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Blood, Vol. 96 No. 3 (August 1), 2000:
pp. 910-916
HEMATOPOIESIS
GATA factor transgenes under GATA-1 locus control rescue
germline GATA-1 mutant deficiencies
Satoru Takahashi,
Ritsuko Shimizu,
Naruyoshi Suwabe,
Takashi Kuroha,
Keigyou Yoh,
Jun Ohta,
Shigeko Nishimura,
Kim-Chew Lim,
James Douglas Engel, and
Masayuki Yamamoto
From the Institute of Basic Medical Sciences and Center for
Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba,
Japan; and Department of Biochemistry, Molecular Biology and Cell
Biology, Northwestern University, Evanston, IL.
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Abstract |
GATA-1 germline mutation in mice results in embryonic
lethality due to defective erythroid cell maturation, and thus other hematopoietic GATA factors do not compensate for the loss of
GATA-1. To determine whether the obligate presence of
GATA-1 in erythroid cells is due to its distinct
biochemical properties or spatiotemporal patterning, we attempted to
rescue GATA-1 mutant mice with hematopoietic GATA factor complementary
DNAs (cDNAs) placed under the transcriptional control of the GATA-1
gene. We found that transgenic expression of a GATA-1 cDNA fully
abrogated the GATA-1-deficient phenotype. Surprisingly, GATA-2 and
GATA-3 factors expressed from the same regulatory cassette also rescued
the embryonic lethal phenotype of the GATA-1 mutation. However,
adult mice rescued with the latter transgenes developed anemia, while
GATA-1 transgenic mice did not. These results demonstrate that the
transcriptional control dictating proper GATA-1 accumulation is the
most critical determinant of GATA-1 activity during erythropoiesis. The
results also show that there are biochemical distinctions among the
hematopoietic GATA proteins and that during adult hematopoiesis the
hematopoietic GATA factors are not functionally equivalent.
(Blood. 2000;96:910-916)
© 2000 by The American Society of Hematology.
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Introduction |
Of the 6 GATA factors, GATA-1, GATA-2, and GATA-3 constitute a distinct
subfamily because they are expressed in hematopoietic lineages and
because they share a similar gene organization. However, the amino acid
sequence of GATA-1 has diverged significantly from that of the GATA-2
and GATA-3 proteins, which are more similar to one
another.1 Within the hematopoietic compartment, GATA-1 is
expressed in erythroid, megakaryocytic, eosinophilic, and mast cells2-5 in addition to Sertoli cells of the
testis.6 The GATA-1 gene is transcribed from the
distal testis first exon (IT) and the proximal erythroid first exon
(IE) in Sertoli cells or hematopoietic cells,
respectively.6,7
Employing a promoter interference approach, we recently generated an
erythroid promoter-specific mutant allele of the GATA-1 gene
(which we called GATA-1.05 because it was expressed at
approximately 5% of wild-type [WT] level8). Because
GATA-1 is located on the X chromosome,9 all male
embryos hemizygous for the mutation died by 12.5 embryonic days (E12.5)
due to arrest of primitive erythropoiesis, as observed in GATA-1-null
mutant embryos.10 Subsequent analyses of GATA-1.05
heterozygous female mice11 or lineage-specific
GATA-1 mutant mice12 showed that GATA-1 was also
vital for terminal megakaryocytic differentiation.
Although the expression of GATA-2 substantially overlaps that of GATA-1
in hematopoietic lineages,2,13 GATA-1 gene
disruption experiments clearly demonstrated that GATA-2 does not
compensate for the loss of GATA-1 function in vivo.8,10
Interestingly, in vitro differentiation of GATA-1-deficient embryonic
stem (ES) cells revealed a 50-fold induction of GATA-2 messenger RNA
(mRNA) in erythroid cells.14,15
The functional significance attributed to differences in the
biochemical activities of GATA family members has been addressed in
cell culture experiments. The first indication that these proteins might have distinct characteristics came from a study in which the
GATA-1, -2, and -3 factors were fused to the human estrogen receptor
hormone binding domain.16 When transfected into primary avian erythroid progenitor cells, GATA-1 and GATA-2 chimeras had starkly opposite hormone-dependent effects on erythroid
differentiation. A number of similar studies have been published since
then generally supporting the notion that each GATA factor differs in
activity in a wide variety of distinct cell types, and hence they may
not be functionally equivalent in vivo.17 In contrast, in
vitro erythroid differentiation of GATA-1-deficient ES cells was
partially restored by expressing various GATA factors, including a
single zinc finger alone.18,19
To resolve the fundamental question of whether GATA-1 possesses unique
functional properties that are distinct from the other hematopoietic
GATA factors, or whether its spatiotemporal patterning and abundance is
the primary requirement for effective erythropoiesis, we attempted to
rescue the GATA-1.05 mutant embryonic lethality in vivo by
expressing different murine GATA factors.
