Inducing Definitive Erythropoiesis From Human Embryonic Stem Cells Through a Novel Intracellular MPL Dimerization Strategy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1172-1172 ◽  
Author(s):  
William Sang Kim ◽  
Yuhua Zhu ◽  
Qiming Deng ◽  
Amanda J. Grieco ◽  
Gautam G. Dravid ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Akt Signaling ◽  
Beta Globin ◽  
Erythroid Progenitors ◽  
Enucleated Rbc ◽  
Definitive Erythropoiesis ◽  
Negative Controls

Abstract Objectives Unlimited self renewal capacity and the ability to differentiate into any cell type make human pluripotent stem cells (PSC) a potential source for the ex vivo manufacture of red blood cells (RBC) for safe transfusion. Current methods of RBC differentiation from PSC suffer from low yields of RBCs, most of which contain embryonic and fetal rather than adult hemoglobins. We have previously shown that dimerization of the intracellular component of MPL (the thrombopoietin receptor), induces expansion of myelo-erythroid progenitors (MEP) from human cord blood as well as their terminal differentiation into enucleated RBC through unique, EPO-independent mechanisms (Parekh et al, 2012). Our goal was to investigate the potential of intracellular MPL dimerization to induce erythropoiesis from human PSC and to identify the signaling pathways activated by this strategy. Methods Human embryonic stem cell (hESC) lines H1 and HES3 were transduced with a lentiviral vector to express the fusion protein F36V-MPL (containing the ligand binding domain F36V and the intracytoplasmic portion of MPL). Dimerization of F36V-MPL was accomplished by addition of the synthetic ligand AP20187 (aka CID) during culture (with or without erythropoietin) on OP9 stroma in the absence of other cytokines. F36V-MPL transduced-hESC that did not receive CID and F36V-transduced hESC cultured with CID served as negative controls. Flow cytometry and Colony Forming Unit (CFU) assays were used to analyze erythroid differentiation. Phosflow and Western Blot were used to analyze cell signaling. MEP generated during hESC differentiation were defined as cells co-expressing GlyA and CD41a/CD42a. Results F36V-MPL dimerization induced significantly more Glycophorin A+ cells (P=0.0001; n=5) and 10-fold higher number of erythroid CFU (P=0.0007; n=15) as compared to negative controls. The effect was consistent across different hESC cell lines. This effect was seen in the absence of any hematopoietic cytokines, including erythropoietin (EPO), a critical cytokine for erythropoiesis and an integral component of all ex vivo PSC erythroid differentiation protocols, indicating that MPL dimerization alone is sufficient to induce erythropoiesis from hESCs. Erythroid output was further enhanced in an additive manner in the presence of EPO (P=0.006; n=5). In order to identify the point at which MPL dimerization affects erythropoiesis, CID was added during differentiation directly from hESC or to isolated MEP generated from hESC. CID and EPO increased the number of MEP compared to untreated controls, demonstrating that MPL dimerization induces the generation of early erythroid progenitors. In addition, CID drove erythroid differentiation from MEP more efficiently than EPO, demonstrated by a significantly higher frequency of total erythroid cells (P=0.02; n=3), and 4-fold increase in yield of enucleated RBC. Globin analysis by HPLC demonstrated that although no detectable beta-globin expression was observed with EPO, CID treatment induced the presence of beta-globin and increased the gamma: epsilon globin ratio, suggesting a shift toward definitive erythropoiesis. Signaling studies found that, unlike the full-length MPL receptor, which activates both STAT5/JAK2 and AKT pathways, F36V-MPL dimerization activated AKT but not STAT5 or JAK2 phosphorylation. PI3K/AKT inhibitors (LY294002 and AKT inhibitor IV) effectively inhibited erythroid differentiation of transduced hESC cultured in the presence of CID (P=0.001; n=4) indicating that MPL dimerization induced erythropoiesis is dependent on AKT signaling. Gene expression analysis by qPCR indicated that MPL dimerization upregulates a network of genes (downstream of AKT signaling) associated with the regulation of cell cycle, apoptosis, and erythroid differentiation, including GATA1, CDKN1A, RB1, VEGFA, and BCL-xL with a corresponding reduction in both apoptosis and cell cycle progression. Conclusion In summary, we have identified a novel EPO-independent approach that is not only more efficient at erythropoiesis but is also able to augment EPO induced erythropoiesis. The mechanistic insights gained from this study opens up potentially new approaches toward the generation of therapeutically relevant quantities of RBCs for transfusion. Disclosures: No relevant conflicts of interest to declare.

