From the Transcription factor GATA-2 has been shown to be
a key regulator in hematopoietic progenitor cells. To elucidate how the
expression of the GATA-2 gene is controlled, we isolated the
mouse GATA-2 (mGATA-2) gene. Transcription of mGATA-2
mRNAs was found to initiate from two distinct first exons, both of
which encode entirely untranslated regions, while the remaining five
exons are shared by each of the two divergent mRNAs. Reverse
transcriptase-polymerase chain reaction analysis revealed that GATA-2
mRNA initiated at the upstream first exon (IS) in
Sca-1+/c-kit+ hematopoietic progenitor
cells, whereas mRNA that initiates at the downstream first exon
(IG) is expressed in all tissues and cell lines that express GATA-2.
While the structure of the IG exon/promoter shows high similarity to
those of the Xenopus and human GATA-2 genes, the IS
exon/promoter has not been described previously. When we examined the
regulation contributing to IS transcription using transient
transfection assays, we found that sequences lying between GATA-2 is a member of the GATA transcription factor family that
binds to a common consensus sequence motif (A/T)GATA(A/G) through a
highly conserved zinc finger DNA binding domain (1). The founding
member of the GATA family is GATA-1 (2-4), whose expression is
restricted to erythroid, megakaryocytic, mast and eosinophil
hematopoietic cell lineages (4-6), and Sertoli cells in the testis (7,
8). Targeted disruption of the gene revealed that loss of GATA-1
function results in a severe deficit in both primitive and definitive
erythropoiesis (9-11).
The tissue distribution of GATA-2 is wider than that of GATA-1,
i.e. GATA-2 is expressed not only in several hematopoietic lineages that also express GATA-1 but in hematopoietic stem and progenitor cells, in the embryonic brain, and in endothelial cells, fibroblasts, kidney, liver, and cardiac muscle (1, 6, 12-14). The
expression profile of GATA-2 during the development of hematopoietic tissues has been studied most extensively in Xenopus and
zebrafish. In Xenopus, low levels of maternal GATA-2 are
present in early embryogenesis (15) while zygotic expression of GATA-2
is localized in the ventro-lateral ectoderm and mesoderm, in blood
cells, and in the central nervous system (16-18). A ventralizing
transforming growth factor (TGF)- Analysis of chimeric mice generated from GATA-2 ( It should be noted that GATA-2 expression is not found in mature
hematopoietic cells (22, 23). In a human bone marrow cell culture
system or in an in vitro ES cell differentiation, GATA-2
expression was down-regulated coordinately with the progression of
erythroid and myeloid cell differentiation (12, 13, 24). Furthermore,
forced expression of GATA-2 in an avian erythroid cell line caused the
proliferation of immature cells and markedly inhibited terminal
erythroid differentiation (25). These results taken together suggested
that the expression of GATA-2 is related to proliferative
versus differentiated capacity of hematopoietic progenitor
cells and that suppression of GATA-2 activity might be essential for
hematopoietic differentiation. Therefore, elucidation of the mechanisms
controlling GATA-2 transcription may offer insight into the earliest
aspects of hematopoietic cellular differentiation and may also give
rise to a means of experimentally manipulating the proliferation
versus differentiation of hematopoietic stem cells.
In this study, we have isolated and characterized the murine GATA-2
gene. We found that GATA-2 expression is regulated by two distinct
promoters. One is used in general in the tissues and cell lines that
express GATA-2, so we refer to this as the IG promoter. The structure
of the IG promoter is homologous to that of the previously reported
Xenopus and human GATA-2 gene promoters (26, 27). The other
promoter regulates the expression of GATA-2 specifically in
hematopoietic cells, thus we refer to this as the IS promoter. Since
this promoter has not been described elsewhere and displays
lineage-restricted utilization, we have also characterized the
regulatory domains that specify IS promoter activity.