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Materials and methods |
Generation of transgenic mice
Transgenic mice were generated by microinjection of DNAs into
fertilized BDF1 eggs using standard procedures.20 Founders were first screened by polymerase chain reaction (PCR) and then verified by Southern blot analysis. The copy number of each line was
determined using a BAS 1500 Mac imaging analyzer (Fuji Film, Tokyo,
Japan). Mice bearing the GATA-1.05 germline mutant
allele8 were bred in clean rooms in the Animal Research
Center at the University of Tsukuba. To rescue GATA-1.05 mice
from embryonic lethality, GATA-1.05 heterozygous female mice
were intercrossed with transgene-positive heterozygous stud mice, and
progenies from these matings were analyzed at the indicated times.
Genotyping of mice
Genomic DNA was analyzed by PCR using the following primers.
Neo gene primers were used for detecting the GATA-1.05
allele, because the mutation was generated by inserting a Neo
gene cassette into the 5' flanking region of the GATA-1
locus.8 Primers for GATA-1, -2, and -3 were used for
detecting the respective transgenes.15 We determined the
sex of transgenic embryos using PCR amplification of the Y
chromosome-specific Zfy-1 gene.21 Southern blots
were performed on the same genomic DNA samples. We used full-length complementary DNAs (cDNAs) of GATA-1 and GATA-3 and used GATA-2 cDNA
without the finger region as probes.
RNA blot analysis
RNA blot analysis was performed as previously
described.6 Total cellular RNA was extracted from spleens
of 3- to 6-month-old mice using RNA extraction reagent, RNAzol
(Tel-Test, Friendswood, TX). RNA samples (10 µg) were electrophresed
on a 1.6% agarose gel and then transferred to nylon membranes
(Zeta-probe, Bio-Rad, Hercules, CA). Full-length cDNAs for each GATA
factor were used as probes.
Western blot analysis
Splenic nuclear extracts from 3- to 6-month-old mice were prepared
as previously described.22 To detect GATA-1 or GATA-2 proteins, N6 6 or RC1.123 monoclonal antibody
was used, respectively.
Electrophoretic mobility shift analysis
The double-stranded GATA oligonucleotide
5'-GCTGATTCCCTTATCTATGCCT
TCCCAGCTGCCTCCCT-3'7 was radiolabeled as probe. For competition experiments, a GATA mutant oligonucleotide
5'-GCTGATTCCCTGGCTTATGCCTTCCCAGCTGCCTCCCT-3' was included. Binding reactions and electrophoresis were carried out as
previously described.1
In vitro colony assays
Neonatal spleens were dispersed into single-cell suspensions and
plated in duplicate in 1.0% methylcellulose in alpha-minimal essential medium containing 30% fetal calf serum, 50-µM
2-mercaptoethanol, 1% bovine serum albumin (Sigma
Chemical Co, St Louis, MO), and Nutridoma-SP (Boerhringer Mannheim,
Mannheim, Germany). A total of 2-U/mL human erythropoietin, 50-ng/mL
murine stem cell factor, 50-ng/mL human thrombopoietin, 10-ng/mL murine
interleukin-3, 10-ng/mL murine granulocyte-macrophage
colony-stimulating factor (GM-CSF), and 10-ng/mL murine interleukin-6
were added as supplements. Cultures were maintained at 37°C under
humidified conditions with 5% carbon dioxide. Colony-forming
units-erythroid (CFU-E) were counted on day 3, and burst-forming
units-erythroid (BFU-E), CFU-GM, and CFU-megakaryocytes (CFU-MEG) were
scored on day 7.
Peripheral blood cell analysis
Mice were bled from the retroorbital plexus, and blood cell indices
were determined by hemocytometer (MEK-6258, Nihon Koden Co, Tokyo,
Japan). Peripheral blood smears were stained with Wright-Giemsa stain.