Efficient Erythropoiesis From Human Embryonic Stem Cells Through Dimerization of Intracellular MPL.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2291-2291
Author(s):  
William Sang Kim ◽  
Gautam G. Dravid ◽  
Yuhua Zhu ◽  
Chintan Parekh ◽  
Qiming Deng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Total Yield ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Akt Signaling ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Enucleated Rbc ◽  
Negative Controls

Abstract Abstract 2291 Objectives: Unlimited self renewal capacity and the ability to differentiate into any cell type make human pluripotent stem cells (PSC) a potential source for the ex vivo manufacture of red blood cells (RBC) for safe transfusion. Current methods of RBC differentiation from PSC suffer from low yields of RBCs, most of which contain embryonic rather than adult or fetal hemoglobins. Therefore, efficient clinical translation of this strategy is critically dependent on the development of novel methods to enhance the generation of functional RBCs from PSC. We have previously shown that dimerization of the intracellular component of MPL (the thrombopoietin receptor), induces expansion of myelo-erythroid progenitors (MEP) from human cord blood as well as their terminal differentiation into enucleated RBC through unique, EPO-independent mechanisms (Parekh et al, 2012). Our goal was to investigate the potential of intracellular MPL dimerization to induce erythropoiesis from human PSC and to identify the signaling pathways activated by this strategy. Methods: Human embryonic stem cell (hESC) lines H1 and HES3 were transduced with a lentiviral vector to express the fusion protein F36V-MPL (containing the ligand binding domain F36V and the intracytoplasmic portion of MPL). Dimerization of F36V-MPL was accomplished by addition of the synthetic ligand AP20187 (aka CID) during culture (with or without erythropoietin) on OP9 stroma in the absence of other cytokines. F36V-MPL transduced-hESC that did not receive CID and F36V-transduced hESC cultured with CID served as negative controls. Flow cytometry and Colony Forming Unit (CFU) assays were used to analyze erythroid differentiation. Phosflow and Western Blot were used to analyze cell signaling. MEP generated during hESC differentiation were defined as cells co-expressing GlyA and CD41a/CD42a. Results: F36V-MPL dimerization induced significantly more Glycophorin A+ cells (P=0.0001; n=5) and 10-fold higher number of erythroid CFU (P=0.0007; n=15) as compared to negative controls. The effect was consistent across different hESC cell lines. The increased yield of erythroid cells was not due to an overall increase in cell proliferation as the total yield of cells was not statistically different between treated and untreated cultures. This effect was seen in the absence of any hematopoietic cytokines, including erythropoietin (EPO), a critical cytokine for erythropoiesis and an integral component of all ex vivo PSC erythroid differentiation protocols, indicating that MPL dimerization alone is sufficient to induce erythropoiesis from hESCs. Erythroid output was further enhanced in an additive manner in the presence of EPO (P=0.0058; n=5). In order to identify the point at which MPL dimerization affects erythropoiesis, CID was added during differentiation directly from hESC or to isolated MEP generated from hESC. CID and EPO increased the number of MEP compared to untreated controls, demonstrating that MPL dimerization induces the generation of early erythroid progenitors. In addition, CID drove erythroid differentiation from MEP more efficiently than EPO, demonstrated by a significantly higher frequency of total erythroid cells (P=0.02; n=3), and 4-fold increase in yield of enucleated RBC. This indicates that CID has a greater effect on terminal erythroid differentiation than EPO. We then investigated the signaling mechanism activated by F36V-MPL dimerization and found that, unlike the full-length MPL receptor, which activates both STAT5/JAK2 and AKT pathways, F36V-MPL dimerization activated AKT but not STAT5 or JAK2 phosphorylation. PI3K/AKT inhibitors (LY294002 and AKT inhibitor IV) effectively inhibited erythroid differentiation of transduced hESC cultured in the presence of CID (P=0.0442; n=2) indicating that MPL dimerization induced erythropoiesis is dependent on AKT signaling. Conclusion: F36V-MPL dimerization during hESC-derived hematopoiesis induces EPO-independent erythroid differentiation through AKT signaling, by both generating erythroid progenitors and promoting maturation of RBC. MPL dimerization also is more potent than EPO in inducing erythropoiesis from hESC and has an additive effect when combined with EPO, making this a potential strategy for the generation of therapeutically relevant levels of functional enucleated RBCs from PSC. Disclosures: No relevant conflicts of interest to declare.