Isolation and Subcloning of Genomic Clones--
A C57Bl2/J mouse
genomic library (CLONTECH) was screened with a
random primed 0.6-kbp SacI fragment, encoding the 5 Southern Blot Hybridization Analysis of Mouse Genomic
DNA--
Genomic DNA (25 µg) was isolated from an adult C57Bl2/J
mouse and digested with BamHI and SacI. After
electrophoresis in a 0.8% agarose gel, the DNA fragments were
transferred hydrodynamically to a Zeta Probe GT membrane (Bio-Rad,
Hercules) using a solution containing 0.4 M NaOH and 0.6 M NaCl. The DNA fragments were cross-linked to the membrane
by UV irradiation and hybridized to a 32P-labeled mGATA-2
cDNA fragment (0.8-kbp HindIII fragment corresponding to
a part of exon VI). Hybridization and washing of the membranes were
performed at 65 °C following the manufacturer protocol.
Cell Culture--
A mouse mast cell line P815 (generously
provided by Dr. A. Ichikawa) and T cell line BW5147 (from ATCC) were
cultured in RPMI 1640 medium supplemented with 10% heat-inactivated
fetal bovine serum (JRH Bioscience). Murine erythroleukemia (MEL) cells
DS19 (from Dr. Shigeru Sassa at the Rockefeller University) were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum. Mouse ProB cell line Ba/F3 (28) was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 10%
WEHI-3B cell (29) conditioned medium.
Fluorescence-activated Cell Sorter (FACS) Analysis--
Bone
marrow cell preparation, antibody staining, and four-color FACS sorting
were performed as described previously (29). Before sorting the
Sca-1+/c-kit+ fraction, cells were reacted with
a mixture of biotinylated rat monoclonal antibodies specific for mouse
differentiation antigens (lin RNA Blotting--
Total RNA (20 µg) was separated on a 1.0%
agarose gel containing formaldehyde and transferred to a Zeta Probe GT
membrane. A NotI-SacI-digested cDNA fragment
(0.6 kbp, containing 118 bp of exon IS, entire exon II, and 233 bp of
exon III), a PstI-SacI-digested genomic DNA
fragment (0.6 kbp, containing IS), and a
BamHI-SacI genomic DNA fragment (0.75 kbp,
containing IG) were used as probes. After exposure, membranes were
stripped and rehybridized to a probe for ribosomal RNA (from the
Japanese Cancer Research Resources Bank).
RT-PCR Analysis of the Expression from IS and IG Exons--
Ten
µg of RNA from normal mouse tissues or total RNA from 1 × 105 cells of each FACS-sorted lineage marker-positive
fraction and from 5 × 104 cells of
Sca-1+/c-kit+ and/or Lin RNase Protection Assay--
Total RNA was extracted from
cultured cell lines and normal mouse tissues using RNAzol B (TEL-TEST)
or ISOGEN (Wako Pure Chemicals, Osaka, Japan). Two mGATA-2 genomic
fragments were subcloned into pBluescript KS(+); one was a 610-bp
PstI-SacI fragment containing the 5 Primer Extension Assay--
An oligonucleotide,
5 Rapid Amplification of cDNA Ends
Assay--
Poly(A)+ RNA was prepared from P815 cells with
Oligotex-dT30 (TaKaRa). 5 Transient Transfection Assay--
Plasmids for functional assay
of the IS promoter and upstream regions were constructed as follows. A
KpnI-NotI fragment that covers Center for Tsukuba Advanced Research
Alliance and Institute of Basic Medical Sciences,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
79 and
61 are critical for the cell type-specific activity of the IS
promoter. DNase I footprinting experiments and electrophoretic mobility
shift assays demonstrated the binding of transcription factors to this
region. These data indicate that the proximal 80 base pair region of IS
promoter is important for the generation of cell type-specific
expression of mGATA-2 from the IS exon.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
family member, BMP-4, induces
Xenopus GATA-2 expression (19). In zebrafish, GATA-2 is
initially detected in the ventral ectoderm (20). High levels of GATA-2
are expressed at the boundary of the embryo and the yolk syncytial
layer, and then by the 2-5 somite stage, in the presumptive
hematopoietic progenitors that border the anterior and posterior of the
embryo (20). Thus, zygotic GATA-2 induction in vertebrates is
coincident with the commencement of hematopoiesis, suggesting that
GATA-2 is intimately involved in the development of hematopoietic cell lineages.
