features
(A-D). The LacZ gene driven by the GPIIb promoter from
598 to +33 (E-H) and the elongation factor-1 promoter (I-L)
were transfected into ES cells with a plasmid carrying the
neomycin-resistant gene. Differentiation induction was carried out
after G418 selection, and the cells were stained with X-gal. Double
staining with AChE was performed to confirm LacZ expression in
megakaryocytes.
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Megakaryocyte-specific gene expression of the GPIIb promoter in
the OP9 system
To ascertain the feasibility of the experimental system, we analyzed
the GPIIb promoter regions encompassing bases 598 to +33
(598/+33), 400 to +33 (400/+33), 100 to +33
(100/+33), and 598 to 400 plus 100 to + 33 (598/400:100/+33). Constructs consisting of the
LacZ reporter gene ligated downstream of the GPIIb promoter and
its derivatives were introduced into ES cells by cotransfection with
the neomycin resistance gene. For a clonal analysis of the LacZ
gene, individual stable transformants were cloned after G418 selection.
Differentiation induction of individual ES cell clones was performed in
the presence of TPO, and the clones that gave rise to
LacZ-positive megakaryocytes were selected. The expression of
LacZ in erythroid lineage cells and macrophages was not
examined in this preliminary selection. Differentiation induction of
these preselected clones was carried out in the presence of TPO, EPO,
or M-CSF (Figure 1). The percentages of LacZ-positive cells in
megakaryocytes, erythroid cells, and macrophages are shown in Table
1. These results show that the shorter the
GPIIb promoter regions were, the lower the proportion of positive
cells. There were no differences in the intensity of the LacZ
staining of the positive cells (data not shown), and therefore
LacZ expression in individual positive cells was all or none.
In other words, the percentage of LacZ-positive cells, but not
the intensity of LacZ staining, could be used reliably as an
index of promoter activity. This all-or-none type of expression will be
discussed later in more detail (see Discussion). The expression of the
LacZ gene driven by the various GPIIb promoters was completely
restricted to megakaryocytes, although the data obtained with the
100/+33 promoter derivative were difficult to interpret because
the percentage of LacZ-positive cells was low, even in
megakaryocytes. In addition, lineage-restricted LacZ expression
was dependent on the promoter activity and not the site of integration
because LacZ gene expression driven by the elongation
factor-1 (EF-1) promoter was observed in all 3 hematopoietic
lineage cells (Table 1, Figure 1). The percentage of
LacZ-positive megakaryocytes tended to decrease during
differentiation when the GPIIb promoter was used. In contrast, no
decrease was observed when the EF-1 promoter was used. The reasons
for the decrease in GPIIb promoter activity and the difference between
these promoters remain unknown.
Although promoter analysis in hematopoietic cells derived from ES cell
clones in the OP9 system is more efficient than promoter analysis in a
transgenic mouse system, clonal analysis after preselection is still
labor intensive. To streamline the process, we pooled ES clones after
drug selection, induced differentiation, and then counted the
LacZ-positive cells. When 5 to 10 preselected clones were
pooled, the percentages of LacZ-positive cells correlated well
with those observed in the clonal analysis described earlier (data not
shown). Next we induced the differentiation of G418-resistant ES cells
in individual wells of 6-well plates without preselection and analyzed
the percentages of LacZ-positive cells. To validate the pool
analysis, we analyzed pools of ES cells with introduced LacZ
genes under the control of various GPIIb promoter derivatives (598/+33, 100/+33, and 598/400:
100/+33). Eight million ES cells were electroporated as
described in Materials and methods and were cultured in 6-well plates.
Individual wells typically contained approximately 50 to 100 independent ES cell clones. The results obtained from pools of
individual wells (Table 2) agreed well with
those of the clonal analysis (Table 1). Pools from individual wells
were used in most subsequent experiments.