Bcl-xL Does Not Rescue Erythroid Colony (CFU-e) Formation in EpoR−/− Progenitors, Suggesting a Cell-Cycle Role for EpoR.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1117-1117
Author(s):  
Ramona Pop ◽  
Srijana Ranjit ◽  
Merav Socolovsky
Keyword(s):  
Cell Cycle ◽  
Fetal Liver ◽  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Erythroid Terminal Differentiation ◽  
Erythroid Maturation ◽  
Semi Solid ◽  
Definitive Erythropoiesis ◽  
Cell Cycle Status

Abstract The essential role of glycoprotein hormone erythropoietin (Epo) and its receptor, EpoR, in erythroid development is well established: both the EpoR−/− and Epo−/− mouse embryos die on embryonic day 13 (E13) due to failure of definitive erythropoiesis in fetal liver (Wu et al. 1995). It has been suggested that Epo’s principal role during erythropoiesis is to protect erythroid progenitors from apoptosis (Koury and Bondurant, Science 1990). Bcl-xL, an anti-apoptotic member of the bcl-2 family, is induced by EpoR signaling in erythroid cells via the Jak2/Stat5 pathway (Silva et al., Blood 1996; Socolovsky et al., Cell, 1999). Bcl-xL is essential for erythroid maturation: bcl-xL−/− embryos die in utero at the same stage as as EpoR−/− mice, lacking definitive erythropoiesis (Motoyama et al., Science 1995; J Exp Med, 1999). Recenlty, it has been shown that over-expression of bcl-xL in primary wild-type erythroblasts confers Epo independence on these cells in vitro and allows them to complete their differentiaion into red blood cells (Dolznig et al., Curr Biol, 2002). Here we reasoned that if the principal function of EpoR signaling is suppression of apoptosis via bcl-xL, it should be possible to rescue all aspects of erythroid differentiation in EpoR−/− fetal liver progenitors by retrovirally-transducing these cells with bcl-xL. We infected EpoR−/− fetal liver progenitors with bicistronic retroviral vectors expressing either bcl-xL or EpoR, each linked via an IRES sequence to a GFP reporter. Control EpoR−/− cells were infected with ‘empty’ bicistronic vector. Infection rates were in excess of 30% for all constructs, and transduced cells were identified for further analysis using GFP fluorescence. We examined terminal differentiation of the transduced EpoR−/− cells over the ensuing 48 hours, using several distinct assays, including their expression of the cell-surface differentiation markers CD71 and Ter119 by FACS, their ability to give rise to CFU-e colonies in semi-solid medium, their cell-cycle status using DNA content analysis and BrdU incorporation, and their maturation and hemoglobinization by diaminobenzidine staining and light microscopy. We found that EpoR−/− progenitors transduced with bcl-xL were protected from apoptosis, and underwent morphological changes characteristic of erythroid maturation, including decreasing cell size, nuclear condensation and expulsion, and accumulation of hemoglobin. These cells also upregulated the erythroid-specific cell surface marker Ter119. However, unlike EpoR−/− cells transduced with EpoR, bcl-xL -transduced cells did not express high levels of CD71, and failed to give rise to CFU-e colonies in semi-solid medium. Instead, they gave rise to small colonies of 6 cells or less. Cell cycle analysis showed that, throughout the 48 hours of erythroid terminal differentiation, the population of bcl-xL-transduced EpoR−/− cells had a lower fraction of cells in S-phase than control, EpoR-transduced EpoR−/− cells. The cell-cycle status of control, terminally-differentiating wild-type erythroid fetal liver progenitors was not altered by transduction with bcl-xL, excluding the possibility that it directly inhibits S-phase. Taken together our results indicate that bcl-xL does not rescue all aspects of erythroid differentiation in EpoR−/− erythroid progenitors. Specifically, the proliferative program during erythroid terminal differentiation is directly dependent on EpoR signaling, and is not simply a default pathway secondary to EpoR’s anti-apoptotic effect.

scholarly journals Decreased PGC1β expression results in disrupted human erythroid differentiation, impaired hemoglobinization and cell cycle exit

