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PubMed1999

Use of altered specificity mutants to probe a specific protein-protein interaction in differentiation: the GATA-1:FOG complex.

GATA-1 and FOG (Friend of GATA-1) are each essential for erythroid and megakaryocyte development. FOG, a zinc finger protein, interacts with the amino (N) finger of GATA-1 and cooperates with GATA-1 to promote differentiation. To determine whether this interaction is critical for GATA-1 action, we selected GATA-1 mutants in yeast that fail to interact with FOG but retain normal DNA binding, as well a compensatory FOG mutant that restores interaction. These novel GATA-1 mutants do not promote erythroid differentiation of GATA-1- erythroid cells. Differentiation is rescued by the second-site FOG mutant. Thus, interaction of FOG with GATA-1 is essential for the function of GATA-1 in erythroid differentiation. These findings provide a paradigm for dissecting protein-protein associations involved in mammalian development.

Amino Acid Sequence↗
PubMed1991

Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1.

The zinc-finger transcription factor GATA-1 (previously known as GF-1, NF-E1 or Eryf 1 binds to GATA consensus elements in regulatory regions of the alpha- and beta-globin gene clusters and other erythroid cell-specific genes. Analysis of the effects of mutations in GATA-binding sites in cell culture and in binding assays in vitro, as well as transactivation studies with GATA-1 expression vectors in heterologous cells, have provided indirect evidence that this factor is involved in the activation of globin and other genes during erythroid cell maturation. GATA-1 is also expressed in megakaryocytes and mast cells, but not in other blood cell lineages or in non-haemopoietic cells. To investigate the role of this factor in haematopoiesis in vivo, we disrupted the X-linked GATA-1 gene by homologous recombination in a male (XY) murine embryonic stem cell line and tested the GATA-1-deficient cells for their ability to contribute to different tissues in chimaeric mice. The mutant embryonic stem cells contributed to all non-haemopoietic tissues tested and to a white blood cell fraction, but failed to give rise to mature red blood cells. This demonstrates that GATA-1 is required for the normal differentiation of erythroid cells, and that other GATA-binding proteins cannot compensate for its absence.

Animals↗
PubMed1995

Regulation of transcription of the human erythropoietin receptor gene by proteins binding to GATA-1 and Sp1 motifs.

Erythropoietin (Epo), the primary regulator of the production of erythroid cells, acts by binding to a cell surface receptor (EpoR) on erythroid progenitors. We used deletion analysis and transfection assays with reporter gene constructs to examine the transcription control elements in the 5' flanking region of the human EpoR gene. In erythroid cells most of the transcription activity was contained in a 150 bp promoter fragment with binding sites for transcription factors AP2, Sp1 and the erythroid-specific GATA-1. The 150 bp hEpoR promoter exhibited high and low activity in erythroid OCIM1 and K562 cells, respectively, reflecting the high and low levels of constitutive hEpoR expression. The GATA-1 and Sp1 binding sites in this promoter lacking a TATA sequence were necessary for a high level of transcription activation. Protein-DNA binding studies suggested that Sp1 and two other CCGCCC binding proteins from erythroid and non-erythroid cells could bind to the Sp1 binding motif. By increasing GATA-1 levels via co-transfection, we were able to transactivate the hEpoR promoter in K562 cells and non-erythroid cells, but not in the highly active OCIM1 cells, although GATA-1 mRNA levels were comparable in OCIM1 and K562. Interestingly, when we mutated the Sp1 site, resulting in a marked decrease in hEpoR promoter activity, we could restore transactivation by increasing GATA-1 levels in OCIM1 cells. These data suggest that while GATA-1 can transactivate the EpoR promoter, the level of hEpoR gene expression does not depend on GATA-1 alone. Rather, hEpoR transcription activity depends on coordination between Sp1 and GATA-1 with other cell-specific factors, including possibly other Sp1-like binding proteins, to provide high level, tissue-specific expression.

Cell Line↗
PubMed1995

Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor.

