Transcriptional Regulation of a Distinct GATA-1 Isoform during Selection of the Mast and Erythroid Lineages.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1600-1600
Author(s):  
Clifford M. Takemoto ◽  
Amir H. Shahlaee ◽  
Ying Ye ◽  
Karen I. Zeller ◽  
Daniela Zablocki ◽  
...  
Keyword(s):  
Mast Cell ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
Mast Cells ◽  
Binding Sites ◽  
Genomic Sequence ◽  
Erythroid Cells ◽  
Mrna Isoform ◽  
In Erythroid Cells

Abstract Current models of hematopoiesis suggest that in early, pluripotent progenitor cells, lineage-specific transcription factors are expressed at low levels. During differentiation, subsets of these transcription factors become dominantly expressed in a lineage-restricted fashion. Understanding how transcription factors are expressed in distinct cell-types is central to defining the regulatory events that occur during lineage selection. GATA-1 is an essential transcriptional regulator for the erythroid and megakaryocyte lineages, while it is absent in neutrophils and monocytes. PU.1, on the other hand, is a critical transcription factor for neutrophils and monocytes, but it is not abundantly expressed in erythroid cells. Although these two factors have been shown to be antagonistic in monocytic and erythroid cells, both GATA-1 and PU.1 are required for the normal development of the mast lineage (Migliaccio et al., 2003, Walsh et al., 2002). Here we show that mast cells express a unique mRNA isoform of GATA-1 that is distinct from the major erythroid/megakaryocyte isoform. It is related, but not identical to the Ib transcript that has been described as a minor expressed form in erythroid cells (Tsai et al., 1991) and as a major expressed form in RNA isolated from CFU-GM primary myeloid cultures (Seshasayee et al., 2000). This GATA-1 mast cell isoform (GATA-1mast) differs from the erythroid/megakaryocyte isoform by a unique, untranslated first exon that is alternatively spliced onto the downstream coding exons. In mast cells, GATA-1mast is expressed from a promoter separate from that utilized in megakaryocytic and erythroid cells. Comparative analysis of genomic sequence of the GATA-1 locus in this region reveals modules of extensive phylogenetic conservation in mammals, including stretches containing both highly conserved PU.1 and GATA binding sites. We have performed chromatin immunoprecipitation studies with GATA-1 antibodies and have defined multiple regions of in vivo binding within the GATA-1 locus in erythroid cells. Addtional studies are underway utilizing the Scanning ChIP procedure (Zeller et al., 2001) to determine in vivo GATA-1, GATA-2, and PU.1 binding sites of these factors to the GATA-1 locus in mast cells. In order to determine whether PU.1 positively regulates the expression of the mast cell GATA-1 isoform, we have examined GATA-1mast expression in PU.1 −/ − cells. PU.1 −/ − fetal liver cells cannot differentiate into mast cells in vitro; reintroduction of PU.1 expression restores mast cell differentiation. We show that PU.1 −/ − cells are deficient in expression of the GATA-1 mast cell mRNA isoform, and reintroduction of PU.1 into the PU.1 deficient cells markedly up-regulates the expression of GATA-1mast. Our findings demonstrate that PU.1 positively regulates a distinct GATA-1 isoform during mast cell differentiation. We propose a model in which GATA factors cooperate with PU.1 to direct cell-specific isoforms of transcriptional regulators during hematopoietic development.