/
) embryonic stem
(ES)1 cells revealed that
GATA-2 (
/
) cells do not contribute to any hematopoietic lineage
(21). GATA-2-null mutant mice die of severe anemia at approximately 10 to 11 days post coitus during embryonic development (21) and manifest a
broad hematopoietic deficit. These studies demonstrated that GATA-2
plays a unique and crucial role in the differentiation of hematopoietic
progenitor cells.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
region of mGATA-2 cDNA, and a 0.8-kbp fragment from the HindIII
site in exon VI to the poly(A) of mGATA-2 cDNA. Both fragments were derived from an mGATA-2 cDNA clone, which we previously isolated from a fetal mouse liver cDNA library
(DDBJ/GenBankTM/EBI accession number
AB000096).2 Hybridization and
washing were carried out as described previously (23). Positive clones
were characterized by restriction enzyme mapping and Southern blot
hybridization, and insert DNA in the positive phages was subcloned into
pBluescript SK(+) vector (Stratagene). Sequences for the exon-intron
boundaries and promoter and upstream regions were determined using a
Taq polymerase cycle sequencing system and an automated DNA
sequencer (Perkin-Elmer Corp.).
) Gr-1, Mac-1, B220, TER119,
CD4 and CD8, and cells positive for these monoclonal antibodies were
removed using streptavidin-conjugated magnetic beads (Biomag;
Perseptive Diagnostics). The monoclonal antibodies used were RA-6B2
(anti-B220), RA3-8C5 (anti-Gr-1, Pharmingen), TER119 (from Dr. Tatsuo
Kina, Kyoto University), M1/70 (anti-Mac1), PK136 (anti-NK1.1),
CD4, CD8, 5a-8 (anti-Thy-1.2, Dainippon Pharmaceutical), ACK-2
(anti-c-kit, from Dr. Shin-ichi Nishikawa, Kyoto University), and Sca-1
(anti-Ly 6A/E, Pharmingen).
fractions were reverse transcribed by Superscript RT (Life
Technologies, Inc.) with random hexamer or oligo-dT primer. Products
were purified by phenol-chloroform extraction, ethanol precipitation,
and one-tenth of the products was used in each PCR reaction. PCR
reactions (35 cycles at 96 °C for 20 s and 68 °C for 60 s) were performed using primer 1 (5
-ACAAAAGCGGCTGTCTGCGCGACG-3
),
primer 2 (5
-CACCCCTATCCCGTGAATCCG-3
), and primer 3 (5
-AGCTGTGCTGCCTCCATGTAGTTAT-3
).
flanking
sequence, IS exon, and the sequence 3
to the IS exon, and the other
was a 760-bp BamHI-SacI fragment containing the
5
flanking region, IG exon, and the sequences 3
to the exon IG. The
constructs were linearized by SpeI and BamHI,
respectively, and a labeled antisense RNA was synthesized from the T7
promoter of the plasmid using T7 RNA polymerase (Promega, Madison).
Total RNA samples (100 µg) from P815 and Ba/F3 cells were hybridized to the labeled transcript for 12 h at 45 °C. The RNA samples
were digested with an RNase mixture (Ambion), and the sizes of the RNA
hybrids were determined by denaturing polyacrylamide gel
electrophoresis. Yeast tRNA (Sigma) and total RNA from DS19 cells were
used as negative controls to detect nonspecific bands. Radiolabeled RNA size markers were synthesized using an RNA marker template set (Wako
Pure Chemicals).
-CCGGCTCCGGCCCCTCTGCATCCTC-3
, was labeled with
[32P]ATP using T4 polynucleotide kinase (Toyobo, Osaka).