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

Animals↗
PubMed1995

Hemoglobin switching in humans is accompanied by changes in the ratio of the transcription factors, GATA-1 and SP1.

BACKGROUND: Understanding the mechanism of developmental regulation of hemoglobin switching has scientific as well as clinical relevance because of the influence of fetal hemoglobin (HbF) production in adulthood on the clinical manifestation of thalassemia and sickle cell anemia. We have previously found that the normal developmental patterns of globin gene expression are recapitulated in an experimental system of primary cultures that support differentiation of erythroid progenitors. We further found that high activities of the transcriptional activators, GATA-1 and SP1, are associated with normal adult erythroid differentiation. MATERIALS AND METHODS: In the present work, we have studied, the activities of GATA-1 and SP1 during differentiation of cultured erythroid progenitors derived from cord blood and from fetal livers, as well as from beta zero-thalassemia patients. RESULTS: The results showed high GATA-1 binding activity and very low SP1 activity in the fetal liver cultures. This pattern was in contrast to cultures derived from normal adult peripheral blood, in which both GATA-1 and SP1 activities were high. Cord blood cultures showed an additive combination of "adult" and "fetal" patterns. The progenitors derived from a beta zero-thalassemia patient with high HbF production showed "fetal" pattern. On the other hand, in cultures of 2 beta zero-thalassemia patients without high HbF, "adult" pattern was observed. CONCLUSIONS: In the present work, we show that human fetal and adult erythroid progenitors are distinct in their transcription factors, and that the commitment to fetal or adult program occurs at a very early differentiation stage. Our studies also demonstrate that under anemic stress, recruitment of fetal progenitors may occur in adulthood.

Adult↗
PubMed1996

Transcriptional and posttranscriptional regulation of erythroid gene expression in anthracycline-induced differentiation of human erythroleukemic cells.

Aclacinomycin (ACLA) and doxorubicin (DOX) were used at subtoxic concentrations to induce erythroid differentiation in the human leukemic cell line K562. Cell hemoglobinization was accompanied by the increased expression of genes encoding gamma-globin and porphobilinogen deaminase (PBGD), an enzyme of heme synthesis. By using run-on assays, ACLA was shown to induce an enhancement of the transcription of erythroid genes, including gamma-globin, PBGD, erythropoietin receptor, and GATA-1 transcription factor. In contrast, in DOX-treated cells, the transcription rate of these genes was unchanged in comparison with control cells. In addition, inhibition of mRNA synthesis with actinomycin D indicated that DOX induced an increased stability of PBGD and GATA-1 mRNAs, whereas ACLA did not affect the half-lives of these mRNAs. Because the increase in erythroid mRNA steady-state level in anthracycline-treated cells was inhibited by cycloheximide, this suggests that transcriptional activation in ACLA-treated cells and mRNA stabilization in DOX-treated cells were dependent on de novo protein synthesis. Finally, GATA-1 protein level was shown to be increased in ACLA-treated but not in DOX-treated cells. These two anthracyclines, although closely related in their structures, appeared to act as differentiation inducers by distinct mechanisms. Indeed, erythroid gene expression was demonstrated to be regulated transcriptionally by ACLA and mainly posttranscriptionally by DOX.

Aclarubicin↗
PubMed1991

Human erythroid 5-aminolevulinate synthase: promoter analysis and identification of an iron-responsive element in the mRNA.