A lineage-specifying negative regulatory element for
megakaryocytes does not exist
To examine which portion(s) of the GPIIb promoter contains the
important regulatory element(s), we created serial 30-bp deletion mutants of the 598/+33 GPIIb promoter and analyzed their
promoter activities (Figure 2). Only 1 mutant promoter induced a significantly higher percentage of positive
cells than the wild-type promoter; this mutant lacked nucleotides
between 118 and 89 ( 118/89). As
discussed later in more detail, recent studies have suggested that the
megakaryocyte-specific expression of GPIIb is regulated by putative
negative regulatory elements, which suppress the expression of GPIIb in
hematopoietic lineages other than megakaryocytes.15,21,22 One of the putative negative regulatory elements was localized in
region 118/93.21 Although the mutant promoter
118/89 significantly increased the level of expression
by up to 150% of the wild-type promoter, the expression of
LacZ was restricted to megakaryocytes (data not shown). To
further analyze this putative negative regulatory element, other
mutated promoters, which were examined in a previous
article,21 were analyzed in the OP9 system. One mutant
promoter harboring a deletion between 148 and 59 and
another with an altered nucleotide between 118 and 93
(GPIIb 118/93m) showed megakaryocyte-specific expression
(Table 1, Figure 3). Combined with the data
obtained from the analysis of the serial 30-bp-deletion mutants, it is
clear that the cell lineage specificity of the GPIIb promoter is not
controlled by a negative regulatory mechanism in our experimental
system.
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| Fig 2.
LacZ expression of serial 30-bp deletion mutants
in megakaryocytes.
The names of the mutants indicate the nucleotides deleted from the
598/+33 wild-type GPIIb promoter. For example,
538/509 shows the data from the promoter lacking the
nucleotides between 538 and 509 bp. These constructs were
cotransfected to undifferentiated ES cells with a plasmid carrying the
neomycin-resistant gene. After G418 selection, ES cells pooled from
individual wells of 6-well plates (see text) were differentiated into
megakaryocytes with OP9 stromal cells in the presence of TPO. The
percentages of LacZ-expressing megakaryocytes were calculated
on day 8 of differentiation induction using double staining with AChE
and X-gal. The results are shown as the relative percentage of LacZ
expression compared with the value for the 598/+33 wild-type
promoter. The data are shown as the mean ± standard deviation. The
data marked with * show significant differences (P < .05
by t test) compared with the wild-type (598/+33)
promoter. The experiment was performed 2 or 3 times, and representative
data are shown.
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| Fig 3.
Megakaryocyte-specific expression of the GPIIb promoter
containing mutations in the putative negative regulatory element.
Hematopoietic cells were generated from ES cells by the addition of TPO
(A, E), EPO (B, F for primitive embryonic erythrocytes and C, G for
definitive adult erythrocytes), and M-CSF (D, H) for megakaryocytes,
erythroid cells, and macrophages, respectively. The wild-type and
mutant GPIIb promoter constructs were transfected and analyzed as
described in Figure 2. The data from the wild-type (598/+33)
promoter (A-D) and the mutated promoter in which the nucleotides
between 148 and 89 (E-H) were deleted are shown.
Promoters lacking 118 to 89 and 148 to 59
show similar results.
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A positive regulatory element is sufficient for
megakaryocyte-specific gene expression
Serial 30-bp deletion analysis revealed a cluster of positive
regulatory elements between 538 and 449. The low activity (LacZ expression) of the 400/+33 promoter (Table 1)
might be attributable to the positive regulatory elements present in 3 successive 30-bp-deleted regions (538/509,
508/479, and 478/449). This 90-bp
element contains an Ets binding motif located at 515
(Ets515) and a GATA motif located at 463
(GATA463), both of which are reported to be critical for full
activity of the promoter.20 Presumably, the elimination of
Ets515, GATA463, or their adjacent elements is
responsible for the low activity of these 3 deletion mutants. To study
the contribution of these motifs to promoter activity, we performed
site-directed mutagenesis of the GATA463 and Ets515 sites
in the wild-type 598/+33 GPIIb promoter. The expression in the
promoter harboring a mutation at the GATA463 site (GATAm) was
only 10% that of the wild-type promoter, whereas the promoter with a
mutated Ets515 binding site (ETSm) exhibited 40% of the
activity of the wild-type GPIIb promoter (Figure
4). The decrease in LacZ expression
driven by the GATAm and ETSm promoters was similar to that of the
corresponding serial 30-bp deletion mutants, 478/449
and 538/509, respectively. In contrast, eliminating
the proximal GATA and Ets binding sites, which are located at 54
and 40, respectively, did not reduce promoter activity (Figure
4). These results indicate that the GATA463 and Ets515
sites, but not the GATA54 and Ets40 sites, are essential
components of the regulatory mechanisms governing high-level expression
of GPIIb in megakaryocytes. However, the lineage-specific expression of
the GPIIb promoter in megakaryocytes is completely maintained even in
the absence of both the GATA463 and Ets515 sites (Table
3).