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Taha Sen ◽  
Jun Chen ◽  
Sofie Singbrant
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Mitochondrial Function ◽  
Iron Homeostasis ◽  
Erythroid Differentiation ◽  
Refractory Anemia ◽  
Cell Cycle Exit ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Terminal Erythroid Differentiation

AbstractProduction of red blood cells relies on proper mitochondrial function, both for their increased energy demands during differentiation and for proper heme and iron homeostasis. Mutations in genes regulating mitochondrial function have been reported in patients with anemia, yet their pathophysiological role often remains unclear. PGC1β is a critical coactivator of mitochondrial biogenesis, with increased expression during terminal erythroid differentiation. The role of PGC1β has however mainly been studied in skeletal muscle, adipose and hepatic tissues, and its function in erythropoiesis remains largely unknown. Here we show that perturbed PGC1β expression in human hematopoietic stem/progenitor cells from both bone marrow and cord blood results in impaired formation of early erythroid progenitors and delayed terminal erythroid differentiation in vitro, with accumulations of polychromatic erythroblasts, similar to MDS-related refractory anemia. Reduced levels of PGC1β resulted in deregulated expression of iron, heme and globin related genes in polychromatic erythroblasts, and reduced hemoglobin content in the more mature bone marrow derived reticulocytes. Furthermore, PGC1β knock-down resulted in disturbed cell cycle exit with accumulation of erythroblasts in S-phase and enhanced expression of G1-S regulating genes, with smaller reticulocytes as a result. Taken together, we demonstrate that PGC1β is directly involved in production of hemoglobin and regulation of G1-S transition and is ultimately required for proper terminal erythroid differentiation.

scholarly journals p53-Independent Cell Cycle and Erythroid Differentiation Defects in Murine Embryonic Stem Cells Haploinsufficient for Diamond Blackfan Anemia-Proteins: RPS19 versus RPL5

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e89098 ◽  
Author(s):  
Sharon A. Singh ◽  
Tracie A. Goldberg ◽  
Adrianna L. Henson ◽  
Sehba Husain-Krautter ◽  
Abdallah Nihrane ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Diamond Blackfan Anemia ◽  
Murine Embryonic Stem Cells

scholarly journals Immature erythroblasts with extensive ex vivo self-renewal capacity emerge from the early mammalian fetus

Blood ◽  
2011 ◽  
Vol 117 (9) ◽  
pp. 2708-2717 ◽  
Author(s):  
Samantha J. England ◽  
Kathleen E. McGrath ◽  
Jenna M. Frame ◽  
James Palis
Keyword(s):  
Stem Cell ◽  
Glucocorticoid Receptors ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Erythroid Progenitors ◽  
Transfusion Therapy ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Erythroid Precursors ◽  
Definitive Erythropoiesis

Abstract In the hematopoietic hierarchy, only stem cells are thought to be capable of long-term self-renewal. Erythroid progenitors derived from fetal or adult mammalian hematopoietic tissues are capable of short-term, or restricted (102- to 105-fold), ex vivo expansion in the presence of erythropoietin, stem cell factor, and dexamethasone. Here, we report that primary erythroid precursors derived from early mouse embryos are capable of extensive (106- to 1060-fold) ex vivo proliferation. These cells morphologically, immunophenotypically, and functionally resemble proerythroblasts, maintaining both cytokine dependence and the potential, despite prolonged culture, to generate enucleated erythrocytes after 3-4 maturational cell divisions. This capacity for extensive erythroblast self-renewal is temporally associated with the emergence of definitive erythropoiesis in the yolk sac and its transition to the fetal liver. In contrast, hematopoietic stem cell-derived definitive erythropoiesis in the adult is associated almost exclusively with restricted ex vivo self-renewal. Primary primitive erythroid precursors, which lack significant expression of Kit and glucocorticoid receptors, lack ex vivo self-renewal capacity. Extensively self-renewing erythroblasts, despite their near complete maturity within the hematopoietic hierarchy, may ultimately serve as a renewable source of red cells for transfusion therapy.

scholarly journals Hematopoietic stem cells differentiate into restricted myeloid progenitors before cell division

