GA Binding Protein (GABP) Is Required for Myeloid Cell Development and Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2713-2713
Author(s):  
Karen C. Drumea ◽  
Zhong-fa Yang ◽  
Alan G. Rosmarin
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Bac Library ◽  
Myeloid Cell ◽  
Cell Development ◽  
Transactivation Domain ◽  
Ets Domain ◽  
Floxed Allele

Abstract GABPα is an ets transcription factor that regulates genes that are required for innate immunity, including CD18 (β2 leukocyte integrin), lysozyme, and neutrophil elastase. GABP consists of two distinct and unrelated proteins that, together, form a functional transcription factor complex. GABPα binds to DNA through its ets domain and forms a multimeric complex by recruiting its partner, GABPβ, which contains the transactivation domain. GABPα is a single copy gene in both the human and murine genomes and it is the only protein that can recruit GABPβ to DNA. We cloned GABPα from a murine genomic BAC library and prepared a targeting vector in which the GABPα ets domain is flanked by loxP recombination sites (floxed allele, designated fl). Mice that bear one intact (and one disrupted copy) of GABPα, i.e. hemizygous mice, are phenotypically normal. Intercrossing of hemizygous mice yielded no nullizygous mice, indicating that homozygous loss of GABPα causes an embryonic lethal defect. To determine the effect of GABPα deletion on myeloid cell development, we bred heterozygous and homozygous floxed mice to mice that bear the interferon-responsive Mx1-Cre transgene, which express Cre in response to injection of the synthetic polynucleotide, poly I-C. Bone marrow cells underwent efficient deletion of GABPα following poly I-C injection; in contrast, other somatic tissues did not efficiently delete the floxed allele. Bone marrow, peripheral blood, and other tissues were examined for cellular morphology and flow cytometry. We compared mice that lack GABPα in bone marrow (i.e. fl/fl Mx1-Cre mice injected with poly I-C) to littermate controls (i.e. fl/fl mice injected with poly I-C). Mice that lack GABPα exhibited a striking and statistically significant decrease in granulocytes and monocytes in bone marrow and peripheral blood, compared with controls; in contrast, there was an increase in erythroid cells in GABPα null bone marrow. This indicates that the loss of GABPα has lineage-specific effects on myeloid cell development. Morphologic analysis indicates that mice which lack GABPα possess more immature granulocytes compared to control mice. Thus, GABP disruption causes a striking loss of myeloid cells in the bone marrow and peripheral blood of mice in a lineage-specific manner. Furthermore, the maturation block of murine granulocytes that is caused by GABPα disruption demonstrates the crucial role of GABP in myeloid differentiation.

GABP Transcription Factor Is Required for Myeloid Cell Development In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 374-374 ◽  
Author(s):  
Zhong-fa Yang ◽  
Karen Drumea ◽  
Alan G. Rosmarin
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
T Cell ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Development ◽  
Wild Type ◽  
Ets Domain

Abstract GABP is an ets transcription factor that regulates genes that are required for innate immunity, including CD18 (β2 leukocyte integrin), lysozyme, and neutrophil elastase. GABP consists of two distinct and unrelated proteins. GABPα binds to DNA through its ets domain and recruits GABPβ, which contains the transactivation domain; together, they form a functional tetrameric transcription factor complex. We recently showed that GABP is required for entry into S phase of the cell cycle through its regulation of genes that are required for DNA synthesis and cyclin dependent kinase inhibitors (Yang, et al. Nature Cell Biol9:339, 2007). Furthermore, GABP is an essential component of a retinoic acid responsive myeloid enhanceosome (Resendes and Rosmarin Mol Cell Biol26:3060, 2006). We cloned Gabpa (the gene that encodes mouse Gabpα) from a mouse genomic BAC library and prepared a targeting vector in which the ets domain is flanked by loxP recombination sites (floxed allele). Deletion of both floxed Gabpa alleles causes an early embryonic lethal defect. In order to define the role of Gabpα in myelopoiesis, we bred floxed Gabpa mice to mice that bear the Mx1-Cre transgene, which drives expression of Cre recombinase in response to injection of the synthetic polynucleotide, poly I-C. Deletion of Gabpa dramatically reduced granulocytes and monocytes in the peripheral blood, spleen, and bone marrow, but myeloid cells recovered within weeks. In vitro colony forming assays indicated that myeloid cells in these mice were derived only from Gabpa replete myeloid precursors (that failed to delete both Gabpa alleles), suggesting strong pressure to retain Gabpα in vivo. We used a novel competitive bone marrow transplantation approach to determine if Gabp is required for myeloid cell development in vivo. Sub-lethally irradiated wild-type recipient mice bearing leukocyte marker CD45.1 received equal proportions of bone marrow from wild type CD45.1 donor mice and floxed-Mx1-Cre donor mice that bear CD45.2. Both the CD45.2 (floxed-Mx1-Cre) and CD45.1 (wild type) bone marrow engrafted well. Mice were then injected with pI-pC to induce Cre-mediated deletion of floxed Gabpa. The mature myeloid and T cell compartments were derived almost entirely from wild type CD45.1 cells. This indicates that the proliferation and/or differentiation of myeloid and T cell lineages requires Gabp. In contrast, B cell development was not impaired. We conclude that Gabpa disruption causes a striking loss of myeloid cells in vivo and corroborates prior in vitro data that GABP plays a crucial role in proliferation of myeloid progenitor cells.