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| Fig 4.
Effect of deletion and mutation of the GATA and ETS
binding sites on GPIIb promoter activity in megakaryocytes.
Wild-type promoter, promoters with mutations in the GATA (GATAm) or Ets
binding site (ETSm), and promoters with deletions of the proximal GATA
and Ets binding sites were analyzed. The percentages of
LacZ-positive cells per AChE-positive cells were calculated on
day 8 of differentiation induction in the presence of TPO.
The results are shown as the relative percentage of LacZ
expression compared with the value for the 598/+33 wild-type
promoter. The data are shown as the mean ± standard deviation.
GATAm and ETSm show significant differences from the wild type
(P < .05 by t test). Each experiment was performed
2 or 3 times, and representative data are shown.
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On the basis of the data shown in Tables 1 and 2 and Figure 2, we
believe that the 598/400 promoter region plays an
important role in expression of the GPIIb gene during normal
megakaryocyte differentiation. To determine whether this region can
confer megakaryocyte-specific activity, we constructed and examined a
fusion of the GPIIb promoter region 598/400 to the
thymidine kinase minimal promoter of herpes simplex virus (TK
promoter). This fusion promoter (598/400:TK) demonstrated
megakaryocyte-specific expression, and the percentage of
LacZ-positive cells with this promoter was similar to that of
the wild-type 598/+33 GPIIb promoter. To delineate the essential region in the 598/400 promoter, we analyzed the activity
of shorter elements within this region. The activities of the promoter regions between 539 and 420 (539/420:TK)
and between 529 and 450 (529/450:TK) were
approximately 60% and 30% of that exhibited by
598/400:TK (Figure 5).
Although the level of LacZ expression decreased in these
constructs, megakaryocyte-specific expression was maintained even in
the 529/450:TK promoter (Table 3). These results suggest
that the GATA463 and Ets515 elements may be sufficient to
confer the cell specificity of GPIIb expression, but insufficient for
full promoter activity.
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| Fig 5.
LacZ gene expression driven by different
promoters in megakaryocytes.
The percentages of LacZ-positive cells in megakaryocytes driven
by various promoters plus the TK-LacZ gene were examined as described
in Figure 2.
The details of individual constructs are explained in the text. Data
are shown as mean ± standard deviation. All of the results except
598/400:TK:LacZ are significantly different from the data
for the wild type (P < .05 by t test). All of the
results except 4 × ETS are significantly different from the
results for TK alone (P < .05 by t test). Each
experiment was performed 2 or 3 times, and representative data are
shown.
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We demonstrated that the region between 598 and 400 of
the GPIIb promoter plays an essential role in megakaryocyte-specific expression. Next we examined whether this promoter element can function
as an enhancer in the context of stable integration and investigated
the positional effects on its expression. Two expression constructs
were created to examine whether the element meets the criteria of an
enhancer, an element that activates gene expression with a reverse
orientation or from the 3' side of the gene. In 1 construct, the
598/400 fragment was inserted in the opposite orientation
upstream of the TK promoter and LacZ
(400/598:TK:LacZ). The other construct contained the
598/400 promoter fragment downstream from the TK promoter
and the LacZ gene in the correct orientation
(TK:LacZ:598/400). These 2 constructs exhibited megakaryocyte-specific activity, although the percentages of positive cells were lower than those observed with the wild-type 598/+33 promoter (Figure 5, Table 3). These results indicate that the 598/400 promoter fragment containing the GATA463
and Ets515 elements governs the lineage-specific expression of
GPIIb in megakaryocytes in a position- and orientation-independent manner.