2018 ◽  
Author(s):  
Tatyana Grinenko ◽  
Anne Eugster ◽  
Lars Thielecke ◽  
Beata Ramazs ◽  
Anja Krueger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Division ◽  
Hematopoietic Stem Cells ◽  
Embryonic Stem ◽  
Erythroid Progenitors ◽  
Multipotent Stem Cells ◽  
Hematopoietic Stem ◽  
Myeloid Progenitors

SummaryHematopoietic stem cells (HSCs) continuously replenish all blood cell types through a series of differentiation steps that involve the generation of lineage-committed progenitors as well as necessary expansion due to repeated cell divisions. However, whether cell division in HSCs precedes differentiation is unclear. To this end, we used an HSC cell tracing approach and Ki67RFP knock-in mice to assess simultaneously divisional history, cell cycle progression, and differentiation of adult HSCs in vivo. Our results reveal that HSCs are able to differentiate into restricted progenitors, especially common myeloid progenitors, restricted megakaryocyte-erythroid progenitors (PreMEs) and pre-megakaryocyte progenitors (PreMegs), without undergoing cell division and even before entering the S phase of the cell cycle. Additionally, the phenotype of the undivided but differentiated progenitors correlated with expression of lineage-specific genes that manifested as functional differences between HSCs and restricted progenitors. Thus, HSC fate decisions appear to be uncoupled from physical cell division. Our results facilitate a better understanding of the mechanisms that control fate decisions in hematopoietic cells. Our data, together with separate findings from embryonic stem cells, suggest that cell division and fate choice are independent processes in pluripotent and multipotent stem cells.

scholarly journals Heme-regulated eIF2α kinase activated Atf4 signaling pathway in oxidative stress and erythropoiesis

Blood ◽  
2012 ◽  
Vol 119 (22) ◽  
pp. 5276-5284 ◽  
Author(s):  
Rajasekhar N. V. S. Suragani ◽  
Roshini S. Zachariah ◽  
Jason G. Velazquez ◽  
Sijin Liu ◽  
Chiao-Wang Sun ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Iron Deficiency ◽  
Signaling Pathway ◽  
Ex Vivo ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Eif2α Phosphorylation ◽  
Eif2α Kinase

Heme-regulated eIF2α kinase (Hri) is necessary for balanced synthesis of heme and globin. In addition, Hri deficiency exacerbates the phenotypic severity of β-thalassemia intermedia in mice. Activation of Hri during heme deficiency and in β-thalassemia increases eIF2α phosphorylation and inhibits globin translation. Under endoplasmic reticulum stress and nutrient starvation, eIF2α phosphorylation also induces the Atf4 signaling pathway to mitigate stress. Although the function of Hri in regulating globin translation is well established, its role in Atf4 signaling in erythroid precursors is not known. Here, we report the role of the Hri-activated Atf4 signaling pathway in reducing oxidative stress and in promoting erythroid differentiation during erythropoiesis. On acute oxidative stress, Hri−/− erythroblasts suffered from increased levels of reactive oxygen species (ROS) and apoptosis. During chronic iron deficiency in vivo, Hri is necessary both to reduce oxidative stress and to promote erythroid differentiation. Hri−/− mice developed ineffective erythropoiesis during iron deficiency with inhibition of differentiation at the basophilic erythroblast stage. This inhibition is recapitulated during ex vivo differentiation of Hri−/− fetal liver erythroid progenitors. Importantly, the Hri-eIF2αP-Atf4 pathway was activated and required for erythroid differentiation. We further demonstrate the potential of modulating Hri-eIF2αP-Atf4 signaling with chemical compounds as pharmaceutical therapies for β-thalassemia.

The cell cycle regulator CDC25A is a target for JAK2V617F oncogene

Blood ◽  
2012 ◽  
Vol 119 (5) ◽  
pp. 1190-1199 ◽  
Author(s):  
Emilie-Fleur Gautier ◽  
Muriel Picard ◽  
Camille Laurent ◽  
Caroline Marty ◽  
Jean-Luc Villeval ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Erythroid Differentiation ◽  
Primary Myelofibrosis ◽  
Initiation Factor ◽  
Proliferative Potential ◽  
Erythroid Progenitors ◽  
Translational Initiation ◽  
Cdc25a Phosphatase