Development of hematopoietic cells lacking transcription factor GATA-1

Development ◽  
1995 ◽  
Vol 121 (1) ◽  
pp. 163-172 ◽  
Author(s):  
L. Pevny ◽  
C.S. Lin ◽  
V. D'Agati ◽  
M.C. Simon ◽  
S.H. Orkin ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Mast Cell ◽  
Cell Differentiation ◽  
Mast Cells ◽  
Blood Cell ◽  
Embryonic Stem ◽  
Sequence Motif ◽  
Erythroid Cells ◽  
Gata Factor

GATA-1 is a zinc-finger transcription factor believed to play an important role in gene regulation during the development of erythroid cells, megakaryocytes and mast cells. Other members of the GATA family, which can bind to the same DNA sequence motif, are co-expressed in several of these hemopoietic lineages, raising the possibility of overlap in function. To examine the specific roles of GATA-1 in hematopoietic cell differentiation, we have tested the ability of embryonic stem cells, carrying a targeted mutation in the X-linked GATA-1 gene, to contribute to various blood cell types when used to produce chimeric embryos or mice. Previously, we reported that GATA-1- mutant cells failed to contribute to the mature red blood cell population, indicating a requirement for this factor at some point in the erythroid lineage (L. Pevny et al., (1991) Nature 349, 257–260). In this study, we have used in vitro colony assays to identify the stage at which mutant erythroid cells are affected, and to examine the requirement for GATA-1 in other lineages. We found that the development of erythroid progenitors in embryonic yolk sacs was unaffected by the mutation, but that the cells failed to mature beyond the proerythroblast stage, an early point in terminal differentiation. GATA-1- colonies contained phenotypically normal macrophages, neutrophils and megakaryocytes, indicating that GATA-1 is not required for the in vitro differentiation of cells in these lineages. GATA-1- megakaryocytes were abnormally abundant in chimeric fetal livers, suggesting an alteration in the kinetics of their formation or turnover. The lack of a block in terminal megakaryocyte differentiation was shown by the in vivo production of platelets expressing the ES cell-derived GPI-1C isozyme. The role of GATA-1 in mast cell differentiation was examined by the isolation of clonal mast cell cultures from chimeric fetal livers. Mutant and wild-type mast cells displayed similar growth and histochemical staining properties after culture under conditions that promote the differentiation of cells resembling mucosal or serosal mast cells. Thus, the mast and megakaryocyte lineages, in which GATA-1 and GATA-2 are co-expressed, can complete their maturation in the absence of GATA-1, while erythroid cells, in which GATA-1 is the predominant GATA factor, are blocked at a relatively early stage of maturation.

Thrombopoietin Regulates Proliferation and Maturation of Murine Mast Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1707-1707
Author(s):  
Giovanni Migliaccio ◽  
Barbara Ghinassi ◽  
Lucia Centurione ◽  
Maria Zingariello ◽  
Lucia Bianchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Cell Differentiation ◽  
Growth Factor ◽  
Mast Cells ◽  
Wild Type ◽  
Connective Tissues ◽  
Immature Cells

Abstract Megakaryocytopoiesis is regulated by extrinsic (interaction of the growth factor thrombopoietin, TPO with its receptor Mpl) and intrinsic (interaction between the trascription factors GATA-1 and Fog-1) factors. The observation that mice impaired for GATA-1 expression (i.e. harbouring the GATA-1low mutation) are defective not only in megakaryocyte maturation but also in mast cell differentiation (Migliaccio et al. J Exp Med197:281, 2003), led us to investigate whether TPO might control mast cell differentiation as well. We first observed that mice genetically unable to responde to TPO (Mplnull mice) express in the connective tissues 5 times more mast cells than their normal littermates. Then, we analysed the effects on mast cell differentiation of in vivo treatment with TPO. Normal mice, and their GATA-1low littermates, were injected i.p. with TPO (100 μg/kg/day per 5 days, kindly provided by Kirin Brewery, Japan) and the number of immature (Toluidinepos) and mature (AlcianBlue/Saphraninepos) mast cells present in the connective tissues of the animals, as well as the frequency of GATA-1pos and TUNELpos mast cells, was evaluated 14 days after treatment. In wild-type animals, TPO reduced the presence of GATA-1 in mast cells (by immuno-histochemistry) and increased the number of immature cells (from 320±28 to 852±60) and of those undergoing apoptosis (from 16±1 to 600±43). In contrast, in GATA-1low animals, TPO-treatment induced the expression of GATA-1 in mast cells while decreased the number of immature cells (from 1100±72 to 427±29) as well as that of apoptotic cells (from 600±45 to 60±2). The role of TPO on mast cell differentiation were further confirmed by the analysis of the effects exerted by the growth factor on in vitro differentiation of bone marrow derived mast cells (BMMC). In these experiments, wild type bone marrow and spleen cells were cultured for 21 days with SCF and IL-3 with or without TPO and BMMC differentiation measured on the basis of the number of cells expressing the phenotype c-kithigh/CD34high and FcεRIpos. In cultures stimulated with SCF and IL-3, all the cells expressed the phenotype c-kithigh/CD34high and FcεRIpos. In contrast, in cultures supplemented also with SCF, IL-3 and TPO, only 25% of the cells were c-kithigh/CD34high and none of them was FcεRIpos. These results establish a role for TPO in the control of mast cell differentiation (possibly by modulating the GATA-1 content of the cells) and unveil further similarities between the mechanism(s) controlling megakaryocyte and mast cell differentiation.