5-Aminolevulinate synthase (ALAS) catalyzes the first step of the heme biosynthetic pathway. cDNA clones for the human erythroid ALAS isozyme were isolated from a fetal liver library. It can be deduced that the erythroid ALAS precursor protein has a molecular weight of 64.6 kd, and is similar in size to the previously isolated human housekeeping ALAS precursor of molecular weight 70.6 kd. The mature mitochondrial forms of the erythroid and housekeeping ALAS isozymes are predicted to have molecular weights of 59.5 kd and 64.6 kd, respectively. The two isozymes show little amino acid identity in their N-terminal signal sequences but have considerable sequence identity in the C-terminal two-thirds of their proteins. An analysis of the immediate promoter of the human erythroid ALAS gene revealed several putative erythroid-specific cis-acting elements including both a GATA-1 and an NF-E2 binding site. An iron-responsive element (IRE) motif has been identified in the 5'-untranslated region of the human erythroid ALAS mRNA, but is not present in the housekeeping ALAS mRNA. Gel retardation experiments established that this IRE motif formed a protein - RNA complex with cytosolic extracts from human K562 cells and this binding was strongly competed with IRE transcripts from ferritin or transferrin receptor mRNAs. A transcript of the ALAS IRE, mutated in the conserved loop of the IRE, did not readily form this protein - RNA complex. These results suggest that the IRE motif in the ALAS mRNA is functional and imply that translation of the mRNA is controlled by cellular iron availability during erythropoiesis.

5-Aminolevulinate Synthetase↗
PubMed1994

Steel factor affects SCL expression during normal erythroid differentiation.

Steel factor is one of the growth factors that controls the proliferation and differentiation of hematopoietic cells and SCL, also known as Tcl-5 or Tal-1, is a transcription factor involved in erythropoiesis. In this report, we studied the role of SCL in the proliferation of human peripheral blood burst-forming unit-erythroid (BFU-E) and the effects of Steel factor on SCL expression in proliferating erythroid cells. BFU-E-derived colonies increase progressively in size, as determined by cell number, from day 7 to day 14 of culture, with the greatest increase in colony size (10-fold expansion) occurring between day 7 and day 10. SCL protein levels in BFU-E-derived cells were highest in day 7 cells and decreased progressively from day 7 to day 14 of culture, suggesting an association of SCL with erythroid proliferation. In contrast, SCL mRNA levels did not decrease significantly between day 7 and day 14 cells, suggesting that posttranscriptional mechanisms are largely responsible for the decrease in SCL protein observed. The role of SCL in Steel factor-induced erythroid proliferation was then examined. In BFU-E-derived colonies cultured with Steel factor, colony size was significantly increased compared to control. In day 7 and day 10 erythroid precursors cultured with Steel factor, SCL protein was increased significantly compared to control. The increase in SCL protein levels in early erythroid precursors stimulated with Steel factor suggests one mechanism through which Steel factor may enhance normal erythroid proliferation. SCL mRNA levels assessed by Northern blot in day 7 cells did not increase significantly in response to Steel factor stimulation, suggesting that posttranscriptional mechanisms may also be important in the increase in SCL protein observed in response to Steel.

Base Sequence↗
PubMed2002

Expression of a human beta-globin transgene in erythroid cells derived from retrovirally transduced transplantable human fetal liver and cord blood cells.

Transfer of therapeutic genes to human hematopoietic stem cells (HSCs) using complex vectors at clinically relevant efficiencies remains a major challenge. Recently we described a stable retroviral vector that sustains long-term expression of green fluorescent protein (GFP) and a human beta-globin gene in the erythroid progeny of transduced murine HSCs. We now report the efficient transduction of primitive human CD34(+) fetal liver or cord blood cells with this vector and expression of the beta-globin transgene in the erythroid progeny of these human cells for at least 2 months. After growth factor prestimulation and then a 2- to 3-day exposure to the virus, 35% to 55% GFP(+) progeny were seen in assays of transduced colony-forming cells, primitive erythroid precursors that generate large numbers of glycophorin A(+) cells in 3-week suspension cultures, and 6-week long-term culture-initiating cells. In immunodeficient mice injected with unselected infected cells, 5% to 15% of the human cells regenerated in the marrow (including the erythroid cells) were GFP(+) 3 and 6 weeks after transplantation. Importantly, the numbers of GFP(+) human lymphoid and either granulopoietic or erythroid cells in individual mice 6 weeks after transplantation were significantly correlated, indicative of the initial transduction of human multipotent cells with in vivo repopulating activity. Expression of the transduced beta-globin gene in human cells obtained directly from the mice or after their differentiation into erythroid cells in vitro was demonstrated by reverse transcriptase-polymerase chain reaction using specific primers. These experiments represent a significant step toward the realization of a gene therapy approach for human beta-globin gene disorders.