Abstract The JAK2V617F mutation is present in the majority of patients with polycythemia vera and one-half of those with essential thrombocythemia and primary myelofibrosis. JAK2V617F is a gain-of-function mutation resulting in constitutive JAK2 signaling involved in the pathogenesis of these diseases. JAK2V617F has been shown to promote S-phase entry. Here, we demonstrate that the CDC25A phosphatase, a key regulator of the G1/S cell-cycle transition, is constitutively overexpressed in JAK2V617F-positive cell lines, JAK2-mutated patient CD36+ progenitors, and in vitro–differentiated proerythroblasts. Accordingly, CDC25A is overexpressed in BM and spleen of Jak2V617F knock-in mice compared with wild-type littermates. By using murine FDC-P1–EPOR and human HEL and SET-2 cell lines, we found that JAK2V617F-induced CDC25A up-regulation was caused neither by increased CDC25A transcription or stability nor by the involvement of its upstream regulators Akt and MAPK. Instead, our results suggest that CDC25A is regulated at the translational level through STAT5 and the translational initiation factor eIF2α. CDC25A inhibition reduces the clonogenic and proliferative potential of JAK2V617F-expressing cell lines and erythroid progenitors while moderately affecting normal erythroid differentiation. These results suggest that CDC25A deregulation may be involved in hematopoietic cells expansion in JAK2V617F patients, making this protein an attracting potential therapeutic target.

GFI-1B Is Increased during Erythroid Differentiation and Appears to Regulate Beta Globin Expression and Differentiation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1623-1623
Author(s):  
Stephen Sawyer ◽  
Jingchun Chen ◽  
Sarah Jacobs-Helber ◽  
Randolph Abutin
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Erythroid Differentiation ◽  
Binding Activity ◽  
Erythroid Cell ◽  
Beta Globin ◽  
Dna Arrays ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
Globin Synthesis

Abstract We identified GFI-1B in DNA arrays as a gene that correlates with erythroid differentiation induced by controlled expression of JunB in the HCD57 erythroid cell line, a novel model for erythroid differentiation we recently developed. JunB expression induces the HCD57 cells to turn bright red from hemoglobin synthesis. Beta Globin, Glycophorin (TER-119), alpha Spectrin and other erythroid marker are also induced by Jun B expression, suggesting the usefulness of the HCD57-JunB cell in studying normal erythroid differentiation. Our initial experiment was designed to identify potential genes that control erythroid differentiation so we analyzed gene expression in HCD57 cells induced by JunB in the first 24-hour period. Whereas the DNA array data found that the expression of all known transcription factors either was unchanged or decreased by induction of JunB and differentiation, GFI-1B RNA initially declined in the first 8 hours but then increased above control at 24 hours. In additional experiments, nuclear GFI-1B protein, and DNA binding activity were found to markedly increase during the late erythroid differentiation, 48 to 72 after induction of JunB to trigger the erythroid differentiation in HCD57 cells. GFI-1B expression and activity also increased when F-MEL cells were induced to differentiate with DMSO. Hemin treatment (to induce globin synthesis) also induced GFI-1B expression and GFI-1B DNA binding in murine HCD57 cells and human erythroid cell lines, UT7 and K562. When we ectopically expressed GFI-1B in human UT7 erythroleukemia cells, we found that elevation of GFI-1B expression reduced the rate of proliferation and markedly enhanced beta-globin synthesis when these cells were treated with hemin. Beta globin mRNA was increased 24 hours earlier in UT7 cell transfected with GFI-1B compared to control UT7 cells, suggesting that up-regulation of GFI-1B is needed for maximum beta globin expression. GFI-1B is selectively expressed in erythroid progenitors and megakaryocytes and was cloned by others using low stringency hybridization with a probe for Growth Factor Independence-1, GFI-1, a gene identified previously as a proto-oncogene induced in IL-2 independent T-cell lymphomas. GFI-1 and GFI-1B both have 5 zinc finger domains in the COOH terminal but are believed to act as transcriptional repressors through a novel 20 amino acid NH terminal domain. The sparse literature on GFI-1B and its homologue, the proto-oncogene GFI-1, suggests these SNAG/zinc finger proteins are repressors of transcription and act in general to prevent differentiation and promote proliferation of hematopoietic progenitor cells independent of normally required growth factors. Our current finding, however, suggests that GFI-1B function is distinct from the hyper-proliferation and anti-differentiation functions of the related GFI-1. Instead, our data suggests a clear role of GFI-1B in erythroid differentiation in agreement with loss of defintive erythropoiesis in GFI-1B-/- mice (recently reported from the Orkin laboratory).

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