Statistical analysis
Statistical analysis was performed using the Student
t test.
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Results |
Generation of IE3.9int-directed GATA transgenic mice
As diagrammed in Figure 1A, the GATA-1,
-2, -3 or mutated GATA-1 (GATA-1 delta C-finger) cDNA was individually
cloned 3' of a GATA-1 genomic fragment that is capable of
conferring the complete GATA-1 expression profile to a reporter
transgene, and the resultant constructs were injected into fertilized
eggs to generate transgenic mice. To monitor the cell types that
expressed the transgene-derived GATA factors, in 1 experiment we
coinjected a green fluorescent protein (GFP) cDNA placed under IE3.9int
transcriptional control.
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| Fig 1.
Transgenic mice bearing IE3.9int-directed
transgenes.
(A) Structure of the GATA-1 gene regulatory region
(IE3.9int) cassette. This plasmid contains 3.9 kilobase
pairs of sequences 5' to the IE exon, the IE exon itself, the
first intron, and a part of the second exon of the mouse GATA-1
gene. The initiation methionine codon in the GATA-1 second exon
was deleted and replaced by a unique NotI site for
subsequent cloning purpose. Restriction enzyme sites are B,
BamHI; E, EcoRI; N, NotI; S,
SacI. (B-D) Genomic Southern blot analyses of
IE3.9int-directed transgenic mice. The transgene-specific bands
(Tg) and endogenous bands(s) (end) are indicated by arrows on the left
of each panel. (E, F) Expression profiles of the IE3.9int-GFP
transgene in bone marrow hematopoietic cells. Strong green fluorescence
is observed in both erythroid cells and megakaryocytes (arrow) (E). In
FACS analysis, most TER119-positive bone marrow cells are also
GFP-positive (F).
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After screening, 4 independent transgenic lines bearing the GATA-1
cDNA, 3 lines with the mutated GATA-1 cDNA, 3 lines carrying GATA-2 and
GFP cDNAs and, finally, 4 lines harboring the GATA-3 transgene were
generated. The GATA-1 cDNA transgenic lines contained between 1 and 4 copies of the transgene (Figure 1B). Similarly, the GATA-2 and GATA-3
transgenic lines contained 3 to 4 and 4 to 13 transgene copies,
respectively (Figure 1C, D). In addition, the "fingerless" GATA-1
cDNA transgenic lines contained 7 (line No. 312) or 2 (line No. 315)
transgene copies (data not shown). We employed multiple transgenic
lines, which varied in copy numbers and expression levels (verified by
reverse transcriptase-PCR) in rescue experiments. Only progeny from F1
or later generations were used in intercross experiments.
Because we coinjected the GATA-2 and GFP expression constructs, which
were under the control of identical GATA-1 regulatory sequences, cells that expressed GATA-2 would also be expected to emit
green fluorescence. Figure 1E shows a typical bone marrow preparation
from such a GATA-2/GFP transgenic mouse. Both megakaryocyte and
numerous erythroid cells emitted intense green fluorescence. Total bone
marrow cells recovered from a GATA-2/GFP transgenic mouse were reacted
with rhodamine-conjugated TER119 antibody and then analyzed by
fluorescence-activated flow cytometry (FACS). Figure 1F shows that many
TER119-positive cells were also positive for GFP. Because normal bone
marrow did not contain any GFP-positive cells (data not shown), these
results demonstrated that the GATA-1 regulatory sequences
examined here reflect the expression profile of the endogenous
GATA-1 gene in vivo. In addition, some cells expressed TER119
but not GFP, suggesting that the GATA-1 gene regulatory
sequences in pIE3.9int do not contain a locus control region-like
activity that is able to overcome transgene integration position effects.