Animals↗
PubMed2005

Interplay of pu.1 and gata1 determines myelo-erythroid progenitor cell fate in zebrafish.

The zebrafish is a powerful model system for investigating embryonic vertebrate hematopoiesis, allowing for the critical in vivo analysis of cell lineage determination. In this study, we identify zebrafish myeloerythroid progenitor cells (MPCs) that are likely to represent the functional equivalent of mammalian common myeloid progenitors. Utilizing transgenic pu.1-GFP fish, real-time MPC differentiation was correlated with dynamic changes in cell motility, morphology, and gene expression. Unlike mammalian hematopoiesis, embryonic zebrafish myelopoiesis and erythropoiesis occur in anatomically separate locations. Gene knockdown experiments and transplantation assays demonstrated the reciprocal negative regulation of pu.1 and gata1 and their non-cell-autonomous regulation that determines myeloid versus erythroid MPC fate in the distinct blood-forming regions. Furthermore, forced expression of pu.1 in the bloodless mutant cloche resulted in myelopoietic rescue, providing intriguing evidence that this gene can function in the absence of some stem cell genes, such as scl, in governing myelopoiesis.

Animals↗
PubMed2000

FLI-1 is a suppressor of erythroid differentiation in human hematopoietic cells.

The FLI-1 oncogene, a member of the ETS family of transcription factors, is associated with both normal and abnormal hematopoietic cell growth and lineage-specific differentiation. We have previously shown that overexpression of FLI-1 in pluripotent human hematopoietic cells leads to the induction of a megakaryocytic phenotype. In this report we show that FLI-1 also acts as an inhibitor of erythroid differentiation. Following the induction of erythroid differentiation, pluripotent cells express reduced levels of FLI-1. In contrast, when FLI-1 is overexpressed in these cells, the levels of erythroid markers are reduced. The ability of FLI-1 overexpressing cells to respond to erythroid-specific inducers such as hemin and Ara-C is also inhibited, and the uninduced cells show a reduced level of the erythroid-associated GATA-1 transcription factor mRNA. Furthermore, expression of a GATA-1 promoter-driven reporter construct in K562 cells is inhibited by co-transfection with a construct expressing FLI-1. Our results support the hypothesis that FLI-1 can act both positively and negatively in the regulation of hematopoietic cell differentiation, and that inhibition of GATA-1 expression may contribute to FLI-1-mediated inhibition of erythroid differentiation.

Cell Differentiation↗
PubMed2002

A minigene containing four discrete cis elements recapitulates GATA-1 gene expression in vivo.

BACKGROUND: The GATA-1haematopoietic enhancer (G1HE), located between 3.9 and 2.6 kb 5' to the haematopoietic first exon, is essential for GATA-1 gene transcription in erythroid cells. However, G1HE is not sufficient to confer tissue specificity on the GATA-1 gene in vivo, indicating that additional regulatory sequences are necessary. RESULTS: We demonstrate here that two other upstream promoter elements containing a double GATA motif or two CACCC boxes are also indispensable for reporter gene expression in erythroid cells in the transgenic mouse. The combination of these three cis-acting regions was sufficient for reporter expression in primitive erythroid cells, as demonstrated by linking the elements together into a 659 bp artificial (GdC) minigene. The minigene activated the transcription of a reporter gene from either the endogenous or an exogenous thymidine kinase promoter, retaining cell type-specificity. The addition of a 320 bp fragment in the first intron to the GdC minigene sustained reporter expression in the definitive stage. Moreover, a line of transgenic mouse that expressed GATA-1 cDNA under the control of the complete 979 bp minigene rescued GATA-1 germ line mutant mice from embryonic lethality. CONCLUSIONS: A combination of four distinct sequence motifs co-operatively serve as a fundamental functional unit for GATA-1 erythroid transcription in vivo.