The labeled primer (1 pmol) was annealed to 50 µg of total RNA from
P815 cells or tRNA for 90 min at 65 °C and then slowly cooled to
room temperature in 150 mM KCl, 10 mM Tris-Cl
(pH 8.3), and 1 mM EDTA. Reverse transcription was
initiated using 10 units of avian myeloblastosis virus reverse
transcriptase (Promega) in a solution containing 50 mM KCl,
23 mM Tris-Cl (pH 8.3), 20 mM
MgCl2, 5 mM dithiothreitol, 0.15 mM
dNTPs and 150 µg of actinomycin-D/ml and incubated for 60 min at
42 °C. The template was then digested with RNase A (2 µg) for 15 min at 37 °C followed by phenol-chloroform extraction, ethanol
precipitation, and size fractionation on a denaturing polyacrylamide
gel. The TaqTrack sequencing system (Promega) was used to generate a
sequence ladder of the IG exon with the same primer.
-Rapid amplification of cDNA ends
(5
-RACE) assay was performed utilizing the AmpliFINDER 5
-RACE system
(CLONTECH). Two µg of poly (A)+ RNA
was reverse transcribed, and mGATA-2 cDNA with the transcription initiation site was amplified using oligonucleotide antisense primer
mG23 (5
-AGCGGCGTGGCTGGTCGGCC-3
, corresponding to nucleotides 211 to
192 of the IS exon) and primers for the tailed linker sequence. Another
antisense primer used for PCR amplification was mG26
(5
-CGCAGGCAGCCGCTTTTGTC-3
, corresponding to nucleotides 124 to 105 of
the IS exon). PCR products were subcloned into a pGEMT vector
(Promega), and their sequences were determined.
6.0 kbp to +177
bp region (transcription start site of IS exon was set as +1) was
ligated to pGL2-Basic plasmid (Promega), which was cleaved with both
KpnI and SacI, resulting in pGL-6.0IS. pGL2-Basic
contains the firefly luciferase (LUC) gene as a reporter. Similarly,
HincII-NotI (
1580 to +177),
EcoRI-NotI (
670 to +177), PstI-NotI (
165 to +177),
SpeI-NotI (
145 to +177), and
SmaI-NotI (
86 to +177) fragments were ligated
to the KpnI/SacI double digested pGL2-Basic,
generating pGL-1580IS, pGL-670IS, pGL-165IS, pGL-145IS, and pGL-86IS,
respectively. pGL-50IS containing
50 to +177 was prepared from the
pGL-165IS using a directional deletion system (TaKaRa).
2.0 kbp upstream from the IG exon,
and from the BamHI site at
482 bp 5
to the IG exon to the
NcoI site were ligated to the
KpnI-XhoI double digested pGL2-Basic
(pGLNot-II, pGL-2.0IG-II, and pGL-482IG-II).
pGL-79IS
IG-II was generated from pGL-79ISIG-II by deleting
SacI fragment. pGL-128IS, pGL-115IS, pGL-79IS, and pGL-61IS,
containing the sequences from
128,
115,
79, or
61 to +177 of IS
exon, respectively, and pGL-125IG, pGL-103IG, pGL-31IG, and pGL-19IG,
containing the sequences from
125,
103,
31, or
19 to +37 of IG
exon, respectively, were prepared by PCR using sequence-specific
oligonucleotides. Internal promoter mutants were constructed by
substituting the sequence between
80 and
35 of pGL-145IS with a
series of overlapping oligonucleotides containing mutations. The
authenticity of these mutations were confirmed by DNA sequencing.
DNase I Footprinting Analysis-- Nuclear extract was prepared from P815 and DS19 cells as described (33), and DNA-binding proteins were purified further by DNA-cellulose (Sigma) column chromatography (34). A fraction eluted with 250 mM ammonium sulfate was dialyzed to 20 mM HEPES (pH 7.9), 5 mM MgCl2, 50 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, and 20% glycerol and was concentrated using Molcut II (Millipore).
DNase I footprinting was performed as described previously (35). The IS promoter and upstream promoter regions (Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared as described in the previous section, and EMSA
was carried out as described (1). Complementary oligonucleotides were
annealed, and the resulting double-stranded DNA was used as probes.
