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Transcription factor GATA-1 is essential for the development of the erythroid lineage. To ascertain whether strict control of GATA-1 expression level is necessary for achieving proper erythropoiesis, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of the GATA-1 gene hematopoietic regulatory domain. We examined the GATA-1 expression level by exploiting the transgenic mice and found 2 GFP-positive hematopoietic progenitor fractions in the bone marrow. One is the GFPhigh fraction containing mainly CFU-E and proerythroblasts, which coexpress transferrin receptor, while the other is the GFPlow/transferrin receptor-negative fraction containing BFU-E. Since the intensity of green fluorescence correlates well with the expression level of GATA-1, these results indicate that GATA-1 is highly expressed in erythroid colony-forming unit (CFU-E) but low in erythroid burst-forming unit (BFU-E), suggesting that the incremental expression of GATA-1 is required for the formation of erythroid progenitors. We also examined GFP-positive fractions in the transgenic mouse spleen and fetal liver and identified fractions containing BFU-E and CFU-E, respectively. This study also presents an efficient method for enriching the CFU-E and BFU-E from mouse hematopoietic tissues. (Blood. 2003;102:3575-3583)

Alas2 encodes the erythroid-specific δ-aminolevulinate synthase (ALAS2 or ALAS-E), the first enzyme in heme biosynthesis in erythroid cells. Mice with theAlas2-null phenotype showed massive cytoplasmic, but not mitochondrial, iron accumulation in their primitive erythroblasts. Because these animals died by day 11.5 in utero, studies of iron metabolism in definitive erythroblasts were not possible using the in vivo model. In this study, embryonic stem (ES) cells lacking theAlas2 gene were induced to undergo differentiation to the definitive erythroblast stage in culture, and the phenotype ofAlas2-null definitive erythroblasts was examined.Alas2-null definitive erythroblasts cell pellets were entirely colorless due to a marked deficiency of heme, although their cell morphology was similar to that of the wild-type erythroblasts. The level of expression of erythroid-specific genes inAlas2-null definitive erythroblasts was also similar to that of the wild-type erythroblasts. These findings indicate thatAlas2-null definitive erythroblasts developed to a stage similar to that of the wild-type erythroblasts, which were also shown to be very similar to the bone marrow erythroblasts in vivo. In contrast, Alas2-null definitive erythroblasts contained 15 times more nonheme iron than did the wild-type erythroblasts, and electron microscopy found this iron to be distributed in the cytoplasm but not in mitochondria. Consistent with the aberrant increase in iron,Alas2-null definitive erythroblasts were more peroxidized than wild-type erythroblasts. These findings suggest that ALAS2 deficiency itself does not interfere with the development of definitive erythroid cells, but it results in a profound iron accumulation and a peroxidized state in erythroblasts.

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-1gene. 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.

Gene targeting experiments have revealed that transcription factors such as c-Myb and GATA-1 play crucial roles during hematopoietic differentiation. c-Myb is necessary in the immature cells of almost every hematopoietic lineage and GATA-1 is essential for the development of the erythroid lineage. In addition, CREB-binding protein (CBP) acts as a transcriptional adapter for various transcription factors, including c-Myb and GATA-1. In this paper, we show that the transcription factors c-Myb and GATA-1 each inhibit the transcriptional activity of the other and that any possible bipartite complexes c-Myb, GATA-1, and CBP could be formed, but the tripartite complex was hardly formed. The exclusive binding of GATA-1 and c-Myb to CBP is probably the molecular basis for the mutual inhibition of their transcriptional activity. Our data suggest that cross-talk between these three factors might be important for hematopoietic differentiation and that CBP functions as a key molecule during the process.

Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

The human beta-globin locus control region (LCR) is required to properly regulate chromatin domain opening, replication timing, and globin gene activation. The LCR contains multiple NF-E2 sites (Maf recognition elements, MAREs) that allow the binding of various basic leucine zipper (bZip) proteins like p45 NF-E2, Nrf1, Nrf2, Bach1, and Bach2, in some cases as obligate heterodimers with a small Maf protein. In addition to the bZip domain, the Bach proteins bear a BTB/POZ domain, which has been implicated in the regulation of chromatin structure. We show here that Bach1 is highly expressed in hematopoietic cells and constitutes one of the two MARE-binding activities in murine erythroleukemic (MEL) cells. We further demonstrate that Bach1/MafK heterodimers interact with each other through the BTB domain, generating a multimeric and multivalent DNA binding complex. These results strongly implicate Bach1/MafK heterodimer as an architectural transcription factor that mediates interactions among multiple MAREs. Such a factor could then provide a model for assembly of the theoretical beta-globin LCR "holocomplex. " Other BTB domain proteins have already been demonstrated to be involved in remodeling chromatin, and thus this class of proteins likely promote the formation of nucleoprotein complexes required to establish the architecture of regulatory domains.

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 themouse 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 −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.

The erythroid-specific isoform of δ-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells, and ALAS-E gene mutations are known to be responsible for x-linked sideroblastic anemia. To study the role of ALAS-E in erythroid development, we prepared mouse embryonic stem (ES) cells carrying a disrupted ALAS-E gene and examined the effect of the lack of ALAS-E gene expression on erythroid differentiation. We found that mRNAs for erythroid transcription factors and TER119-positive cells were increased similarly both in the wild-type and mutant cells. In contrast, heme content, the number of benzidine-positive cells, adult globin protein, and mRNA for β-major globin were significantly decreased in the mutant cells. These results were confirmed using another ES differentiation system in vitro and suggest that ALAS-E expression, hence heme supply, is critical for the late stage of erythroid cell differentiation, which involves hemoglobin synthesis.

To elucidate the in vivo function of GATA-1 during hematopoiesis, we specifically disrupted the erythroid promoter of the GATA-1 gene in embryonic stem cells and generated germ line chimeras. Male offspring of chimeras bearing the targeted mutation were found to die by 12.5 days post coitus due to severe anemia while heterozygous females displayed characteristics ranging from severe anemia to normal erythropoiesis. When female heterozygotes were crossed with transgenic males carrying a reporter gene, which specifically marks primitive erythroid progenitors, massive accumulation of undifferentiated erythroid cells were observed in the yolk sacs of the GATA-1-mutant embryos, demonstrating that GATA-1 is required for the terminal differentiation of primitive erythroid cells in vivo.

To understand the regulatory mechanism of erythropoietin (EPO) receptor (EPOR) gene expression, the effect of EPO on the steady-state level of EPOR mRNA was examined using the human EPO-dependent cell line UT-7 as a model system. We found that the treatment of UT-7 cells with EPO resulted in a transient decrease of the EPOR mRNA level. This transient downregulation was also induced by stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF ), another stimulator of UT-7 cell growth. These results raised the possibility that EPOR gene expression is in part related to cell growth. Moreover, it was found that EPO-induced downregulation of EPOR mRNA level was preceded by a transient downregulation of GATA-1 mRNA. To examine the relationship between the expression of EPOR, GATA-1, and GATA-2 mRNA levels and the cell cycle, logarithmically growing UT-7 cells were centrifugically fractionated according to the cell-cycle phase. Both EPOR and GATA-1 mRNA levels, but not the GATA-2 mRNA level, concomitantly decreased at the G0/G1 phase and increased at the S and G2/M phases. An electrophoretic mobility shift assay (EMSA) showed that in EPO-stimulated UT-7 cells, the dynamic changes in EPOR gene expression paralleled the GATA-1 DNA-binding activity to the oligonucleotide probe containing a GATA-binding site located at the promoter region of the EPOR gene. These findings suggest that the regulation of EPOR mRNA level is mainly associated with GATA-1 gene expression in UT-7 cells undergoing proliferation, and that these serial events are under the control of, or related to, the cell cycle.