Animals↗
PubMed2000

ABC-me: a novel mitochondrial transporter induced by GATA-1 during erythroid differentiation.

Transcription factor GATA-1 is essential for normal erythropoiesis. GATA-binding sites are consistently found in promoters or enhancers of genes expressed selectively in erythroid cells. To discover novel GATA-1-regulated genes, we searched for GATA-1-activated transcripts in G1E cells, an erythroid line derived from GATA-1(-) embryonic stem cells. By subtractive analysis, we identified a new ATP-binding cassette (ABC) transporter that is strongly and rapidly induced by GATA-1. This protein, named ABC-me (for ABC-mitochondrial erythroid), localizes to the mitochondrial inner membrane and is expressed at particularly high levels in erythroid tissues of embryos and adults. ABC-me is induced during erythroid maturation in cell lines and primary hematopoietic cells, and its overexpression enhances hemoglobin synthesis in erythroleukemia cells. The ABC proteins participate in diverse physiological processes by coupling ATP hydrolysis to the transport of a variety of substrates across cell membranes. We speculate that ABC-me, a newly identified erythroid-expressed ABC superfamily member, may mediate critical mitochondrial transport functions related to heme biosynthesis.

ATP-Binding Cassette Transporters↗
PubMed1993

Expression of the chicken GATA factor family during early erythroid development and differentiation.

The DNA motif WGATAR has been identified within transcriptional regulatory domains of globin and other erythroid-specific genes and the activator proteins that bind to this regulatory element, the GATA factors, belong to a multi-gene family that is expressed in chicken erythroid cells. Here we show that, as in chickens, multiple members of the GATA factor family are expressed in human and murine erythroid cells. During the early stages of chicken embryogenesis (well before blood island formation), each of the GATA family members is transcribed with a unique temporal and spatial pattern. In the primitive erythroid lineage, transcription of the embryonic epsilon-globin gene parallels GATA-1 expression while the switch to beta-globin transcription in definitive erythroid cells is directly preceded by a pronounced increase in GATA-3 accumulation. The timing and pattern of expression of these different mRNAs during avian erythroid development and differentiation suggests that temporally regulated changes in GATA factor expression are required for vertebrate hematopoiesis.

Animals↗
PubMed1998

Measurement of the cell proliferation rate of bone marrow erythroid precursors by flow cytometry: initial applications to multiple myeloma.

To investigate the pathophysiology of anemia, a two-color flow cytometric method was developed that measures the proliferative rate of the marrow erythroid cells (EPR). The method uses a monoclonal antibody, RC17.2, to identify erythroid precursors and propidium iodide to determine the %S-phase. This technique was then used to test the hypothesis that a decrease in the proliferative rate of the marrow erythroid precursors contributes to the anemia of multiple myeloma. The EPR was determined on the marrow aspirate from 56 patients and the mean EPR was 31.2% (median, 31: range, 14-55). Patients with anemia (n = 36) had a median EPR of 27% compared to 35% for those patients with a normal Hgb (p = < 0.001); however, there was no difference in the % marrow erythroid precursors (p = 0.96) or % marrow plasma cells (p = 0.08) between the two groups. These results suggest that one possible cause for the anemia of myeloma is a decrease in the EPR. This flow cytometric technique may also be useful in studying other anemias.

Anemia↗
PubMed2006

Delta-4 Notch ligand promotes erythroid differentiation of human umbilical cord blood CD34+ cells.