Novel Functional Roles for Ten-Eleven-Translocation 2 (Tet2) in Normal and Leukemic Growth of Mast Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 775-775
Author(s):  
Raghuveer Mali ◽  
Holly Rene Martin ◽  
Baskar Ramdas ◽  
Lakshmi Palam ◽  
Valeria Visconte ◽  
...  
Keyword(s):  
Mast Cell ◽  
Cell Differentiation ◽  
Mast Cells ◽  
Cell Development ◽  
Dna Demethylation ◽  
Human Diseases ◽  
Kit Mutation ◽  
Altered Expression ◽  
Kit Receptor

Abstract KIT receptor signaling plays an important role in mast cell development. Gain-of-function mutations in KIT receptor have been identified in human diseases including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM) and acute myeloid leukemia (AML). Although KIT mutations found in GIST are sensitive to imatinib, KIT mutation (KITD816V) found in 90% of SM patients is imatinib-resistant and currently no therapies are available to treat the human diseases associated with this mutation. Our recent studies have identified Ten-Eleven-Translocation 2 (TET2) mutations in ~23% of SM patients and are associated with poor prognosis and overall survival. TET2 is a methylcytosine dioxygenase that plays a vital role in active DNA demethylation. Recent studies suggest that patients with mutations in TET2 and KITD816V develop more aggressive form of mastocytosis with worse prognosis. Although it is known that TET2 and KITD816V cooperate in SM patients, it is not clear how they cooperate with each other and what is the physiologic role of TET2 in normal mast cell development. We show that loss of Tet2 results in impaired maturation of mast cells in vivo and in bone marrow-derived mast cells (BMMC) compared to WT controls, which is associated with reduction in 5-hmc levels compared to WT BMMCs. We also observed reduction in the expression of mast cell-specific genesincluding Mast cell proteinase-5 (MCP-5), Mast cell proteinase-6 (MCP-6) and Carboxypeptidase A (CPA). To determine the mechanism behind altered mast cell differentiation in Tet2-/- BMMCs, we performed RNA-seq analysis in WT and Tet2-/- mast cells and observed altered expression of various genes involved in development of mast cells including Kit, FcεR1, Mitf, Notch, and Myc. We further confirmed altered expression of Mitf, Gata-2, and PU.1 in Tet2-/- BMMCs compared to WT BMMCs by western blotting. Since Tet2 regulates DNA demethylation, we tested whether altered BMMC differentiation in Tet2-/- mice is due to enhanced DNA methylation. We treated WT or Tet2-/- BM cells for 3 weeks with vehicle or 5-azacytidine (hypomethylating agent) and analyzed mast cell differentiation. Treatment with 5-azacytidine completely corrected the defective mast cell differentiation in Tet2-/- cells to WT levels. These results suggest that Tet2 plays a significant role in mast cell differentiation by regulating the expression of critical transcription factors including Mitf, Gata-2 and PU.1. We next analyzed the growth of Tet2-/- BMMCs in response to cytokines. Tet2-deficient BMMCs show enhanced cytokine mediated growth compared to WT BMMCs. Hyper-proliferation of Tet2-/- BMMCs is associated with reduced expression of tumor suppressor, PTEN, whose promoter is hypermethylated and a concomitant increase in the activation of the PI3K/AKT pathway. Since loss of function TET2 mutations have been observed in SM patients in addition to KITD816V mutation, we tested whether loss of Tet2 cooperates with KIT mutation in vitro and in vivo. Tet2-deficiency or knockdown in conjunction with the expression of KIT mutation resulted in significantly enhanced growth compared to cells bearing KIT mutation alone or lacking Tet2 expression. Likewise in human mastocytosis xenograft model, significantly enlarged tumors were observed in NSG mice transplanted with human mastocytosis cell line bearing the KITD816V mutation (HMC1.2) and knockdown of TET2 compared to HMC1.2 cells bearing only the KITD816V mutation. The cooperation between loss of Tet2 and KIT mutation was associated with further increase in PI3K/AKT activation and pharmacologic inhibitor treatment with a PI3K inhibitor GDC-0941 (Pan PI3K), but not TGX221 (p110β-specific) or IC87114 (p110δ-specific), significantly reduced the hyper-proliferation of Tet2-/- BMMCs and cell lines as well as primary BM blasts derived from SM patients bearing the KITD816V mutation. Consistently, combined loss of p110α and p110δ subunits of PI3K resulted in the most profound growth repression in oncogenic KIT bearing BM cells, but did not correct altered differentiation in Tet2-/- BMMCs. Taken together our results suggest that combinational therapy involving 5-azacytidine (which corrects the impaired mast cell differentiation) and PI3K inhibitor (which corrects the excessive proliferation) is a better therapeutic option for treating human mastocytosis patients bearing TET2 and KIT mutations. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inactivation of thrombin by a complex between rat mast-cell protease 1 and heparin proteoglycan

