Regulation and function of transcription factor GATA-1 during red blood cell differentiation

Development ◽  
1996 ◽  
Vol 122 (12) ◽  
pp. 3839-3850 ◽  
Author(s):  
K. Briegel ◽  
P. Bartunek ◽  
G. Stengl ◽  
K.C. Lim ◽  
H. Beug ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcription Factor ◽  
Cell Differentiation ◽  
Blood Cell ◽  
Progenitor Cells ◽  
Red Blood Cell ◽  
Nuclear Translocation ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Induction Of Differentiation

The tissue-specific transcription factor GATA-1 is a key regulator of red blood cell differentiation. One seemingly contradictory aspect of GATA-1 function is that, while it is abundant in erythroid progenitor cells prior to the onset of overt differentiation, it does not significantly activate known GATA-1 target genes in those cells. To investigate the mechanisms underlying GATA-1 function during the transition from early to late erythropoiesis, we have examined its expression and activity in normal avian erythroid progenitor cells before and after induction of differentiation. In these primary progenitor cells, GATA-1 protein was predominantly located in the cytoplasm, while induction of differentiation caused its rapid relocalization to the nucleus, suggesting that nuclear translocation constitutes an important regulatory step in GATA-1 activation. As an alternative way of addressing the same question, we also ectopically expressed a GATA-1/estrogen receptor fusion protein (GATA-1/ER) in red blood cell progenitors, where nuclear translocation of, and transcriptional activation by, this hybrid factor are conditionally controlled by estrogen. We found that hormone-activated GATA-1/ER protein accelerated red blood cell differentiation, and concomitantly suppressed cell proliferation. These phenotypic effects were accompanied by a simultaneous suppression of c-myb and GATA-2 transcription, two genes thought to be involved in the proliferative capacity of hematopoietic progenitor cells. Thus, GATA-1 appears to promote differentiation in committed erythroid progenitor cells both by inducing differentiation-specific genes and by simultaneously suppressing genes involved in cell proliferation.

g-Globin Induction in Human Erythroid Progenitor Cells by Histone Deacetylase Inhibitor OSI-2040γ.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2700-2700
Author(s):  
Heather M. Rogers ◽  
Constance Tom Noguchi
Keyword(s):  
Cell Proliferation ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Progenitor Cells ◽  
Zinc Finger ◽  
Critical Role ◽  
Erythroid Differentiation ◽  
Erythroid Progenitor ◽  
Zinc Finger Transcription Factor ◽  
Erythroid Progenitor Cells