The GATA463 and Ets515 elements are not
sufficient for megakaryocyte-specific expression
To examine whether the sequences flanking the GATA463 and
Ets515 sites are sufficient to mediate megakaryocyte-specific gene expression, we tested the double-stranded oligonucleotides encompassing each site and the 13 bp flanking each end of the sites for
their ability to direct the expression of LacZ in hematopoietic cells (Figure 5). These oligonucleotides were linked upstream from a
LacZ reporter gene driven by the TK promoter. Four tandem repeats of the GATA-site oligonucleotide (4 × GATA:TK) drove
LacZ expression not only in megakaryocytes, but also in
erythroid cells (Table 3, Figure 6). The
percentage of LacZ-positive erythroid cells was slightly lower
than that of LacZ-positive megakaryocytes. However, no
LacZ-positive macrophages could be detected. In contrast to the
4 × GATA-TK promoter, the 4 × ETS-TK promoter did not
drive significant expression of LacZ in megakaryocytes,
erythroid cells, or macrophages. (Table 3, Figure 6).
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| Fig 6.
LacZ expression in erythroid and megakaryocytic
lineages driven by the 4 × GATA promoter.
Hematopoietic cells were generated from ES cells by the addition of TPO
(A, E, I), EPO (B, F, J for primitive embryonic erythrocytes and C, G,
K for definitive adult erythrocytes), and M-CSF (D, H, L) for
megakaryocytes, erythroid cells, and macrophages, respectively. The
wild-type (598/+33) (A-D), the 4 × GATA (E-H), and the
4 × ETS promoters (I-L) were transfected into ES cells;
differentiation induction was carried out; and the cells were stained
with X-gal. Double staining with AChE was performed to confirm
LacZ expression in megakaryocytes (A, E, I). The
4 × GATA promoter was active in megakaryocytes (E), primitive
embryonic erythrocytes (F), and definitive adult erythrocytes (G), but
was inactive in macrophages (H).
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c-Ets-1 is not involved in the regulation of
the GPIIb promoter
As shown in Figure 4, we demonstrated that the
Ets515 site plays an important role in the transcriptional
activity of the GPIIb gene. One previous study reported that c-Ets-1,
which is a member of the Ets family of transcription factors, can bind to this element and regulate expression. It was suggested that c-Ets-1
is important for megakaryocytic differentiation.20 To determine whether c-Ets-1 is involved in regulating GPIIb promoter activity, we investigated wild-type 598/+33 GPIIb promoter
activity in c-ets-1 null ES cells (Table
4).35 Megakaryocytic
differentiation from c-ets-1 null ES cells was similar to that
from control ES cells. Moreover, the level of LacZ expression
did not differ between megakaryocytes generated from
c-ets-1+/+ and
c-ets-1/ES
cells, suggesting that at least in our system, c-Ets-1 does not play a
role in megakaryocytic differentiation, or if it does, then its role is
redundant.
View this table:
[in this window]
[in a new window]
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Table 4.
Clonal analysis of LacZ-positive cells after
differentiation induction from c-ets-1+/+ and
c-ets-1/ ES cells
|
|
| |
Discussion |
We have established a new experimental system to analyze
promoter activity with a combination of reporter gene analysis and in
vitro hematopoietic differentiation from ES cells. This experimental system can be used as an alternative to transgenic mice. We analyzed megakaryocyte-specific expression of the GPIIb promoter region between
nucleotides 598 and 400 using LacZ reporter gene
constructs. Our data show that this 598 to 400 region
functions as an enhancer that is necessary and sufficient for
megakaryocyte-specific expression and that no lineage-specific negative
regulatory element exists from 598 to +33.