The Ets Transcription Factor, GABP, Is Required by Myeloid Progenitors for Normal Myeloid Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 256-256
Author(s):  
Zhongfa Yang ◽  
Karen Drumea ◽  
Junling Wang ◽  
James Cormier ◽  
Alan G. Rosmarin
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Transcriptional Repressor ◽  
Myeloid Cell ◽  
Myeloid Differentiation ◽  
Ets Domain

Abstract Abstract 256 GABP transcription factor regulates genes that are required for innate immunity. GABP is an obligate tetrameric protein complex that contains two molecules of GABPa, which binds to DNA through its ets domain, and two molecules of GABPb, which contains a transcription activation domain. GABP is an essential component of a multiprotein enhanceosome that is required for retinoic acid-dependent myeloid gene transcription. Disruption in mouse embryo fibroblasts of Gabpa, the mouse gene that encodes mouse Gabpa, causes profound cell cycle arrest at the G1-S boundary, due to reduced expression of DNA Polymerase a and Thymidylate Synthase, which are required for DNA synthesis, and of Skp2, a ubiquitin ligase that controls degradation of the cyclin-dependent kinase inhibitors (CDKIs), p21 and p27. Thus, GABP is a key regulator of the cell cycle. In order to define the role of GABP in myeloid differentiation, we generated mice in which exons that encode the Gabpa ets domain are flanked by loxP recombination sites, and bred these floxed mice to mice that bear the Mx1-Cre transgene. Their progeny were treated with pI-C and Gabpa was efficiently deleted in hematopoietic cells of these Gabpa−/− mice. As controls for all experiments, mice that bear Mx1-Cre but which lack the floxed Gabpa allele were also injected with pI-C. Within days, the peripheral blood white blood cell count fell in the Gabpa−/− mice compared to the controls; half of the Gabpa−/− mice died within two weeks. Gabpa−/− mice exhibited a striking loss of Gr1+, CD11b+ cells in the peripheral blood, spleen, and bone marrow. Myeloid cells of Gabpa−/− mice were immature, morphologically dysplastic, and demonstrated aberrant patterns of myeloid gene expression. Bone marrow from Gabpa−/− mice formed reduced numbers of in vitro myeloid colonies in the presence of G-CSF, M-CSF, or GM-CSF; cells isolated from in vitro colonies from Gabpa−/− mice exhibited a strong bias toward macrophage-like morphology. Multicolor flow cytometry revealed a loss of granulocyte-monocyte committed progenitor cells (GMPs) in the bone marrow of Gabpa−/− mice, and these progenitors expressed aberrant patterns of key transcription factors. Especially notable in Gabpa−/− GMPs was reduced expression of Gfi-1, a transcriptional repressor that is mutated in some congenital neutropenic syndromes, and whose genetic disruption causes abnormalities in granulocyte development. We used chromatin immunoprecipitation (ChIP) to identify ets sites in the Gfi-1 promoter that are bound by GABP in vivo. We conclude that GABP is required for proliferation or survival of committed myeloid progenitor cells and for normal maturation of granulocytes. We hypothesize that defects in myeloid cell proliferation and differentiation associated with Gabpa disruption are caused, at least in part, by its regulation of the Gfi-1 transcriptional repressor. Furthermore, we propose that the regulation of Gfi-1 by GABP constitutes a key regulatory pathway in myeloid cell development. Disclosures: No relevant conflicts of interest to declare.

Nrf2, a Critical Regulator of Oxidative Stress, Is Required for HSC Function and Cytokine Response.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1492-1492
Author(s):  
Akil Merchant ◽  
Anju Singh ◽  
Giselle Joseph ◽  
Qiuju Wang ◽  
Ping Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Increased Sensitivity ◽  
Marrow Cells