Abstract An important treatment strategy for sickle cell anemia is increasing fetal hemoglobin (HbF) in circulating erythrocytes. OSI-2040 (Apicidin), a fungus-derived cyclic tetra-peptide, induces g-globin production in K562 cells. The effect of increasing doses of OSI-2040 (from 7.5 to 750 nM) was determined on cultures of primary human hematopoietic progenitor cells stimulated with erythropoietin (EPO). Cell proliferation and differentiation, globin production, and erythroid transcription factors expression were examined. At concentrations 7.5 nM - 75 nM there was minimal decrease in cell proliferation with little change in % benzidine positive cells after 12 days of culture with EPO. As OSI-2040 concentration increased above 75 nM, cell proliferation and % benzidine positive cells decreased, with concentrations of 300 and 750 nM being highly toxic, reducing cell number by 75% or more. Analysis of globin gene expression indicates that low to mid concentrations of OSI-2040 increase g-globin, with the peak increase occurring at 75 nM, while the highest concentrations (300 and 750 nM) suppress g-globin. OSI-2040 decreases b-globin expression with the highest concentrations resulting in the greatest decreases. The g/(g+b) ratio increases with increasing OSI-2040 concentration reaching a value of 4-fold and greater for concentrations of 75 nM or more, partially a consequence of the suppression of b-globin expression, particularly at higher concentrations. Although the g/(g+b) ratio is relatively high at the highest concentrations of OSI-2040 (300 and 750 nM), it is at a cost in overall globin production and cell toxicity. Hemoglobin expression is determined primarily at the transcription level. We found that OSI-2040 affects expression of select transcription factors, GATA-1, GATA-2, SCL/Tal-1 and EKLF, which are critical for erythroid differentiation. Peak EPO induction of GATA-1, a zinc-finger transcription factor essential for survival and differentiation of erythroid progenitor cells, is delayed with OSI-2040 treatment. OSI-2040 also delays expression of SCL/Tal-1, a basic-helix-loop-helix transcription factor that positively regulates erythroid differentiation and is required for the production of mature erythrocytes. In addition, there is a delay in the induction of EKLF, a zinc-finger transcription factor necessary for induction of b-globin in adult erythroid cells that acts by direct binding to the b-globin promoter. With increasing OSI-2040 concentrations, there is a dose-dependent decrease in overall levels of GATA-1, SCL/Tal-1 and EKLF. GATA-2, a member of the GATA-family that plays a critical role in the survival of early erythroid progenitor cells and is down-regulated with EPO stimulation, shows a slight delay in its reduction at 75 and 150 nM but overall is not greatly affected by OSI-2040. Thus, OSI-2040 concentration is crucial in optimizing the production of HbF. As we have also observed with hydroxyurea, the greatest increase in the g/g+b ratio is at high concentrations of OSI-2040 (300 and 750 nM), up to 25-fold, and is a consequence of reductions in both b- and g-globin. In contrast, a mid-level concentration (75 nM) yields a 2.5–4 fold increase in the g/(g+b) ratio with little or no cytotoxicity. These data suggest that like hydroxyurea, OSI-2040 may be effective in inducing HbF and may be a useful therapeutic alternative.

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

1995 ◽  
Vol 15 (6) ◽  
pp. 3147-3153 ◽  
Author(s):  
G A Blobel ◽  
C A Sieff ◽  
S H Orkin
Keyword(s):  
Bone Marrow ◽  
Estrogen Receptor ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Erythroid Cell ◽  
High Dose ◽  
Dependent Manner ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

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.

UCP2 Modulates Cell Proliferation through the MAPK/ERK Pathway during Erythropoiesis and Has No Effect on Heme Biosynthesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5372-5372
Author(s):  
Alvaro A Elorza ◽  
Brigham B Hyde ◽  
Hanna Mikkola ◽  
Sheila Collins ◽  
Orian S Shirihai
Keyword(s):  
Cell Proliferation ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Mitochondrial Protein ◽  
Erythroid Progenitor ◽  
Heme Synthesis ◽  
Erythroid Progenitor Cells ◽  
Deficient Mice

Abstract UCP2, an inner membrane mitochondrial protein, has been implicated in bioenergetics and Reactive Oxygen Species (ROS) modulation. UCP2 has been previously hypothesized to function as a facilitator of heme synthesis and iron metabolism by reducing ROS production. While UCP2 has been found to be induced by GATA1 during erythroid differentiation its role in erythropoiesis in vivo or in vitro has not been reported thus far. Here we report on the study of UCP2 role in erythropoiesis and the hematologic phenotype of UCP2 deficient mouse. In vivo we found that UCP2 protein peaks at early stages of erythroid maturation when cells are not fully committed in heme synthesis and then becomes undetectable at the reticulocyte stage. Iron incorporation into heme was unaltered in erythroid cells from UCP2 deficient mice. While heme synthesis was not influenced by UCP2 deficiency, mice lacking UCP2 had a delayed recovery from chemically induced hemolytic anemia. Analysis of the erythroid lineage from bone marrow and fetal liver revealed that in the UCP2 deficient mice the R3 (CD71high/Ter119high) population was reduced by 24%. The count of BFU-E and CFU-E colonies, scored in an erythroid colony assay, was unaffected, indicating an equivalent number of early erythroid progenitor cells in both UCP2 deficient and control cells. Ex-vivo differentiation assay revealed that UCP2 deficient c-kit+ progenitor cells expansion was overall reduced by 14% with population analysis determining that the main effect is at the R3 stage. No increased rate of apoptosis was found indicating that expansion rather than cell death is being compromised. Reduced expansion of c-kit+ cells was accompanied by 30% reduction in the phosphorylated form of ERK, a ROS dependent cytosolic regulator of cell proliferation. Analysis of ROS in UCP2 null erythroid progenitors revealed altered distribution of ROS resulting in 14% decrease in cytosolic and 32% increase in mitochondrial ROS. Restoration of the cytosolic oxidative state of erythroid progenitor cells by the pro-oxidant Paraquat reversed the effect of UCP2 deficiency on cell proliferation in in vitro differentiation assays. Together, these results indicate that UCP2 is a regulator of erythropoiesis and suggests that inhibition of UCP2 function may contribute to the development of anemia.