Probes used for DNA-binding assays correspond to 85 to
51
(5
-GGCACCCTCCTGCCCCCCTGCGGCGTTCCCTCCCC-3
). Poly(dA-dT/dA-dT) was used
as a nonspecific competitor, and 100-fold excesses of unlabeled
double-stranded oligonucleotides were used as sequence-specific competitors. Sequences of the mutant oligonucleotides used as competitors were identical to those used in the LUC mutant reporter constructs.
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RESULTS |
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Structure and Organization of mGATA-2 Gene--
To begin to
analyze the regulatory mechanisms controlling transcription in the
mGATA-2 gene, genomic DNA fragments containing the gene were cloned and
characterized. Using the 5 end region and the 3
untranslated region
(UTR) of an mGATA-2 cDNA clone, which was previously isolated from
a fetal liver cDNA library, a mouse
genomic library was screened, and the recovered recombinants were
plaque-purified. Twelve independent mGATA-2 bacteriophage
genomic
clones were characterized by restriction enzyme site mapping and
Southern blot hybridization. The genomic phage clones were found to be
largely overlapping and to cover the entire coding region of the
mGATA-2 locus of approximately 14 kbp (Fig.
1A).
|
Tissue-specific Utilization of the IS and IG Exons/Promoters-- To elucidate how the promoters preceding the two individual first exons might be differentially utilized, we performed a series of expression analyses. First, RNA blot analysis showed that, of the cell lines examined, GATA-2 mRNA was expressed abundantly in P815 mastocytoma (Fig. 2, lane 1) and Ba/F3 proB lymphocyte (lane 2). In contrast, GATA-2 mRNA levels were undetectable in the DS19 murine erythroleukemia (lane 3) and BW5147 T lymphocyte (lane 4) cell lines. When we carried out additional analyses using IS or IG exon-specific probes, the experiment showed that IS mRNA was transcribed abundantly in P815 cells but only poorly in Ba/F3 cells, whereas IG mRNA is expressed in both P815 and Ba/F3 cells (Fig. 2). RNA blot analysis using these three probes all showed the presence of two mRNAs for mouse GATA-2. In this regard, we previously found the presence of two GATA-2 mRNAs in human and presented evidence indicating that the two bands reflect alternative usage of two polyadenylation consensus signals (23). Since the two polyadenylation consensus signals are also conserved in the mouse GATA-2 gene (see above), we assume that the usage of multiple polyadenylation signals is also true for the mouse GATA-2 gene.
|
|
Transcription Initiation Sites for the Alternate mGATA-2 First Exons-- To identify the transcription initiation sites of the IS and IG exons, RNase protection assays were performed using RNA samples recovered from P815, Ba/F3, and DS19 cells. We found that both the IS and IG probes were protected in P815 cells (Fig. 4A) and in Ba/F3 cells (upon longer exposure; data not shown). The sizes of the major protected fragments of the IS and IG probes were estimated to be 280 bp (Fig. 4A, lane 4) and 216 bp (lane 11), respectively. There were several extra bands protected by both probes, suggesting the existence of minor transcription start sites; in particular, the IG protection probe gave rise to bands around 270 bp (lane 11).
|
|
Structure of the Promoter and Upstream Regions of the IS and IG Exons-- We next determined the sequences of the promoter and upstream regions of both the IS and IG exons (Fig. 5). We analyzed these sequences by TFSEARCH4 which searches within sequenced fragments versus the TFMATRIX transcription factor binding site profile data base by E. Wingender, R. Knueppel, P. Dietze, and H. Karas (GBF-Braunschweig). We found a number of possible transcription factors binding sites, including some expressed uniquely in hematopoietic cells, in this search and in other literature searches (described under "Discussion"). These positions of these transcription factor binding sites are indicated in Fig. 5.