Transcription factor GATA-1 was first identified in erythroid cells, but was later shown to also be expressed in Sertoli cells of the mouse testis. GATA-1 transcription in testis initiates from a different first exon (exon IT) than the erythroid mRNA (transcribed from exon IE). To begin to address the question of how expression of GATA-1 might be differentially regulated in Sertoli and erythroid cells, we have cloned and determined the structure of the IT promoters of both the rat and mouse GATA-1 genes. The transcription regulatory mechanism(s) controlling the synthesis of exon IT-derived mRNA was investigated by transfection of wild-type and mutant reporter genes, with and without co-transfected GATA factor expression plasmids, into either fibroblasts or Sertoli cell lines. Two GATA binding sites in the IT promoter were found to be required for GATA factor-mediated activation in fibroblasts: GATA-IT-directed reporter gene expression was activated only after co-transfection with GATA-1, implying that transcriptional activation of GATA-1 in the testis might be at least partially mediated through these GATA regulatory elements. We also found that the endogenous GATA-1 gene was silent in primary culture and two different Sertoli cell lines, and that the repression of co-transfected GATA-1 reporter genes could not be relieved by forced expression of GATA-1 in Sertoli cells. Thus the GATA-IT promoter may be under the control of a regulatory network in Sertoli cells which involves both positive and negative regulation of transcription, and conserved GATA motifs found in the IT promoter may be required for transducing these effects.

Phenotypic characteristics of blasts were studied in a Down's infant with transient abnormal myelopoiesis (TAM). Two major subpopulations were identified: (1) CD33+CD42b+ cells with platelet peroxidase activity, the commitment of which to megakaryocytic lineage was supported by an increased expression of GATA-1 mRNA; (2) CD33+CD34+CD7+CD4+ cells with immature ultrastructure, which could be either immature megakaryocytic or myeloid cells with aberrant differentiation. Mixed colonies containing megakaryocytes and monocyte/macrophages in the peripheral blood suggested the presence of progenitors common to these subpopulations. These results may indicate that subpopulations of blasts with phenotypic diversity could be derived from aberrant common progenitors to megakaryocytic and myeloid lineages in this patient.

We retrospectively investigated the feasibility of the apheresis procedure for red blood cell (RBC) reduction with a closed-bag system. We also sought to determine the optimal processing volume for the maximal recovery of hematopoietic progenitor cells (HPC). Twelve bone marrow (BM) harvests were processed for major ABO-incompatible allogeneic transplantation and one BM harvest was processed for autologous transplantation. The processing was performed through seven apheresis cycles with a two-bag system using COBE Spectra Version 6.1. The mean recovery rates were compared in the products after four cycles and seven cycles of BM processing. Mean cell recovery rates were 79.2% (67.6–97.5%) and 87.3% (68.9–111.9%) for the mononuclear cells (MNC) and 84.5% (69.4–109.5%) and 92.0% (79.0–107.7%) for the CD34+ cells after four and seven cycles, respectively. A mean of 96.3% (93.0–98.1%) of the RBCs were finally removed. The yield of CD34+ cells after seven cycles of processing (median: 10.35 × 107 cells) was 7.9% greater than that after four cycles of processing (median: 9.65 × 107 cells), exhibiting a less-than-significant enhancement in yield. The CD34+ cell contents recovered in the concentrates up to four cycles (r = 0.989) and up to seven cycles (r = 0.993) were strongly correlated with the original content of the CD34+ cells. Engraftment was obtained in all patients except one patient infused with purified CD34+ cells. This latter result confirmed the hematopoietic potential of the cell populations recovered. Granulocyte recovery (defined as an absolute neutrophil cell count ≧ 500/μL for a period of three consecutive days) ranged from 8 to 25 days (median: 16 days) post-transplantation. No hemolytic reaction was observed in any of the patients. Our results confirmed the efficacy of BM processing cycles with the COBE Spectra device. However, we could not conclude that the large-volume apheresis for BM processing significantly enhanced the yields of HPC. The final recovery of CD34+ cells after processing could be predicted from the CD34+ cell content of the original collected marrow. J. Clin. Apheresis 2006. © 2006 Wiley-Liss, Inc.