Abstract Abstract 1492 Poster Board I-515 Previous studies have established an important role for reactive oxygen species (ROS) in regulating the function and life-span of hematopoietic stem cells (HSC). Nuclear factor erythroid-2–related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates cellular responses to ROS and detoxification pathways implicated in chemoresistance, however, its role in normal stem cells is unknown. We analyzed Nrf2null mice and found increased total bone marrow cellularity, cKit+Sca1+Lin− (KSL) stem-progenitor cells, and long-term quiescent HSC (CD34−KSL) compared to wild type mice (p<0.05). Transplantation of equal numbers of KSL cells from Nrf2wt and Nrf2null resulted in a five-fold decrease in peripheral blood chimerism from Nrf2null derived cells at 16 weeks (15% wild type vs. 3% null, p<0.05). Unlike other models of deficiencies in genes associated with ROS handling, such as ATM or the FoxO family of transcription factors, basal ROS levels were not elevated in Nrf2null HSC. However, Nrf2null bone marrow cells demonstrated increased sensitivity to induced oxidative stress and in vitro treatment with H2O2 resulted in a 2 fold decrease in colony formation in methylcellulose. We also examined the in vivo sensitivity of Nrf2null cells to oxidative stress by irradiating (400 rads) stably chimeric mice 20 weeks following transplantation with either Nrf2wt or Nrf2null HSC. Mice receiving Nrf2null HSC demonstrated a 50% decrease in peripheral blood chimerism at 4 months following radiation compared to no change in Nrf2wt recipients (p<0.05) confirming that loss of Nrf2 leads to increased sensitivity to oxidative stress. Microarray gene expression analysis from Nrf2wt and Nrf2null mice revealed down regulation of the G-CSF cytokine receptor in Nrf2null HSC and suggested that defective cytokine signaling may contribute to the HSC dysfunction seen in Nrf2null bone marrow cells. To test this hypothesis, we attempted to rescue the function of Nrf2null HSC by treating mice with exogenous G-CSF. Nrf2wt and Nrf2null mice were treated with one week of daily G-CSF and then HSC were harvested and transplanted. In contrast to the defects in engraftment of untreated Nrf2null HSC, there was no significant difference in peripheral blood chimerism following transplantation of G-CSF treated Nrf2wt or Nrf2null HSC, thus demonstrating that G-CSF treatment could rescue the HSC defect in mutant mice. In conclusion, the Nrf2 transcription factor appears to be a novel and essential regulator of normal HSC function through the modulation of oxidative stress response and cytokine signaling. Disclosures: No relevant conflicts of interest to declare.

The Transcription Factor IRF8 is a Key Transcription Factor for Basophil Development

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1197-1197
Author(s):  
Daisuke Kurotaki ◽  
Haruka Sasaki ◽  
Naoki Osato ◽  
Izumi Sasaki ◽  
Chika Kaneda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Promoter Regions ◽  
Retroviral Transduction ◽  
Pass Through ◽  
Myeloid Progenitors

Abstract Basophils are the rarest granulocytes circulating in the peripheral blood. They play critical roles in anti-parasite Th2-type immune responses and chronic allergic disorders. The developmental pathway for basophils has been recently demonstrated; myeloid progenitors pass through common myeloid progenitors, granulocyte-monocyte progenitors, granulocyte-committed progenitors (GPs), and basophil-committed progenitors (BaPs) in the bone marrow. BaPs then give rise to mature basophils. However, our understanding of how this pathway is regulated remains still elusive. Interferon Regulatory Factor-8 (IRF8), a hematopoietic cell-specific IRF transcription factor, is essential for the development of monocytes, dendritic cells, and eosinophils, while it inhibits neutrophil differentiation. Its role in the development of basophils has yet to be analyzed. In this study, we investigated whether IRF8 has any role in the development of the basophil lineage. We found that Irf8–/– mice displayed a severe reduction of basophil counts in the bone marrow, peripheral blood and spleen compared to wild-type (WT) mice. Irf8–/– mice retained GPs but lacked BaPs. Cell transfer experiments revealed that the defect of basophil development in Irf8–/– mice resides in bone marrow cells. We utilized IRF8-GFP chimera knock-in mice to examine IRF8 protein expression in the basophil lineage at a single cell level. We found that GPs, but not BaPs and mature basophils, expressed IRF8. Furthermore, purified Irf8–/– GPs failed to efficiently give rise to basophils in vitro. These results indicate that IRF8 acts at the stage of GPs in a cell-intrinsic manner. To understand the mechanism by which IRF8 promotes basophil development, we performed transcriptome analysis of purified GPs from WT and Irf8–/– mice by microarray. Because IRF8 is no more expressed in BaPs, we envisaged that IRF8 acts by inducing downstream transcription factors in GPs. The expression of several transcription factor genes such as Gata2 and Spib was reduced in Irf8–/– GPs compared to WT GPs. Analysis of DNA motifs in the promoter regions of genes downregulated in Irf8–/– GPs predicted that GATA transcription factor(s) may act downstream of IRF8. Indeed, retroviral transduction of GATA2, known to be essential for basophil development, into Irf8–/– hematopoietic progenitor cells rescued basophil differentiation in vitro. On the other hand, Spib–/– mice showed no obvious defects in basophil development. Taken together, these results suggest that the IRF8-GATA2 axis in GPs critically regulates basophil development. Disclosures: No relevant conflicts of interest to declare.