In the IS promoter, a TATA box-like AAATAAAAA element was found between positionTrans-activation Activity of the IS Promoter in P815 Cells--
We
next attempted to localize cis-acting elements involved in the
determination of the hematopoietic cell-specific expression of the IS
promoter. We first examined six constructs bearing rather large
deletions of 5 sequence information containing the IS promoter. The
largest construct in this analysis is pGL-6.0IS, containing 6.0 kbp
upstream from the transcription initiation site and 177 bp of exon IS
(to a NotI site in the IS exon). The shortest construct we
examined was pGL-50IS, in which the LUC reporter gene was driven by a
proximal promoter fragment extending only to
50 relative to the
beginning of exon IS. These reporters were transfected into P815 cells.
LUC activity was determined using total cellular extracts and
normalized for transfection efficiency based on a cotransfected pENL
-galactosidase control plasmid.
|
IS and IG Promoter Activities in P815 Cells--
We performed a
second set of transient transfection assays using constructs containing
sequences lying between the IS promoter and the common second exon
transfected into P815 cells (Fig.
7A). Transfection of reporter
plasmid pGL-79ISIG-II, containing the IS promoter (79 bp), IS exon,
and a part of the second exon, resulted in almost identical LUC
activity to that generated by pGL-482IG-II, indicating that genuine IS
promoter activity is comparable with the IG promoter activity in P815
cells. pGL-79ISIG-II typically generated lower LUC activity than did
pGL-482IG-II. Thus the region upstream from the
482 to IS exon has no
stimulating activity in P815 cells (Fig. 7A).
|
Cell-type Specificity of Minimal IS Promoter Activity-- To test for cell-type specificity of IS promoter activity, the reporter gene activities of pGL-145IS and pGL-50IS were determined in several culture cell lines. LUC activity generated by pGL-145IS in DS19 and BW5147 cells were 27 and 11% of that generated in P815 cells, respectively, whereas LUC activity in Ba/F3 cells was 99% of that in P815 cells (Fig. 8). The former two cell lines do not express GATA-2 mRNA to a detectable level (see Fig. 2). These differences were not observed when we transfected pGL-50IS construct into the same cell lines. These results indicate that the IS promoter indeed contributes primarily, if not exclusively, to the determination of cell-type specificity of mGATA-2 expression from the IS exon.
|
The Functional Cis-elements Are Protected in DNase I Footprinting
Analysis--
We next tried to identify transcription factors that
might bind to the IS promoter. To this end, we first carried out DNase I footprinting analysis of these domains using nuclear extracts prepared from P815 (GATA-2 high) or DS19 (GATA-2 low) cells. As shown
in Fig. 9A, two regions
(downstream from 54 and from
61 to
79) were protected using a
sense probe in combination with P815 nuclear extracts (lanes
4-6). Several hypersensitive sites, another hallmark of specific
protein-DNA interactions, were also observed at
51,
68,
75, and
80 in this experiment. In contrast, the DS19 nuclear extract
protected only the region downstream from
54 and did not produce
strong hypersensitive sites (lanes 9-11). When we carried
out the footprinting experiment with the antisense probe, the P815
nuclear extract protected the region proximal of
54 and
65 to
75
from DNase I digestion, whereas the DS19 nuclear extract did not
protect these regions effectively (Fig. 9B). Since the DS19
nuclear extract was purified by the same procedure as the P815 nuclear
extract and since we used identical amounts of nuclear protein in
comparable lanes in this experiment, these results suggest that a
transcription regulatory complex is formed more efficiently with the
P815 nuclear extract than that with DS19 nuclear extract. The two
regions protected by the P815 nuclear extract (i.e. proximal
to
54 and
61 to
79) show very good coincidence with the regions
that are important in the transient transfection assays (see
above).
|
Competitive Transcription Factor Binding to GC-rich Sequences in
the IS Promoter--
To further investigate transcription factor
binding to the IS promoter region, we next performed EMSA using the
P815 and DS19 nuclear extracts. A probe spanning 85 to
51 (Fig.
10A) formed several major
bands (bands a to d, see lane 1 of
Fig. 10B and lane 8 of Fig. 10C).