GATA-1 rescues GATA-1.05 mutant embryonic lethality
To verify that the GATA-1 genomic fragment used in this
study was fully sufficient for complete GATA-1 regulation in
erythroid cells, we first asked whether a GATA-1 cDNA placed under its
regulatory control could abrogate the GATA-1.05 mutant
phenotype. We examined 95 pups from 12 litters obtained from 3 independent GATA-1 transgenic lines (Nos. 782, 801, and 831), which
contained approximately 2, 4, and 3 transgene copies, respectively, and
recovered 11 newborns of compound mutant (G1R) genotype (Table
1A). The results thus indicate that when
GATA-1 is expressed under the transcriptional control of the IE3.9int
genomic sequence, GATA-1.05 transgene-positive male embryos can
overcome the lethality due to the germline mutation. A similar
phenotypic rescue was not observed in compound mutant male neonates
bearing a truncated GATA-1 cDNA, which had the carboxyl finger
(C-finger) of GATA-1 deleted (Table 1B); nor was rescue evident during
embryogenesis at E13.5 (data not shown).
GATA-2 and GATA-3 can substitute for GATA-1 in erythropoiesis in
vivo
To ascertain whether heterotypic hematopoietic GATA factors could
functionally replace GATA-1 in erythropoiesis, we generated transgenic
lines expressing either GATA-2 or GATA-3 cDNA under GATA-1 gene
transcriptional control. Because GATA-1.05 hemizygous males do
not survive beyond E12.5,8 we looked for preliminary evidence of rescue at E13.5. Figure 2A
shows representative genotyping results of offspring from an intercross
between a GATA-1.05/X female and an IE3.9int-GATA-2
transgenic male (line No. 620, which had 4 transgene copies).
Significantly, embryo No. 4, which was positive for all 3 (Neo,
Zfy-1, and GATA-2 transgene) markers, was still alive
and displayed grossly normal morphology at E13.5 (G2R; Figure 2C).
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| Fig 2.
Rescue of hematopoiesis in GATA-1.05 mouse by
transgenic expression of GATA-2.
(A) Genotyping of GATA-1.05::GATA-2 Tg
compound mutant mice. Three sets of primers were used for the
genotyping. Primer sets for the neomycin resistance gene (Neo)
and Zfy-1 gene were used for detecting GATA-1.05 allele
and sex of the embryos, respectively. Another set of primers was used
to detect the GATA-2 transgene. Numbers represent sibling
embryos from a single litter. (B-D) Gross appearance of a litter of
E13.5 embryos from the mating of GATA-1.05 heterozygous female
mouse with GATA-2 Tg(+) male mouse. An embryo with
GATA-1.05/Zfy-1(+)/GATA-2 Tg(-) genotype was found dead at
E13.5 and showed signs of necrosis (B). In contrast, an embryo of
GATA-1.05(+)/Zfy-1(+)/GATA-2 Tg(+) genotype was apparently
healthy (G2R mouse; C), albeit slightly smaller in size, compared with
WT male embryo (D).
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In the absence of the GATA-2 transgene, GATA-1.05 hemizygous
male embryos did not survive beyond E12.5 (Figure 2B) and exhibited significant necrosis by E13.5. We analyzed 8 litters of 46 embryos between gestational days 13.5 and 16.5 (data not shown) as well as 75 newborns from 12 litters using GATA-2-expressing transgenic line No.
620 (Table 2) and found that all live males
had the GATA-2 transgene. As expected, no transgene-negative
GATA-1.05 male was recovered at birth. Similar results were
observed when 2 other GATA-2 transgenic lines (No. 625, 3 copies; No.
99, 4 copies; Table 2) were used in similar breeding paradigms. These data indicated that GATA-2, when expressed under GATA-1 transcriptional control, could rescue the GATA-1.05 mutation.
We next asked whether a GATA-3 transgene could similarly overcome the
mid embryonic lethal effect of the GATA-1.05 mutation. Unlike
GATA-1 and GATA-2, GATA-3 is not expressed in erythroid or
megakaryocytic lineage cells at significant levels; therefore, this
experiment would constitute a cardinal test for possible functional
redundancy among the hematopoietic GATA family members. Two independent
GATA-3 transgenic lines (Nos. 390 and 820, bearing approximately 13 and
9 transgene copies, respectively) were mated with GATA-1.05
heterozygous female mice. Significantly, both GATA-3 transgenic lines
were able to rescue GATA-1.05 hemizygotic males (GATA-3; Table
2). These results unequivocally demonstrated that GATA-2 and GATA-3,
when expressed under the regulatory control of the GATA-1
locus, could overcome the lethal phenotype caused by the GATA-1
germline loss of function mutation.