Roles of MITF for development of mast cells in mice: effects on both precursors and tissue environments

Blood ◽  
2004 ◽  
Vol 104 (6) ◽  
pp. 1656-1661 ◽  
Author(s):  
Eiichi Morii ◽  
Keisuke Oboki ◽  
Katsuhiko Ishihara ◽  
Tomoko Jippo ◽  
Toshio Hirano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Mast Cell ◽  
Mast Cells ◽  
Normal Tissue ◽  
Bone Marrow Cells ◽  
Cell Development ◽  
Skin Transplantation ◽  
Marrow Cells

Abstract The mutant tg/tg mice, which do not express mi transcription factor (MITF), lack mast cells in most tissues. Since MITF is expressed in both mast cells and tissues where mast cells develop, there is a possibility that the tg/tg mice may show abnormalities in both mast cell precursors and tissue environments. We examined this possibility by bone marrow and skin transplantation. When bone marrow cells of tg/tg mice were transplanted to W/Wv mice that possess normal tissue environment, mast cells did not develop in all tissues examined. The number of developing mast cells in the skin of W/Wv mice was much lower when grafted to tg/tg recipients than when grafted to normal (+/+) recipients. These results indicated that mast cell precursors of tg/tg mice were defective. When bone marrow cells of +/+ mice were transplanted, the number of developing mast cells was significantly lower in examined tissues of tg/tg recipients than in those of W/Wv recipients, suggesting that the tissue environment for mast cell development was defective in tg/tg mice. MITF appeared essential for the function of both mast cell precursors and tissue environments for their development. (Blood. 2004;104:1656-1661)

scholarly journals Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2276-2285 ◽  
Author(s):  
Maria De La Luz Sierra ◽  
Paola Gasperini ◽  
Peter J. McCormick ◽  
Jinfang Zhu ◽  
Giovanna Tosato
Keyword(s):  
Bone Marrow ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Cell Function ◽  
Myeloid Cell ◽  
Cxcr4 Expression ◽  
Negative Regulator ◽  
Hematopoietic Stem

The mechanisms underlying granulocyte-colony stimulating factor (G-CSF)–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood remain elusive. We provide evidence that the transcriptional repressor growth factor independence-1 (Gfi-1) is involved in G-CSF–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We show that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4, a chemokine receptor essential for the retention of hematopoietic stem cells and granulocytic cells in the bone marrow. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also probably promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function.

scholarly journals Adult Mice With Targeted Mutation of the Interleukin-11 Receptor (IL11Ra) Display Normal Hematopoiesis

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2148-2159 ◽  
Author(s):  
Harshal H. Nandurkar ◽  
Lorraine Robb ◽  
David Tarlinton ◽  
Louise Barnett ◽  
Frank Köntgen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
White Blood Cells ◽  
Null Mutation ◽  
Wild Type ◽  
Normal Numbers ◽  
Interleukin 11 ◽  
Marrow Cells

Abstract Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the α-chain (IL-11Rα). Two genes potentially encode the IL-11Rα: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11Rα, we have generated mice with a null mutation of IL11Ra (IL11Ra−/−) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra−/− bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra−/− mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra−/− mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra−/− and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra−/− and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.

scholarly journals Analysis of interferon-inducible genes in cells of chronic myeloid leukemia patients responsive or resistant to an interferon-alpha treatment

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2337-2342
Author(s):  
IM Clauss ◽  
B Vandenplas ◽  
MG Wathelet ◽  
C Dorval ◽  
A Delforge ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Interferon Alpha ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Healthy Controls ◽  
Ifn Alpha ◽  
Inducible Genes ◽  
Marrow Cells

Recombinant human interferon-alpha (IFN-alpha) can induce a hematologic remission in patients with chronic myeloid leukemia. However, some patients are resistant and others develop late resistance to the IFN- alpha treatment. To understand the molecular mechanism of this resistance, we have analyzed the expression of 10 IFN-inducible genes in the cells of three resistant patients, two responsive patients, and six healthy controls. Northern blot hybridizations showed that all the genes were induced in in vitro IFN-alpha treated peripheral blood cells of the patients and healthy controls. These genes were also inducible in peripheral blood and bone marrow cells of two out of two resistant patients administered an injection of IFN-alpha. We conclude that the resistance to the IFN-alpha treatment of the chronic myeloid leukemia patients we studied is not due to (1) the absence of induction of any of the 10 IFN-inducible genes we studied, including the low-molecular- weight 2′-5′oligoadenylate synthetase; (2) the presence of an antagonist of IFN-alpha in the peripheral blood or bone marrow cells; and (3) the presence of neutralizing anti-IFN-alpha antibodies.

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