These bands could be competed with a 100-fold excess of unlabeled
competitor (lane 2 of Fig. 10B), indicating that
the binding of transcription factors to this probe is specific.
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DISCUSSION |
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Although transcription factor GATA-2 is essential for hematopoiesis (21), its expression is not restricted to hematopoietic lineages. In hematopoietic cells, the expression level of GATA-2 changes dramatically during progressive cell differentiation (12, 39). This suggested that mechanisms could exist for regulating the expression of GATA-2 specifically in the hematopoietic lineage. Consistent with this expectation, we report here the presence and activity of an independent hematopoietic promoter in the mGATA-2 gene. The IS promoter is used specifically in hematopoietic progenitors, whereas the IG promoter seems to be responsible for the expression of GATA-2 in various tissues but also including hematopoietic cells. While we found the structure of the IG promoter to be quite homologous to the previously reported human and Xenopus GATA-2 promoters (26, 27), the IS promoter was identified for the first time here.
Determination of the complete structure of the mGATA-2 gene revealed an organization resembling that of the other GATA-1/2/3 factors so far defined (23, 26, 27, 40, 41) (Fig. 11). The restriction enzyme sites within the mGATA-2 gene determined in this study coincide completely with the map previously reported by Tsai and colleagues (21). Genes for vertebrate GATA-1/2/3 factors are generally composed of six exons. In addition, both GATA-1 (7) and GATA-2 (this study) genes contain two alternative first exons (Fig. 11). The chicken GATA-5 gene, which belongs to another subfamily of GATA factors (GATA-4/5/6), also contains two alternative untranslated first exons (42). The use of multiple first exons/promoters has been found in a number of transcription factor genes that are essential for hematopoietic cell development. For instance, the SCL/TAL-1 gene contains Ia and Ib first exons/promoters, and the Ia promoter has erythroid-specific activity (31, 43). The p45 NF-E2 gene also has two promoters, i.e. fetal (f) and adult (a), although use of these promoters is not exclusively restricted (44). We found that GATA-1 expression is regulated by both an erythroid-specific (IE) and Sertoli-specific (IT) first exons/promoters (7, 32, 45). These observations show that, in many cases, animals have repeatedly adopted the same simple strategy to achieve specific expression of lineage-restricted transcription factors: the use of alternative promoters.
|
GATA-2 cDNAs were originally cloned from a chicken erythroid cell
cDNA library (1), from a human endothelial cell cDNA library
(36, 46), and from a stage 17 Xenopus embryonic cDNA library (16). In contrast, we have cloned the IS promoter-derived cDNA from a hematopoietic fetal liver cDNA library. We would
speculate, based on the expression studies presented here, that the
IS-type cDNA clone may not exist in the former libraries. Our
preliminary mRNA in situ analysis demonstrates that
GATA-2-positive cells increase temporally in the fetal liver and
neonatal spleen, both of which are hematopoietic organs. Thereafter,
however, GATA-2-positive cells are only rarely detected in various
mouse tissues, suggesting that the level of GATA-2 mRNA is high
when hematopoietic progenitors are rapidly
expanding.5 An assessment of
the trans-activation activity in a transgenic mouse system, using
constructs containing both the mGATA-2 promoter driving the expression
of the Escherichia coli -galactosidase gene, is now
ongoing in this laboratory. Our preliminary data show that the reporter
gene expression in the hematopoietic stem cell fraction of bone marrow
cells and in aorta/gonad/mesonephros region of mouse embryo is mainly
driven by IS promoter.5
The basic transcriptional activity of the IS promoter was found to be
localized within the proximal 80 bp. Specific transcription factor
interactions to the 85 to
50 region of the IS promoter were
identified through DNase I footprinting analysis and EMSA using nuclear
extracts from P815 cells. Whereas EMSA competition experiments with
mutated oligonucleotides showed that transcription factors binding to
the
73 to
70 sequence are responsible to the IS promoter activity,
no consensus DNA-binding sequence of known transcription factors has
been identified for this sequence.