The OP9 system is useful for promoter analysis in
hematopoietic cells
Recently, some in vitro ES cell-based hematopoietic differentiation
systems have been developed. Most systems use the formation of embryoid
bodies for the initial step of differentiation
induction.25,26 Although embryoid body formation allows
analysis of the frequency of hematopoietic precursors, it is
insufficient to obtain reasonable numbers of mature hematopoietic
cells. We have demonstrated that large numbers of erythrocytes (both
primitive embryonic and definitive adult erythrocytes), megakaryocytes,
and macrophages are propagated by the addition of the appropriate
growth factors. More than 4 × 105 cells of
individual lineages were induced from 104 ES cells. The
combination of the differentiation induction system and LacZ
promoter analysis enabled us to analyze hematopoietic lineage-specific
gene expression more easily and efficiently than in a transgenic animal
study.39 The LacZ expression pattern in individual
positive cells was all or none, and the percentage of
LacZ-positive cells could be used reliably as an index of
promoter activity. Patterns consistent with all-or-none responses have been observed in in vivo studies in which reporter gene
expression has been analyzed at the single-cell
level.40,41 One reason for the lack of larger differences
in LacZ intensity might be the relatively small numbers of
LacZ gene copies integrated into ES cells. Although we did not
examine all of the clones, only a few copies were integrated under the
electroporation conditions used in this study.
The other advantage of this experimental system is the easy genetic
manipulation of ES cells. Our analysis of
c-ets-1/ ES cells suggests that the
role of different genes in regulating different promoter regions can be
examined easily using double knockout ES cells. Lien et
al42 examined regulation of the human gp91-phox gene using
a combination of transfer of YAC clones into ES cells and
differentiation induction into myeloid cells by the embryoid body
method. Gene expression of gp91-phox was monitored by staining with a
monoclonal antibody against gp91-phox, but lineage-specific expression
was barely examined. The complexity of this system may have precluded
examination of lineage-specific expression. Our data show that not only
large DNA fragments, such as YAC, but also small promoter fragments,
such as those used in a transient expression system, can be well
characterized using our experimental system. Therefore, this new method
should facilitate the study of hematopoietic lineage-specific expression.
A negative regulatory element in the GPIIb promoter
So far, 3 studies of GPIIb gene regulation have defined "silencer
domains" in its promoter region.15,21,22 Two studies analyzed the human GPIIb gene and the other examined the rat GPIIb gene. The location of the silencer element differed in these 3 studies.
Fong and Santoro15 determined that 2 sites (198 to 178 bp and 124 to 99 bp) were involved in the
silencer effect in the human GPIIb promoter (598 to +32 bp) by
examining PMA-induced differentiation of K562 cells into
megakaryocytes. Prandini et al21 also detected silencer
elements in the human GPIIb promoter (120 to 116 bp and
102 to 93 bp) that partially overlapped the element
detected in the study of Fong and Santoro.15 This element
was also described in another erythro-megakaryocytic human leukemic
cell line, HEL. These studies of the human GPIIb promoter suggest a
common silencer site around 120 to 116 bp with the sequence 5'-ATGAG-3'. Prandini et al21
suggested that the sequence 5'-ATGAG-3' is found in the
5'-flanking regions of several megakaryocyte-specific promoters
and may be a common silencer of these genes. However, this region is
not conserved in the rat and mouse GPIIb promoter regions. In our
study, the human GPIIb promoter deletion mutant 118/89, which removes part of the putative negative
regulatory element, had higher transcriptional activity than the
control promoter in megakaryocytes. But lineage-specific expression of the promoter was not affected by the deletion at all. In contrast to
the previous study,21 a promoter mutated at the negative regulatory element, GPIIb 118/93m, did not show lineage
abrogation. Our study suggests that a negative regulatory element might
exist in the region 118 to 89. However, the activity of
this element is restricted to megakaryocytes and does not function to
determine lineage-specific gene expression. Unexpectedly, the deletion
mutant (208/179) lacking the other putative negative
regulatory element at 198 to 178 bp showed significantly
reduced expression. This result suggests that the region from
198 to 178 bp may control expression positively rather
than negatively. The other putative negative regulatory element
reported by Shou et al22 is a nonconsensus SP1-binding site
between 145 and 125 of the rat GPIIb promoter. Deletion
mutants lacking the human homologous region, 178/149 and 148/119, did not demonstrate significantly
different transcriptional activity compared with the control promoter.