OBJECTIVE: Important roles of Notch signaling have been demonstrated in hematopoiesis. In many cases, activation of the Notch pathway leads to the inhibition of differentiation of immature precursors, suggesting a potential role in self-renewal promotion. However, the function of Notch and Notch ligands is not so straightforward because it is considerably dependent on cytokine context. In this study, we analyzed effects of one Notch ligand, Delta-4, whose function is less clear than others, such as Delta-1 and Jagged-1 and -2. METHODS: CD34(+) cells isolated from human umbilical cord blood were cocultured with a Delta-4-expressing murine stromal cell line, SC9-19, and induced to erythroid differentiation by adding stem cell factor and erythropoietin. To examine the involvement of Delta-4, we utilized stromal cell subclones expressing Delta-4 protein at higher or lower level than parental SC9-19 by plasmid transfection. Erythroid maturation was examined by surface phenotype (CD34 and glycophorin A) and cytospin morphology. Recombinant human Delta-4 protein was prepared to analyze direct effects of Delta-4. RESULTS: Under erythroid lineage-inducing conditions, we found that the increase in Delta-4 expression of SC9-19 promoted erythroid differentiation whereas the decrease in Delta-4 expression inhibited it. Morphologic examination as well as colony formation analysis supported this observation. Moreover, the experiment using recombinant Delta-4 provided direct evidence of the Delta-4 activity found in coculture system. CONCLUSIONS: By modifying Delta-4 expression of the stromal cells and using the recombinant protein, we demonstrated that Delta-4 had a differentiation promoting activity for human primitive hematopoietic cells into erythroid lineage.

Animals↗
PubMed1997

A common precursor for primitive erythropoiesis and definitive haematopoiesis.

The generation of blood cells, haematopoiesis, in the mouse embryo begins with the development of primitive nucleated erythroid cells in the yolk sac followed by the appearance of precursors for multiple definitive haematopoietic lineages. The later developing lineages arise from multipotential stem cells, but the relationship of primitive erythroid cells to these other haematopoietic populations is unknown. Using an in vitro embryonic stem (ES) cell differentiation system, we show that primitive erythrocytes and other haematopoietic lineages arise from a common multipotential precursor that develops within embryoid bodies generated from differentiated ES cells. In response to vascular endothelial growth factor and c-kit ligand these precursors give rise to colonies containing immature cells (blasts) expressing marker genes characteristic of haematopoietic precursors. Many blast colonies also expressed betaH1 and beta major globins but not Brachyury, a mesodermal marker. Kinetic analysis demonstrated that the blast colony-forming cells represent a transient population, preceding the establishment of the primitive erythroid and other lineage-restricted precursors. This precursor population may represent the earliest stage of embryonic haematopoietic commitment.

Animals↗
PubMed1995

A newly established megakaryoblastic/erythroid cell line that differentiates to red cells in the presence of erythropoietin and produces platelet-like particles.

In August, 1992, we established a leukemic cell line (NS-Meg) from a patient in megakaryoblastic transformation of Philadelphia chromosome-positive chronic myeloid leukemia. The NS-Meg cells were positive for alpha-naphthyl acetate esterase and periodic acid-Schiff (PAS) staining and for surface CD4, CD7, CD13, CD34, CD41a, and glycophorin A antigens. Ultrastructurally, the cells had alpha-granules, demarcation membranes, and platelet peroxidase activity. The NS-Meg cells spontaneously produced platelet-like particles which contained alpha-granules, mitochondria and dense bodies, strongly suggesting platelet production. Erythropoietin (Epo), granulocyte/macrophage colony stimulating factor(GM-CSF), and interleukin 3 (IL-3) promoted the growth of NS-Meg cells. Phorbol-12-myristate-13-acetate increased the expression of both CD41a and CD61 antigens. Ten-day exposure to Epo induced mature erythroblasts and red cells. These benzidine-positive cells were positive for hemoglobin F staining. Untreated NS-Meg cells expressed mRNA for the Epo receptor (EpoR), for GATA-1, and for alpha 1, alpha 2 and gamma globin genes. These results indicate that NS-Meg cells undergo terminal differentiation of both megakaryocytic and erythroid lineages. This cell line should be a very useful tool for the investigation of both megakaryocytic and erythroid maturation.

Adult↗