In this regard, we noticed that transcription factors Sp1, MZF-1 (47),
CTCF (48), or MAZ/ZF87 (49) has the possibility to bind to the 58 to
48 sequence. Mutational analysis of the IS promoter revealed that
this region is also responsible for mGATA-2 IS promoter activity. It is
interesting to note that MZF-1 is supposed to regulate the expression
of c-myb and CD34, both of which are expressed in
undifferentiated hematopoietic cells (47, 50), and overexpression of
MZF-1 inhibits hematopoietic cell development from ES cells (50). These
properties are similar to those of GATA-2. We also have noticed the
presence of three CCCTC sequences in the IS promoter (
81 to
87,
58 to
54, and
50 to
45). These motifs resemble the cis-elements
in the c-myc promoter, to which several transcription
factors, such as Sp1, ZF78, MAZ, hnRNPK, and CNBP, can bind (48, 51,
52). As c-myc controls differentiation, proliferation, and apoptosis of
hematopoietic cells (53), factors binding to the CCCTC sequence may
also be related to mGATA-2 gene expression in hematopoietic
progenitors.
A 31-bp pyrimidine-rich sequence
(5-TCTGCGCCGCTTTCTGCCCCCTCCTGCCCTC-3
,
3831 to
3800 relative
to translation start site) in the zebrafish GATA-2 gene was recently
reported to be responsible for neuronal expression of the gene (54),
although the position of the first exons as well as the transcription
initiation sites has not been firmly identified in the zebrafish GATA-2
gene. Interestingly, when the plasmid containing the 7.3-kbp region
upstream from the translation start site is linked to a modified green
fluorescent protein (GFP), the plasmid expresses GFP in the
intermediate cell mass, the early hematopoietic tissue of the
zebrafish. Deletion of 2.5 kbp from the 5
end of the 7.3-kbp plasmid
nearly abolishes the GFP expression specifically in the posterior end
of the intermediate cell mass. Since the IS promoter is active in early
hematopoietic progenitors in mouse bone marrow, we speculate that the
upstream 2.5-kbp region may contain a first exon homologous to the
mouse IS exon.
We demonstrated here the presence of two mGATA-2 gene promoters. Of the two promoters, the IS promoter is expressed specifically in hematopoietic lineage cells. Detailed analysis of the cis-acting elements in transfection assays and protein-DNA interaction assays indicated that transcription factors binding to the IS promoter play an important role in determining its specificity. Since GATA-2 is essential for the development of all hematopoietic lineage cells, detailing the mechanisms that regulate GATA-2 gene expression may allow new insights and perspectives into hematopoietic stem cell biology.
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ACKNOWLEDGEMENTS |
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We thank Drs. K.-C. Lim, S. Takahashi, K. Umesono, M. E. Walmsley, and R. Yu for help and Drs. A. Ichikawa, T. Kina, S.-I. Nishikawa, and S. Sassa for cell lines and antibodies.
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FOOTNOTES |
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* This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture and Japanese Society for Promotion of Sciences (RFTF96I00202).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB007371 and AB009272.
¶ To whom correspondence should be addressed. Tel.: 81-298-53-3111; Fax: 81-298-53-6965; E-mail: masiya{at}igaku.md.tsukuba.ac.jp.
1 The abbreviations used are: ES, embryonic stem; IS, upstream first exon; IG, downstream first exon; bp, base pair(s); kbp, kilobase pair(s); FACS, fluorescence-activated cell sorter; RT, reverse transcriptase; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; UTR, untranslated region; EMSA, electrophoretic mobility shift assay.
2 N. Suwabe, N. Minegishi, and M. Yamamoto, unpublished data.
3 H. Ishihara and M. Yamamoto, unpublished observations.
4 Version 1.3© 1995 by Yutaka Akiyama at the World Wide Web site http://www.pdap1.tre.rwcp.or.jp/research/db/TFSEARCH.html.
5 J. Ohta, N. Minegishi, and M. Yamamoto, unpublished observation.
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