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. 2002 Jun 17;21(12):3060-9.
doi: 10.1093/emboj/cdf301.

GATA-2 and GATA-2/ER display opposing activities in the development and differentiation of blood progenitors

Kenji Kitajima  1 , Masaaki Masuhara, Takumi Era, Tariq Enver, Toru Nakano
Affiliations

GATA-2 and GATA-2/ER display opposing activities in the development and differentiation of blood progenitors

Kenji Kitajima et al. EMBO J. .

Abstract

GATA-2 is a zinc finger transcription factor essential for the development of hematopoiesis. While GATA-2 is generally considered to play an important role in the biology of hematopoietic stem and progenitor cells, its function within these compartments is not well understood. Here we have employed both conditional expression of GATA-2 and conditional activation of a GATA-2/estrogen receptor (ER) chimera to examine the effect of enforced GATA-2 expression in the development and differentiation of hematopoietic progenitors from murine embryonic stem cells. Consistent with the phenotype of GATA-2 null animals, conditional expression of GATA-2 from a tetracycline-inducible promoter enhanced the production of hematopoietic progenitors. Conditional activation of a GATA-2/ER chimera produced essentially opposite effects to those observed with conditional GATA-2 expression. GATA-2 and GATA-2/ER differ in their binding activities and transcriptional interactions from other hematopoietic-associated transcription factors such as c-Myb and PU.1. While we have exploited these differences in activity to explore the transcriptional networks underlying hematopoietic cell fate determination, our results suggest that care should be taken in interpreting results obtained using only chimeric proteins.

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Figures

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Fig. 1. Conditional expression of GATA-2 and EGFP. (A) Constructs of GATA-2/IRES–EGFP and control IRES–EGFP. TetO stands for the tetracycline-responsive element. (B and C) FACS and western blot analyses of the time course of EGFP and GATA-2 expression in ES cells after being deprived of tetracycline. (D) Morphological change of ES cells by depriving them of tetracycline. (E) Expression of primitive endoderm differentiation markers, HNF-4 and collagen type IV, 4 days after the expression of GATA-2, GATA-1 and activated GATA-2/ER. Expression of GATA-2 and GATA-1 was induced by depriving the cells of tetracycline. The activated GATA-2/ER was obtained by depriving the cells of tetracycline and by the addition of β-estradiol (βE). The primer set for collagen type IV was used for the RT(–) control. (F) Expression of EGFP in TER-119-expressing erythroid cells induced from ES cells. The cells were deprived of tetracycline from day 5 of induction and the data were obtained 3 days later.
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Fig. 2. Effect of induced GATA-2 on immature hematopoietic colony formation at day 7 of induction. A total of 105 day 5 induced cells were seeded onto the OP9 stroma cell layer and the cells were cultured in the presence or absence of tetracycline. All the analyses were carried out at day 7 of induction. (A) Relative number of colonies in control and Tet-regulated GATA-2 clones. Data are shown as the mean ± SD of six samples (*P < 0.01 by t-test). (B) Numbers of cells in individual colonies in the presence or absence of tetracycline. GATA-2 increased the number of cells significantly (*P < 0.01 by t-test). (C) Numbers of colonies in the culture containing various concentrations of tetracycline. (D) Typical hematopoietic colonies in the presence (left panels) or absence (right panels) of tetracycline. A colony grown underneath the OP9 stroma cells with a cobble stone appearance (pseudoemperipolesis) is shown in the right lower panel. (E) Number of colonies showing pseudoemperipolesis. Data are shown as the mean ± SD of six samples (*P < 0.01 by t-test).
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Fig. 3. Number of hematopoietic colonies at day 15 and surface marker profile of day 18 induced cells. (A) Hematopoietic colonies at day 15 of induction. Data are shown as the mean ± SD of six samples (*P < 0.01 by t-test). (B) c-Kit and Sca-1 expression on the cells persistent at day 18 of induction with the expression of GATA-2. The majority of EGFP-positive cells expressed c-Kit and Sca-1, markers of immature hematopoietic cells.
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Fig. 4. Numbers and lineage marker profile of the cells induced from ES cells in the presence of EPO or TPO. (A–C) Numbers of cells at days 8 and 11 in the presence of EPO or TPO as described. GATA-2 expression increased the numbers of cell significantly (*P < 0.01 by t-test). Data are shown as the mean ± SD of six samples. (D–F) Surface expression of lineage markers TER-119 and platelet GPV.
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Fig. 5. ‘Lineage switch’ from macrophage to megakaryocyte by GATA-2. (A) Morphology of the cells at day 9 of induction in the presence of M-CSF. The characteristics of macrophages were lost in the absence of tetracycline. (B and C) Expression of Mac-1 and platelet GPV in the presence or absence of tetracycline. (D) Cell numbers of control (P > 0.05 by t-test) and GATA-2 clones (*P < 0.01) in the presence or absence of tetracycline at day 9 of induction with M-CSF. (E) Western blot analysis of GATA-1, PU.1 and GATA-2 of control E14tg2a ES cells and GATA-2 clones in the presence or absence of tetracycline.
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Fig. 6. Effects of GATA-1 and the activated GATA-2/ER on hematopoietic colony formation and differentiation. (A) Effects on day 7 colony formation. A significant reduction in the cell number by the enforced expression of GATA-2/ER was observed in the presence of β-estradiol (*P < 0.01 by t-test). (B) Effects of GATA-1 and the activated GATA-2/ER on erythrocyte production in the presence of EPO. GATA-1 and the activated GATA-2/ER gave rise to a significant increase and decrease of the erythroid lineage cells, respectively (*P < 0.01, #P < 0.05 by t-test). Data are shown as the mean ± SD of six samples. (C) Western blot analyses of GATA-2 and GATA-2/ER in the day 8 differentiation-induced cells. (D and E) Expression of Mac-1 and platelet GPV at day 9 of culture in the presence of M-CSF. (F) Expression of megakaryocyte-specific acetylcholine esterase by overexpression of GATA-1 and GATA-2.
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Fig. 7. Differential binding activity and transcriptional activity of GATA-2 and the activated GATA-2/ER. (A) Co-immunoprecipitation analysis of c-Myb and PU.1. 293T cells were transfected with expression plasmids of FLAG-GATA-2 and c-Myb (lane 1), FLAG-GATA-2/ER and c-Myb (lane 2), FLAG-GATA-2 and PU.1 (lane 3) and FLAG-GATA-2/ER and PU.1 (lane 4). β-estradiol was added in the experiments of FLAG-GATA-2/ER (lanes 2 and 4); however, the data were essentially the same in the absence of β-estradiol (data not shown). Total cell lysates were immunoprecipitated with anti-FLAG antibody. The precipitated protein was electrophoresed by SDS–PAGE, blotted and hybridized with the anti-FLAG antibody (upper panels), anti- c-Myb antibody (lower panels of lanes 1 and 2) and anti-PU.1 antibody (lower panels of lanes 3 and 4). (B) Reporter gene analysis of various promoters. Reporter luciferase genes are as described in each panel. G2 and ER stand for the transfection of the GATA-2-expressing plasmid and the GATA-2/ER-expressing plasmid, respectively. When the GATA-2/ER expression plasmid was transfected, β-estradiol was added to activate the protein. An 8 µg aliquot of individual expression plasmids and reporter gene plasmids was transfected into CV-1 cells. Data are shown as the mean ± SD of nine samples (*P < 0.01; #P < 0.05 by t-test).
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