1994 ◽  
Vol 299 (2) ◽  
pp. 507-513 ◽  
Author(s):  
G Pejler ◽  
K Söderström ◽  
A Karlström
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Binding Sites ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Macromolecular Complex ◽  
Mast Cell Protease ◽  
Peritoneal Mast Cells ◽  
Terminal Amino

Rat peritoneal mast cells were shown to inactivate thrombin rapidly. The thrombin-inactivating activity was purified to homogeneity by a combination of anion-exchange chromatography and h.p.l.c. on a Superdex 75 column. The purified thrombin inactivator had an apparent molecular mass of 29 kDa and an N-terminal amino acid sequence identical to rat mast-cell protease 1 (RMCP-1). After labelling of the mast cells in vivo with 35SO4(2-), RMCP-1 was recovered in a macromolecular complex with [35S]heparin proteoglycans. Dissociation of RMCP-1 from the heparin proteoglycans by Superdex 75 chromatography in the presence of 2 M NaCl resulted in a marked loss of the thrombin-inactivating activity displayed by the enzyme. When RMCP-1 was reconstituted with either endogenous [35S]heparin proteoglycans or standard pig mucosal heparin, the enzyme regained its thrombin-inactivating properties. Affinity chromatography of endogenous [35S]heparin on matrix-linked RMCP-1 demonstrated that all of the heparin molecules contained high-affinity binding sites for the mast-cell protease. In contrast, the endogenous mast-cell heparin showed low affinity for antithrombin, a protease inhibitor involved in the regulation of coagulation enzymes.