Transcription factor GATA-1 plays an important role in gene regulation during the development of erythroid cells. Several reports suggest that GATA-1 plays multiple roles in survival, proliferation, and differentiation of erythroid cells. However, little is known about the relationship between the level of GATA-1 expression and its nature of multifunction to affect erythroid cell fate. To address this issue, we developed in vitro embryonic stem (ES) culture system by using OP9 stromal cells (OP9/ES cell co-culture system), and cultured the mutant (GATA-1.05 and GATA-1-null) and wild type (WT)ES cells, respectively. By using this OP9/ES cell co-culture system, primitive and definitive erythroid cells were developed individually, and we examined how expression level of GATA-1 affects the development of erythroid cells. GATA-1.05 ES-derived definitive erythroid cells were immature with the appearance of proerythroblasts, and highly proliferated, compared with WT and GATA-1-null ES-derived erythroid cells. Extensive studies of cell cycle kinetics revealed that the GATA-1.05 proerythroblasts accumulated in S phase and expressed lower levels of p16(INK4A) than WT ES cell-derived proerythroblasts. We concluded that GATA-1 must achieve a critical threshold activity to achieve selective activation of specific target genes, thereby influencing the developmental decision of an erythroid progenitor cell to undergo apoptosis, proliferation, or terminal differentiation.

Members of the small Maf family of transcription factors play important roles in hematopoiesis. Using transgenic assays, we discovered a tissue-specific enhancer 3′ to the mafK gene. This enhancer directs mafK transcription in hematopoietic as well as in developing cardiac muscle cells, and was thus designated the hematopoietic and cardiac enhancer of mafK (HCEK). Only two of four GATA consensus motifs identified within HCEK contributed to enhancer activity, and both of these sites were required for both cardiac and hematopoietic transcriptional activation. The expression profile of MafK significantly overlapped that of GATA-1 in hematopoietic cells and of GATA-4/-6 in cardiac tissues. Each of these GATA factors bound with high specificity to both of the critical GATA sites in HCEK. Hence, the mafK gene is regulated by different GATA proteins in the hematopoietic and cardiac compartments through the same two GATA-binding sites in HCEK. These data provide the first in vivo demonstration that distinct members of a related transcription factor family activate the tissue-specific expression of a single target gene using the same cis-regulatory element.

A region located at kbp -3.9 to -2.6 5' to the first hematopoietic exon of the GATA-1 gene is necessary to recapitulate gene expression in both the primitive and definitive erythroid lineages. In transfection analyses, this region activated reporter gene expression from an artificial promoter in a position- and orientation-independent manner, indicating that the region functions as the GATA-1 gene hematopoietic enhancer (G1HE). However, when analyzed in transgenic embryos in vivo, G1HE activity was orientation dependent and also required the presence of the endogenous GATA-1 gene hematopoietic promoter. To define the boundaries of G1HE, a series of deletion constructs were prepared and tested in transfection and transgenic mice analyses. We show that G1HE contains a 149-bp core region which is critical for GATA-1 gene expression in both primitive and definitive erythroid cells but that expression in megakaryocytes requires the core plus additional sequences from G1HE. This core region contains one GATA, one GAT, and two E boxes. Mutational analyses revealed that only the GATA box is critical for gene-regulatory activity. Importantly, G1HE was active in SCL(-/-) embryos. These results thus demonstrate the presence of a critical network of GATA factors and GATA binding sites that controls the expression of this gene.