scholarly journals Inhibition of Immature Erythroid Progenitor Cell Proliferation by Macrophage Inflammatory Protein-1α by Interacting Mainly With a C-C Chemokine Receptor, CCR1

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 605-611 ◽  
Author(s):  
Shao-bo Su ◽  
Naofumi Mukaida ◽  
Jian-bin Wang ◽  
Yi Zhang ◽  
Akiyoshi Takami ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Inflammatory Protein ◽  
Cd34 Cells ◽  
Macrophage Inflammatory Protein ◽  
Marrow Cells

Abstract Several lines of evidence indicate that macrophage inflammatory protein-1α (MIP-1α) modulates the proliferation of hematopoietic progenitor cells, depending on their maturational stages. To clarify the mechanisms for the modulation of hematopoiesis by this chemokine, we examined the expression of a receptor for MIP-1α, CCR1, on bone marrow cells of normal individuals using a specific antibody and explored the effects of MIP-1α on in vitro erythropoiesis driven by stem cell factor (SCF) and erythropoietin (Epo). CCR1 was expressed on glycophorin A-positive erythroblasts in addition to lymphocytes and granulocytes. CCR1+ cells, isolated from bone marrow mononuclear cells (BMMNCs) using a cell sorter, comprised virtually all erythroid progenitor cells in the BMMNCs. Moreover, MIP-1α inhibited, in a dose-dependent manner, colony formation by burst-forming unit-erythroid (BFU-E), but not by colony forming unit-erythroid (CFU-E), in a methylcellulose culture of purified human CD34+ bone marrow cells. Although reverse-transcription polymerase chain reaction (RT-PCR) showed the presence of CCR1, CCR4, and CCR5 transcripts in CD34+ cells in BM, anti-CCR1 antibodies significantly abrogated the inhibitory effects of MIP-1α on BFU-E formation both in a methylcellulose culture and in a single cell proliferation assay of purified CD34+ cells. Although the contribution of CCR4 or CCR5 cannot be completely excluded, these results suggest that MIP-1α–mediated suppression of the proliferation of immature, but not mature erythroid progenitor cells, is largely mediated by CCR1 expressed on these progenitor cells.

scholarly journals Deletion of Mtg16, a Target of t(16;21), Alters Hematopoietic Progenitor Cell Proliferation and Lineage Allocation

2008 ◽  
Vol 28 (20) ◽  
pp. 6234-6247 ◽  
Author(s):  
Brenda J. Chyla ◽  
Isabel Moreno-Miralles ◽  
Melissa A. Steapleton ◽  
Mary Ann Thompson ◽  
Srividya Bhaskara ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Progenitor Cell ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Cell Fate Decisions ◽  
Erythroid Progenitor Cells ◽  
Progenitor Cell Proliferation