GATA-1 Forms Distinct Activating and Repressive Complexes in Erythroid Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 356-356
Author(s):  
John Strouboulis ◽  
Patrick Rodriguez ◽  
Edgar Bonte ◽  
Jeroen Krijgsveld ◽  
Katarzyna Kolodziej ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Chromatin Remodeling ◽  
Gene Activation ◽  
Erythroid Differentiation ◽  
Specific Gene ◽  
Erythroid Cells ◽  
In Erythroid Cells

Abstract GATA-1 is a key transcription factor essential for the differentiation of the erythroid, megakaryocytic and eosinophilic lineages. GATA-1 functions in erythropoiesis involve lineage-specific gene activation and repression of early hematopoietic transcription programs. GATA-1 is known to interact with other transcription factors, such as FOG-1, TAL-1 and Sp1 and also with CBP/p300 and the SWI/SNF chromatin remodeling complex in vitro. Despite this information the molecular basis of its essential functions in erythropoiesis remains unclear. We show here that GATA-1 is mostly present in a high (> 670kDa) molecular weight complex that appears to be dynamic during erythroid differentiation. In order to characterize the GATA-1 complex(es) from erythroid cells, we employed an in vivo biotinylation tagging approach in mouse erythroleukemic (MEL) cells1. Briefly, this involved the fusion of a small (23aa) peptide tag to GATA-1 and its specific, efficient biotinylation by the bacterial BirA biotin ligase which is co-expressed with tagged GATA-1 in MEL cells. Nuclear extracts expressing biotinylated tagged GATA-1 were bound directly to streptavidin beads and co-purifying proteins were identified by mass spectrometry. In addition to the known GATA-1-interacting transcription factors FOG-1, TAL-1 and Ldb-1, we describe novel interactions with the essential hematopoietic transcription factor Gfi-1b and the chromatin remodeling complexes MeCP1 and ACF/WCRF. Significantly, GATA-1 interaction with the repressive MeCP1 complex requires FOG-1. We also show in erythroid cells that GATA-1, FOG-1 and MeCP1 are stably bound to repressed genes representing early hematopoietic (e.g. GATA-2) or alternative lineage-specific (e.g. eosinophilic) transcription programs, whereas the GATA-1/Gfi1b complex is bound to repressed genes involved in cell proliferation. In contrast, GATA-1 and TAL-1 are bound to the active erythroid-specific EKLF gene. Our findings on GATA-1 complexes provide novel insight as to the critical roles that GATA-1 plays in many aspects of erythropoiesis by revealing the GATA-1 partners in the execution of specific functions.

scholarly journals Transcriptional up-regulation of the mouse cytosolic glutathione peroxidase gene in erythroid cells is due to a tissue-specific 3' enhancer containing functionally important CACC/GT motifs and binding sites for GATA and Ets transcription factors.

1993 ◽  
Vol 13 (10) ◽  
pp. 6290-6303 ◽  
Author(s):  
J O'Prey ◽  
S Ramsay ◽  
I Chambers ◽  
P R Harrison
Keyword(s):  
Transcription Factors ◽  
Glutathione Peroxidase ◽  
Binding Sites ◽  
Erythroid Cell ◽  
Transcriptional Level ◽  
Erythroid Cells ◽  
Peroxidase Gene ◽  
Cytosolic Glutathione ◽  
Low Expression ◽  
In Erythroid Cells

Nuclear run-on experiments have shown that the high level of expression of the mouse cytosolic glutathione peroxidase mRNA in erythroid cells is due to up-regulation of the gene at the transcriptional level. Studies of the chromatin structure around the cytosolic glutathione peroxidase gene have revealed a series of DNase I hypersensitive sites (DHSS) in the 3' flanking region of the gene in erythroid and other high-expression tissues that are lacking in low-expression cells, in addition to a DHSS over the promoter region in both high- and low-expression tissues. Functional transfection experiments have demonstrated that one of the 3' DHSS regions functions as an enhancer in erythroid cells but not in a low-expression epithelial cell line; and site-directed mutagenesis and footprinting experiments reveal that the activity of the erythroid cell-specific enhancer requires a cluster of binding sites for the CACC/GT box factors and the GATA and Ets families of transcription factors.