Compound mutant mice express transgene-derived GATA proteins
To determine the expression levels of transgene-derived GATA factors
in these rescued animals, RNA blot analysis was performed on RNAs
prepared from G1R, G2R, and G3R transgenic spleens. Transcripts derived
from the GATA-1, -2, or -3 transgenes were abundant in the spleens of
all compound mutant mice (Figure 3A). As
expected, endogenous GATA-1 RNA (G1-end) was not detected in the G2R or G3R mice.
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| Fig 3.
Expression of transgene in each rescued mouse.
(A) RNA blot analyses of rescued GATA-1.05 compound mutant
mice. Total cellular RNA was extracted from the spleens of G1R (line
No. 782), G2R (line No. 620), G3R (line No. 390), or WT mice. The
transcript sizes of endogenous and transgenic RNA transcripts are as
follows: endogenous GATA-1 (G1-end) is 1.9 kilobases (kb); transgene
(G1 Tg) of GATA-1 is 2.1 kb; endogenous GATA-2 (G2-end) is 3.5 and 2.9 kb; transgene (G2 Tg) of GATA-2 is 2.3 kb; transgene of GATA-3 (G3 Tg)
is 2.7 kb. A  -actin probe was used as the internal control. (B, C)
Western blot analysis of G1R or G2R lines. To examine protein levels of
transgene-derived GATA factors, splenic nuclear extracts prepared from
various transgenic lines (indicated above the lanes) were subjected to
Western blot analysis using anti-GATA-1 N6 monoclonal antibody (B) or
anti-GATA-2 RC1.1 monoclonal antibody (C). For controls, nuclear
extract from MEL cells (+) or IE3.9int-GFP mouse spleen was used. (D)
EMSA of transgenic GATA factors. Radiolabeled GATA probe was incubated
without (lane 1) or with splenic nuclear extracts from a WT mouse
(lanes 2-3), G1R line No. 782 (lanes 4-6), G2R line No. 620 (lanes
7-9), and G3R line No. 390 (lanes 10-12) in the presence of cold GATA
(lanes 3, 5, 8, 11) or mutated GATA (lanes 6, 9, 12) oligonucleotides.
G1R and G3R were similar to WT, but G2R was significantly higher than
WT.
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To determine the protein level in each transgenic line, we
performed Western blot analysis on splenic extracts of each
GATA-1 or GATA-2 transgenic line. As evident in Figures 3B and 3C, the protein levels varied among the lines; however, all lines could reverse the lethal phenotype of GATA-1.05 mutation. G2R-line
620 expressed GATA-2 protein more than 20-fold higher than G2R-line 625. In the case of GATA-3 transgenic lines, transgene-derived mRNA
levels in both lines were almost identical (data not shown).
The DNA binding activity of the transgene-derived GATA factors was
examined by electrophoretic mobility shift analysis (EMSA) using spleen
nuclear extracts of the compound G1R, G2R, and G3R mutant animals
(Figure 3D). The GATA motif binding activity in G1R or G3R spleens was
comparable to that detected in WT spleens (G1R, 146; G3R, 243; and WT,
145 arbitrary densitometric units, respectively). In the case of G2R,
the GATA motif binding activity was 10-fold greater (1430 units) than
that in the WT spleens, probably because the protein extract was
prepared from the highest GATA-2-expressing line, line No. 620 (Figure
3C). Because the affinity of all 3 GATA factors for a GATA-1-preferred
binding site is similar,24 this experiment demonstrates
that the transgene-derived GATA protein was either similar in abundance
to (ie, GATA-3) or 10 times more than (ie, GATA-2) endogenous GATA-1
protein levels.
Hematopoietic colony activity in G2R mice
As an independent measure of whether hematopoiesis was normal in the
G2R newborns, we conducted in vitro progenitor colony assays. The
colony-forming activity of G2R newborns was found to be almost normal
when compared with that of WT littermates (Table
3). Because we had previously shown that
the CFU-E activity of GATA-1.05 hemizygous embryos at E11.5 was
less than 5% of that of the WT embryos,8 this result
indicated that the CFU-E activity of G2R newborns had been fully
restored. Interestingly, there were more BFU-E colonies in the spleen
cultures of animals carrying the GATA-2 transgene than in WT newborns
(31 ± 5.2 colonies/105 vs 12 ± 6.5
colonies/105 spleen cells, respectively). Thus, the GATA-2
transgene fully restored hematopoietic colony-forming activity in the
GATA-1.05 genetic background.