ABSTRACT While a number of DNA binding transcription factors have been identified that control hematopoietic cell fate decisions, only a limited number of transcriptional corepressors (e.g., the retinoblastoma protein [pRB] and the nuclear hormone corepressor [N-CoR]) have been linked to these functions. Here, we show that the transcriptional corepressor Mtg16 (myeloid translocation gene on chromosome 16), which is targeted by t(16;21) in acute myeloid leukemia, is required for hematopoietic progenitor cell fate decisions and for early progenitor cell proliferation. Inactivation of Mtg16 skewed early myeloid progenitor cells toward the granulocytic/macrophage lineage while reducing the numbers of megakaryocyte-erythroid progenitor cells. In addition, inactivation of Mtg16 impaired the rapid expansion of short-term stem cells, multipotent progenitor cells, and megakaryocyte-erythroid progenitor cells that is required under hematopoietic stress/emergency. This impairment appears to be a failure to proliferate rather than an induction of cell death, as expression of c-Myc, but not Bcl2, complemented the Mtg16 − / − defect.

Hydroxyurea Cytotoxicity and Fetal Hemoglobin Induction.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3607-3607
Author(s):  
Heather M. Rogers ◽  
Xiaobing Yu ◽  
Constance Tom Noguchi
Keyword(s):  
Transcription Factor ◽  
Progenitor Cells ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Cell Toxicity ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
High Concentrations ◽  
Globin Gene Expression

Abstract An important treatment strategy for sickle cell anemia is to increase fetal hemoglobin (HbF) in circulating erythrocytes. We use hydroxyurea (HU) as a model compound to induce HbF in adult human erythroid progenitor cells to examine the relationship between cell toxicity and globin gene expression. HU inhibits ribonucleotide reductase and its use is limited by hematopoietic toxicity at high dose. Cultures of primary human hematopoietic progenitor cells were stimulated with erythropoietin (EPO) and the effect of increasing doses of HU (from 1 to 200 mM) was determined on cell proliferation and differentiation, globin production, and erythroid transcription factors expression. At the lowest concentration (1 mM) we observed a minimal increase in cell proliferation with little change in % benzidine positive cells after 12 days of culture with EPO. As HU concentration increased, proliferation and % benzidine positive cells decreased, with concentrations of 100 and 200 mM being highly toxic, reducing cell number by 10 fold or more. Analysis of globin gene expression indicates that low concentrations of HU increase both g-globin and b-globin, resulting in only a modest increase in the g/(g+b) ratio compared with control. The g/(g+b) ratio increases with increasing HU concentration reaching a value of 0.25 or greater for concentrations of 50 mM or more, and approaching 1.0 at 200 mM, a consequence of the suppression of b-globin expression. This concentration of HU also inhibited g-globin expression, so that although the g/(g+b) ratio is quite high, it is at a cost in overall globin production and cell toxicity. Hemoglobin expression is determined primarily at the transcription level. We examined expression of GATA-1, GATA-2, SCL/Tal-1 and EKLF as regulatory proteins critical to erythropoiesis. We found that HU affects expression of select transcription factors associated with erythroid differentiation. EPO induction of GATA-1, a zinc-finger transcription factor required for survival and differentiation of erythroid progenitor cells, is delayed with HU, and the peak level of GATA-1 decreases at mid- and high concentrations, falling by 10 fold or more at 100 mM or greater. At the lowest concentration (1 mM) GATA-1 increases higher than the control. HU also delays EPO induction of SCL/Tal-1, a basic-helix-loop-helix transcription factor that positively regulates erythroid differentiation and is required for the production of mature erythrocytes, and EKLF, a zinc-finger transcription factor necessary for induction of b-globin in adult erythroid cells that acts by direct binding to the b-globin promoter. At the lowest concentration (1 mM), the delay in EPO induction of SCL/Tal-1 and EKLF is followed by a marked increase leading to peak levels greater than the control. At mid- and high concentrations, overall levels of SCL/Tal-1 and EKLF are reduced. GATA-2, a member of the GATA-family that plays a critical role in proliferation and survival of early erythroid progenitor cells, is down-regulated with EPO stimulation and is not markedly affected by HU. Therefore, HU concentration is crucial in optimizing the production of HbF. At low levels, HU increases both b- and g-globin resulting in small increases in g/(g+b) ratio, while at high concentrations the maximal increases in g/(g+b) ratio are concomitant with cytotoxicity. These data explain in part the importance of the maximum tolerated dose to achieve maximum increase in %HbF in hydroxyurea therapy.μμμγβγγβγγβμμβγγγβμμμβγγγβγγβββ