Transcriptional up-regulation of the mouse cytosolic glutathione peroxidase gene in erythroid cells is due to a tissue-specific 3' enhancer containing functionally important CACC/GT motifs and binding sites for GATA and Ets transcription factors

1993 ◽  
Vol 13 (10) ◽  
pp. 6290-6303
Author(s):  
J O'Prey ◽  
S Ramsay ◽  
I Chambers ◽  
P R Harrison
Keyword(s):  
Transcription Factors ◽  
Glutathione Peroxidase ◽  
Binding Sites ◽  
Erythroid Cell ◽  
Transcriptional Level ◽  
Erythroid Cells ◽  
Peroxidase Gene ◽  
Cytosolic Glutathione ◽  
Low Expression ◽  
In Erythroid Cells

Nuclear run-on experiments have shown that the high level of expression of the mouse cytosolic glutathione peroxidase mRNA in erythroid cells is due to up-regulation of the gene at the transcriptional level. Studies of the chromatin structure around the cytosolic glutathione peroxidase gene have revealed a series of DNase I hypersensitive sites (DHSS) in the 3' flanking region of the gene in erythroid and other high-expression tissues that are lacking in low-expression cells, in addition to a DHSS over the promoter region in both high- and low-expression tissues. Functional transfection experiments have demonstrated that one of the 3' DHSS regions functions as an enhancer in erythroid cells but not in a low-expression epithelial cell line; and site-directed mutagenesis and footprinting experiments reveal that the activity of the erythroid cell-specific enhancer requires a cluster of binding sites for the CACC/GT box factors and the GATA and Ets families of transcription factors.

In-vitro and in-vivo Evaluation of Anti-asthmatic Activity of Eugenia jambolana bark

2021 ◽  
pp. 3337-3342
Author(s):  
Bhong Prabha N. ◽  
Naikawade Nilofar. S. ◽  
Mali Pratibha. R. ◽  
Bindu Madhavi. S.
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Animal Models ◽  
Aqueous Extract ◽  
Guinea Pigs ◽  
Bark Extract ◽  
Eugenia Jambolana ◽  
In Vivo Animal Models

Objectives: The present study designed to evaluate the Antiasthmatic activity of aqueous extract of bark of Eugenia Jambolana (AEEJ) on in vitro and in vivo animal models. Materials and methods: Different in vitro and in vivo animal models was used to study the anti asthmatic activity as isolated goat tracheal chain preparation, Acetylcholine and Histamine induced bronconstriction in guinea pigs, effect of drug extract on histamine release from mast cell was checked by clonidine-induced mast cell degranulation, and milk-induced eosinophilia and leukocytosis. Results: In-vitro study on goat tracheal chain preparation revealed that aqueous extract of Eugenia jambolana (AEEJ)bark exerted antagonistic effect on the histamine induced contraction. (P<0.05) The guinea pigs when exposed to 0.2% histamine aerosol showed signs of progressive dyspnoea leading to convulsions. AEEJ significantly prolonged the latent period of convulsions (PCT) as compared to control following the exposure of histamine (0.2%) aerosol (P<0.01). The observation of present study indicates aqueous extract of Eugenia jambolana shows significant inhibition of milk induced eosinophilia and leukocytosis. Group of animals pretreated with aqueous Eugenia jambolana bark extract showed significant reduction in degranulation of mast cells when challenged with clonidine. The prevention of degranulation process by the aqueous Eugenia jambolana bark extract (P<0.01) indicates a possible stabilizing effect on the mast cells, indicating mast cell stabilizing activity. Conclusions: Thus, AEEJ showed antihistaminic, mast cell stabilizing and protective in guinea pigs against histamine induced PCD, reduced eosinophilia and leukocytosis and hence possesses potential role in the treatment of asthma.

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