Here again, lineage-specific expression of these deletion mutants was
observed. In summary, our study does not provide any evidence for a
lineage-specifying negative regulatory element in the GPIIb promoter,
suggesting that only positive regulation is necessary and sufficient
for megakaryocyte-specific gene expression.
There are 2 major differences between our experimental system and other
systems. First, the types of cells are different. Our system used ES
cells and their differentiating progeny within 3 hematopoietic
lineages. The differentiation process and developmental characteristics
of this in vitro differentiation system are considered very similar to
those seen in ontogeny. Meanwhile, all the studies reporting negative
regulatory elements in the GPIIb promoter were performed using
transient expression in human erythro-megakaryocytic leukemic cell
lines. However, leukemic cell lines such as HEL are not necessarily
representative of normal megakaryocytes because they express the gene
products of other hematopoietic lineages. We believe that using a
totipotent ES cell line may avoid the limitations inherent in such
leukemic cell lines.
The second issue is the species of cells examined. The discrepancies
between our observations and those of others may be attributed to
species differences of the cells used. It seems unlikely, however, that
the discrepant report of an Sp-1-binding putative negative regulatory
element by Shou et al22 is due to species differences because this promoter region is conserved in the human, rat, and mouse,
and the homology of Sp-1 among these species is very high. It is
difficult to evaluate the other 2 putative negative regulatory elements
reported because those promoter elements are not conserved in the human
and mouse. However, tissue-specific expression elements are generally
well conserved among species, allowing expression analysis of human
genes in transgenic mouse systems. It also may be possible that the
difference between transient transfection and stable transformation
caused some of the discrepancy.
Combinatorial regulation of a cis-regulatory
element containing GATA and Ets binding sites
The promoter region between bases 598 and 400 of the
human GPIIb promoter showed full and lineage-specific expression. In contrast, the 4 × GATA:TK promoter exhibited activity
(LacZ expression) not only in megakaryocytes but also in
erythroid cells. However, this promoter did not produce activity in
macrophages. Thus, GATA-1, which is the only member of the GATA
transcription family expressed in erythroid and megakaryocytic
lineages, can drive the expression of this promoter in both lineages.
The promoter containing both GATA463 and Ets515
(529/450:TK) has megakaryocyte-specific expression.
Taken together, our data suggest that some member(s) of the ets
gene family, rather than GATA factors, function to restrict expression
to the megakaryocyte lineage. It is not yet known which member of the
ets gene family is critical for megakaryocyte-specific gene
expression. Candidates are c-Ets-1, Spi-1, Fli-1, and
PU.1, all of which are expressed in
megakaryocytes.23,43,44 The data using c-ets-1
double knockout ES cells demonstrated that c-Ets-1 is not essential for
the lineage-specific expression.
The observation that the promoter regions of many
megakaryocyte-specific genes contain both GATA and Ets binding sites
underlines the importance of these 2 binding sites.21
However, there is no general rule concerning the orientation, the
surrounding nucleotide sequence, and the length separating these 2 sites. Our study shows that adjacent regions around the binding sites
and the region flanked by these 2 sites influence transcriptional
activity. The regulation of GPIIb gene expression may be more complex
than the many studies to date have suggested.
| |
Acknowledgments |
We thank Dr Shigekazu Nagata, Kirin Brewery Co Ltd, and Snow Brand Co
Ltd for the kind gift of EF1- promoter, human recombinant EPO and
TPO, and human recombinant M-CSF; and Drs Janusz Kabarowski and Anne B. Satterthwaite for critical reading of the manuscript.
| |
Footnotes |
Submitted June 28, 1999; accepted October 4, 1999.
Supported in part by grants from the Ministry of Education, Science,
Sports and Culture; Japanese Society for Promotion of Sciences
(JSPS-RFTF98L01101); the Cell Science Research Foundation; and the
Osaka Foundation for Promotion of Clinical Immunology.
Reprints: Toru Nakano, Department of Molecular Cell Biology,
Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita 565-0871, Osaka, Japan; e-mail:
tnakano{at}biken.osaka-u.ac.jp.
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.
| |
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Abstract
Introduction