Excessive Isolated Erythrocytosis Linked to Induction of SCL/Tal1 and Erythropoietin Receptor Expression.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1244-1244
Author(s):  
Heather M. Rogers ◽  
Josef T. Prchal ◽  
Babette B. Weksler ◽  
Xiaobing Yu ◽  
Constance Tom Noguchi
Keyword(s):  
Cell Proliferation ◽  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Jak2 V617f ◽  
Erythropoietin Receptor ◽  
Jak2 V617f Mutation ◽  
Erythroid Progenitor ◽  
Cell Numbers ◽  
Erythroid Progenitor Cells ◽  
V617f Mutation

Abstract The JAK2 V617F mutation has been detected in more than 90% polycythemia vera patients and is associated with increased erythropoietin (EPO) sensitivity or EPO independence of erythroid progenitor cells during early erythropoiesis. To determine if other molecular lesions give rise to increased erythrocytosis and realizing that the JAK2 V617F mutation may not be the initiating event in polycythemia vera, we identified an individual with isolated, excessive erythrocytosis who showed erythroid precursor hypersensitivity to EPO; no mutations on sequencing genes for erythropoietin receptor (EPO-R), VHL, or HIF-1 IRE; normal serum EPO level; normal hemoglobin function and p50; normal cardiac, hepatic and pulmonary function; no JAK2 V617F mutation and no EPO independent BFU-E. While it was not certain that this individual’s erythrocytosis was lifelong, other relatives had increased hemoglobin levels. The cause of this individual’s increased erythropoiesis was investigated using EPO-stimulated cultures of hematopoietic progenitor cells isolated from peripheral blood. As with normal controls, cell numbers were not increased after 12 days of incubation with low levels of EPO (<0.1 U/ml). With 1U/ml of EPO, cell proliferation was similar to control early in erythropoiesis but increased markedly after 8 days so that by day 12 cell numbers were 3-fold greater than in control cultures with 95% benzidine positive cells compared with 76% for control and 82–87% for JAK2 V617F positive erythroid progenitor cells. Analyses of transcription factor expression revealed that induction of GATA-1 and down regulation of GATA-2 were similar to control. However, SCL/Tal1 and EKLF markedly increased beginning at day 8 to 10 and continued to rise during late erythropoiesis in contrast to control and JAK2 V617F positive cultures in which SCL/Tal1 and EKLF peaked at day 10 and decreased with late erythroid differentiation. Since increased beta-globin expression is concomitant with induction of EKLF, the rise in EKLF may account for the marked increase in benzidine positive cells from our subject. Moreover, expression of EPO-R followed the continuing rise in SCL/Tal1, increasing by 3.5 fold at day 12 compared with control cultures. This high EPO-R expression is consistent with the dramatic increase in cell proliferation during late erythropoiesis. Using forced expression of SCL/Tal1, reporter gene assay and gel mobility shift analysis in erythroid cells, we determined that SCL/Tal1 is able to bind to E-boxes located 3′ of the EPO-R proximal promoter and to activate transcription. Although EPO stimulation of erythroid progenitor cells activates EPO-R, we found that forced expression of EPO-R in primary erythroid progenitor cells in the presence of EPO increases expression of GATA-1 as well as SCL/Tal1 and EKLF, providing further evidence that specific increase in SCL/Tal1 but not GATA-1 must precede induction of EPO-R in this case of excessive erythrocytosis. These data demonstrate a link between high level induction of SCL/Tal1 expression and increased erythrocytosis and suggest a mechanism that contributes to increased erythropoietin sensitivity during late erythropoiesis.

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