G2R and G3R mice display abnormal adult hematopoiesis
To determine whether the G2R or G3R mice exhibited adult phenotypes,
erythropoiesis was examined. While there were no significant differences in the hematocrits of WT mice with or without transgene copies, there was a severe decrease of the hematocrit in GATA-2 and
GATA-3 rescued mice. The hematocrits of both compound mutant mice were
low, and the severity of the consequent anemia in the G3R was more
apparent than in that of G2R mice (Figure
4). All rescued mice lived beyond 6 months,
and the mean hematocrit values of G2R and G3R were constant over that
time. Compared with the peripheral blood smears of G1R mice (Figure
5A), G2R and G3R mice displayed markedly
abnormal blood cell morphology. In G2R and G3R mice, nucleated red
blood cells as well as erythrocytes containing Howell-Jolly bodies and
polychromatic erythrocytes were observed (Figure 5B, C). Marked
reticulocytosis was observed in G2R and G3R mice (Figure 5E, F).
Anisocytosis was detected in scanning electron microscopic analysis of
G3R (Figure 5I). Peripheral blood cells from transgenic mice that had
no germline mutation displayed normal morphology (data not shown).
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| Fig 4.
Hematocrits of rescued mice.
Hematocrits (Ht) of WT mice carrying no (WT) or GATA-1 (G1 Tg), GATA-2
(G2 Tg), or GATA-3 (G3 Tg) transgenes and depicted alongside that of
compound mutant G1R (line No.782, open circle; No. 801, closed circle),
G2R (line No. 620, open circle; No. 625, closed circle; No. 99, gray
circle), and G3R (line No. 390, open circle; No. 820, closed circle)
adult mice. Each dot represents the Ht of an individual mouse. Ht
values of 1- to 2-month-old mice, 3- to 6-month-old mice, and 6- to
20-month-old mice are shown.
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| Fig 5.
Peripheral blood analysis of rescued GATA-1.05
mice.
Blood samples were obtained from 5-week-old mice. While peripheral
blood from a G1R line No. 782 mouse (A) contains normal red blood
cells, the peripheral blood from G2R line No. 620 (B) and G3R line No.
390 (C) contains nucleated red blood cells. Reticulocytosis is evident
in G2R (E) and G3R (F) but not in G1R (D). (G-I) Scanning electron
microscopy of peripheral red blood cells. Anisocytosis is observed in
G3R (I). Original magnifications in panels G, H, and I are
× 10 000, × 2300, and × 4300, respectively.
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Hemocytometric analysis revealed that G2R and G3R animals displayed
normocytic and normochromic anemia and thrombocytopenia, although white
blood cell counts were similar (Table 4).
In addition, there was no significant difference in peripheral blood
indices between WT mice with or without GATA transgene(s). These data strongly indicate that the phenotypes observed in GATA-2 or GATA-3 rescued mice are not due to overexpression of the GATA-2 or GATA-3 proteins or to silencing of transgene expression.
Hence, we concluded that the GATA-1 molecule specifies functions in
adult erythropoiesis that cannot be completely complemented by the
GATA-2 or GATA-3 proteins. The nature of these functions remains to be elucidated.
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Discussion |
In this study, we comprehensively evaluated the efficacy of
substituting other hematopoietic GATA factors for the erythroid functions of GATA-1 in vivo. G1R compound mutant males do not succumb
to embryonic lethality as do GATA-1.05 mutant males. This result provides compelling evidence that the IE3.9int genomic fragment
contains sequences that can elicit proper physiologic levels of
GATA-1 gene transcription. To our surprise, GATA-2 and GATA-3
transgenes could also fully compensate for the loss of GATA-1 function
in utero, resulting in normal embryonic and perinatal hematopoiesis. These data thus illustrate that while precise
transcriptional regulation of the GATA-1 gene is vital for
normal erythropoiesis, the identity of the GATA factor is
inconsequential for prenatal hematopoiesis.
Because we employed a GATA-1 knockdown mutant allele that
retained only 5% of the WT GATA-1 mRNA levels, it remained formally possible that residual endogenous GATA-1 activity could taint our
conclusions. However, based on the following lines of evidence, we
believe this to be unlikely. First, there was no induction above the
undetectable level of endogenous GATA-1 transcripts in the G2R
or G3R compound mutant mice. Second, GATA-1 was not detectable by
immunostaining in the fetal livers of G2R compound mutant embryos (data
not shown). Third, the GATA-1.05 mutant phenotype was not
rescued by the expression of a modified GATA-1 transgene that was
missing the C-finger, which is required for site-specific DNA binding.