The difficulty and uncertainty encountered in diagnosing a systemic mycosis often lead to a delay in starting antifungal therapy. We reported a disseminated infection of multiple fungal isolates including Fusarium species during donor leukocyte transfusion (DLT) after allogeneic bone marrow transplantation in a 20-year-old woman with a refractory leukemia. Skin lesions are the feature of Fusarium and occur in the early period of the infection. In this case, during immunosuppression state after DLT, she presented with the whole body ache and erythematous lesions which appeared rapidly on her trunk and extremities. While administration of amphotericin B was started, her condition was further deteriorated and she died. Autopsy materials revealed that she had multiple fungal infection with different isolates, including Aspergillus and Candida in the brain, lung and liver, but not in the skin. With the immunohistochemical staining with specific antibody, Fusarium or Aspergillus infection was identified from the biopsy skin or autopsy brain, respectively. This rapid and specific immunohistochemical method may be useful for the diagnosis and treatment of invasive fungal infection without delay.

To elucidate the contributions of GATA-1 to definitive hematopoiesis in vivo, we have examined adult mice that were rendered genetically defective in GATA-1 synthesis (Takahashi et al, J Biol Chem272:12611, 1997). Because the GATA-1 gene is located on the X chromosome, which is randomly inactivated in every cell, heterozygous females can bear either an active wild-type or mutant (referred to asGATA-1.05) GATA-1 allele, consequently leading to variable anemic severity. These heterozygous mutant mice usually developed normally, but they began to die after 5 months. These affected animals displayed marked splenomegaly, anemia, and thrombocytopenia. Proerythroblasts and megakaryocytes massively accumulated in the spleens of the heterozygotes, and we showed that the neomycin resistance gene (which is the positive selection marker in ES cells) was expressed profusely in the abnormally abundant cells generated in the GATA-1.05 mutant females. We also observed hematopoiesis outside of the bone marrow in the affected mutant mice. These data suggest that a small number of GATA-1.05 mutant hematopoietic progenitor cells begin to proliferate vigorously during early adulthood, but because the cells are unable to terminally differentiate, this leads to progenitor proliferation in the spleen and consequently death. Thus, GATA-1 plays important in vivo roles for directing definitive hematopoietic progenitors to differentiate along both the erythroid and megakaryocytic pathways. The GATA-1 heterozygous mutant mouse shows a phenotype that is analogous to human myelodysplastic syndrome and thus may serve as a useful model for this disorder.

Background While mast cells have been previously shown to express both GATA-1 and GATA-2 mRNAs, individual functions for these related factors during their course of differentiation within the mast cell lineage have not yet been defined. To address this question, the expression of GATA-1 and GATA-2 mRNAs and proteins were examined in three mouse mast cell progenitor lines as well as in mast cells isolated from both wild-type and GATA-1-deficient mice. Results Both mast cell progenitor lines, as well as primary mouse bone marrow-derived mast cells (BMMCs) and peritoneal mast cells (PMCs) were examined by RNA blotting and immunological analyses. GATA-2 protein was abundantly expressed in all three mast cell lines and in BMMCs, but only weakly in some of PMCs. In contrast, GATA-1 protein was expressed in PMCs and BMMCs after culture in the presence of IL-3 and SCF. We also found the presence of Alcian blue staining-positive but berberine staining-negative mast cells in the skin of mice heterozygous to GATA-1 knock-down allele. Conclusion These results suggest that the expression of GATA factor-dependent genes is regulated by GATA-2 during mast cell development and that GATA-1 is required for the specification of differentiated mast cell phenotypes.

GATA-1 protein is thought to be a positive regulator of erythroid differentiation. However, ectopic expression of a conditional GATA- 2/estrogen receptor chimera was shown to inhibit erythroid differentiation in a hormone-dependent manner, suggesting the negative regulation of erythroid differentiation by GATA-2 protein. Accordingly, we reasoned that the quantitative balance of GATA-1 and GATA-2 protein might affect erythroid differentiation. In this report, we performed specific and quantitative measurements of GATA-1 and GATA-2 protein in a new erythroid cell line, SAM-1, after treatment with 12-O- tetradecanoylphorbol 13-acetate (TPA). On the basis of these measurements, we show that TPA-induced arrest of erythroid differentiation is coupled with the upregulation of GATA-2 protein, as well as the downregulation of GATA-1 protein. Our results suggest that it is the precise quantitative balance of GATA-1 and GATA-2 protein that regulates erythroid differentiation.