Thus, the rescue of the GATA-1.05 germline mutation is
absolutely dependent on the expression of functional, DNA binding, transgene-derived GATA factor, and the residual level of endogenous GATA-1 in the GATA-1.05 mutant background never became
empirically significant.
Several precedents illustrate that related transcription factors are
often functionally interchangeable in vivo. Knock-in experiments in the
mouse have shown that myogenin can replace Myf5 in rib cage development
and that En-2 can replace En-1 in midbrain and hindbrain
development.25,26 These studies indicate that each locus
has acquired distinctive gene transcriptional regulatory modules that
are responsible for eliciting the unique spatial and temporal
expression profiles, and it is this attribute that makes each of these
factors indispensable in vivo.
In contrast to the present report, a human GATA-3 cDNA inserted into
the mouse GATA-1 locus showed incomplete rescue of the GATA-1-null lethal phenotype.27 Consistent with this
result, a murine GATA-2 cDNA inserted into the mouse GATA-1
locus also could not completely rescue erythroid differentiation in an
in vitro ES cell differentiation assay (our unpublished observations). We suggest that the molecular basis for the partial rescue in these
experiments is probably complex. It may be due, at least in part, to
insufficient accumulation of gene-targeted GATA protein compared with
the normal level of GATA-1. Indeed, we showed here that GATA-2 and
GATA-3 could rescue the GATA-1 knockdown mouse from embryonic lethality
when they were expressed under the regulatory influence of the
GATA-1 gene.
It should be noted that in the transgenic approach employed here, the
protein levels of transgenic GATA-2 and GATA-3 were similar to or
higher than the level of GATA-1 in WT erythroid cells. These results
suggest that GATA-2 and GATA-3 proteins or mRNAs may be subject to more
stringent posttranslational regulation than is GATA-1 in erythroid
cells. If so, a gene-targeted replacement strategy may not always be
most suitable for comparing the biological functions of related factors
in vivo.
The relative stability of GATA-1, -2, and -3 in erythroid cells may
well have biological significance. The mice rescued with the GATA-2 and
-3 transgenes suffered from anemia and abnormal hematopoiesis in
adulthood. Considering that, in the hematopoietic cells of these
transgenic mice, GATA-2 and GATA-3 were expressed at levels similar to
or higher than that of endogenous GATA-1 in WT mice and that the mean
hematocrit value of older G2R and G3R mice were constant during their
foreshortened lifespans, the anemic phenotype in adult animals is
probably a consequence of the distinct biochemical natures of the
individual GATA proteins.
In summary, transgenic expression of GATA-2 and GATA-3 under
GATA-1 transcriptional regulatory influence indeed rescued the GATA-1.05 mutant mouse from embryonic lethality, indicating
that precise transcriptional regulation of the GATA-1 gene is
most critical for its function in erythropoiesis. The assay system employed here is immediately applicable to the analysis of other transcription factor families and may prove to be especially useful for
evaluating the genuine contributions of presumed redundancy among
different family members and for revealing subtle functional differences among related members of protein families.
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Acknowledgments |
We would like to thank Drs Noriko Kajiwara, Fumihiro Sugiyama, Naomi
Kaneko, and Kenichi Yagami for help and discussion.
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Footnotes |
Submitted October 26, 1999; accepted March 24, 2000.
Supported in part by grants-in-aid from the Ministry of Education,
Science, Sports and Culture, the Japanese Society for Promotion of
Sciences (RFTF), Core Research for Evolutional Sciences and Technology,
and the National Institutes of Health (GM 28 896).
Reprints: Masayuki Yamamoto, Center for TARA and Institute of
Basic Medical Institute, University of Tsukuba, 1-1-1 Tennodai, Tsukuba
305-8577, Japan; e-mail: masi{at}tara.tsukuba.ac.jp.
The publication costs of this
article were defrayed in part by
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and solely to indicate this fact,
this article is hereby marked
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in accordance with 18 U.S.C.
section 1734.
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Top
Abstract
Introduction