scholarly journals Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line.

1997 ◽  
Vol 17 (3) ◽  
pp. 1642-1651 ◽  
Author(s):  
M J Weiss ◽  
C Yu ◽  
S H Orkin
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Zinc Finger ◽  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Transactivation Domain ◽  
Stage Of Development ◽  
Erythroid Maturation

The zinc finger transcription factor GATA-1 is essential for erythropoiesis. In its absence, committed erythroid precursors arrest at the proerythroblast stage of development and undergo apoptosis. To study the function of GATA-1 in an erythroid cell environment, we generated an erythroid cell line from in vitro-differentiated GATA-1- murine embryonic stem (ES) cells. These cells, termed G1E for GATA-1- erythroid, proliferate as immature erythroblasts yet complete differentiation upon restoration of GATA-1 function. We used rescue of terminal erythroid maturation in G1E cells as a stringent cellular assay system in which to evaluate the functional relevance of domains of GATA-1 previously characterized in nonhematopoietic cells. At least two major differences were established between domains required in G1E cells and those required in nonhematopoietic cells. First, an obligatory transactivation domain defined in conventional nonhematopoietic cell transfection assays is dispensable for terminal erythroid maturation. Second, the amino (N) zinc finger, which is nonessential for binding to the vast majority of GATA DNA motifs, is strictly required for GATA-1-mediated erythroid differentiation. Our data lead us to propose a model in which a nuclear cofactor(s) interacting with the N-finger facilitates transcriptional action by GATA-1 in erythroid cells. More generally, our experimental approach highlights critical differences in the action of cell-specific transcription proteins in different cellular environments and the power of cell lines derived from genetically modified ES cells to elucidate gene function.

315 MELATONIN EFFECTS ON THE PROLIFERATION AND DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 254
Author(s):  
Y.-M. Yoo ◽  
E.-B. Jeung
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Zinc Finger ◽  
Cellular Proliferation ◽  
Ex Vivo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Melatonin Treatment ◽  
Proliferation And Differentiation

Mouse embryonic stem (ES) cells constitute a versatile biological system that can facilitate major advances in the fields of cell and developmental biology. Several studies have been performed to determine whether melatonin can affect ex vivo and in vitro proliferation and differentiation of stem cells (mesenchymal and neural stem cells derived human, rats, and mice), but its effect on ES cells is largely unknown. Thus, we further examined in this study the effects of melatonin at biological or pharmacological concentrations (100 or 200 μM) on the proliferation and differentiation of ES cells (ES-E14TG2a cells) using an in vitro culture system (n = 3) by Western blot analysis and real-time PCR. We found that melatonin at 100 and 200 μM resulted in cellular proliferation and phosphorylation of ERK and Akt, respectively. Melatonin treatment also increased Bcl-2 expression and suppressed Bax gene expression and increased phosphorylation of GSK α/β. The transcription factor Oct-4, which contains the POU (N-terminal to homeobox) domain, and the transcription factor Sox2, the zinc finger transcription factor Zfp206, and the zinc finger gene REX-1 (Znf42), which contain the high mobility group domain, are all important for cellular pluripotency and preimplantation development. In this study, melatonin (100 μM) treatment induced Oct-4 and REX-1 expression at day 1 but not at days 2 and 3. In addition, Sox2 and Zfp206 expressions were not altered following melatonin treatment. Taken together, these results suggest that melatonin may affect Akt phosphorylation and stem cell proliferation at biological or pharmacological concentrations.

scholarly journals Transcription Factor Lbx1 Expression in Mouse Embryonic Stem Cell-Derived Phenotypes

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Stefanie Schmitteckert ◽  
Cornelia Ziegler ◽  
Liane Kartes ◽  
Alexandra Rolletschek
Keyword(s):  
Transcription Factor ◽  
Developmental Stages ◽  
Troponin T ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Cardiac Cells ◽  
Cell Stage

Transcription factor Lbx1 is known to play a role in the migration of muscle progenitor cells in limb buds and also in neuronal determination processes. In addition, involvement of Lbx1 in cardiac neural crest-related cardiogenesis was postulated. Here, we used mouse embryonic stem (ES) cells which have the capacity to develop into cells of all three primary germ layers. Duringin vitrodifferentiation, ES cells recapitulate cellular developmental processes and gene expression patterns of early embryogenesis. Transcript analysis revealed a significant upregulation ofLbx1at the progenitor cell stage. Immunofluorescence staining confirmed the expression of Lbx1 in skeletal muscle cell progenitors and GABAergic neurons. To verify the presence of Lbx1 in cardiac cells, triple immunocytochemistry of ES cell-derived cardiomyocytes and a quantification assay were performed at different developmental stages. Colabeling of Lbx1 and cardiac specific markers troponin T, α-actinin, GATA4, and Nkx2.5 suggested a potential role in early myocardial development.

Mouse GATA-4: a retinoic acid-inducible GATA-binding transcription factor expressed in endodermally derived tissues and heart

1993 ◽  
Vol 13 (4) ◽  
pp. 2235-2246
Author(s):  
R J Arceci ◽  
A A King ◽  
M C Simon ◽  
S H Orkin ◽  
D B Wilson
Keyword(s):  
Transcription Factor ◽  
Retinoic Acid ◽  
Zinc Finger ◽  
Intestinal Epithelium ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Primitive Endoderm ◽  
Developmentally Regulated ◽  
The Family

We report the cDNA cloning and characterization of mouse GATA-4, a new member of the family of zinc finger transcription factors that bind a core GATA motif. GATA-4 cDNA was identified by screening a 6.5-day mouse embryo library with oligonucleotide probes corresponding to a highly conserved region of the finger domains. Like other proteins of the family, GATA-4 is approximately 50 kDa in size and contains two zinc finger domains of the form C-X-N-C-(X17)-C-N-X-C. Cotransfection assays in heterologous cells demonstrate that GATA-4 trans activates reporter constructs containing GATA promoter elements. Northern (RNA) analysis and in situ hybridization show that GATA-4 mRNA is expressed in the heart, intestinal epithelium, primitive endoderm, and gonads. Retinoic acid-induced differentiation of mouse F9 cells into visceral or parietal endoderm is accompanied by increased expression of GATA-4 mRNA and protein. In vitro differentiation of embryonic stem cells into embryoid bodies is also associated with increased GATA-4 expression. We conclude that GATA-4 is a tissue-specific, retinoic acid-inducible, and developmentally regulated transcription factor. On the basis of its tissue distribution, we speculate that GATA-4 plays a role in gene expression in the heart, intestinal epithelium, primitive endoderm, and gonads.

scholarly journals Mouse GATA-4: a retinoic acid-inducible GATA-binding transcription factor expressed in endodermally derived tissues and heart.

1993 ◽  
Vol 13 (4) ◽  
pp. 2235-2246 ◽  
Author(s):  
R J Arceci ◽  
A A King ◽  
M C Simon ◽  
S H Orkin ◽  
D B Wilson
Keyword(s):  
Transcription Factor ◽  
Retinoic Acid ◽  
Zinc Finger ◽  
Intestinal Epithelium ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Primitive Endoderm ◽  
Developmentally Regulated ◽  
The Family

We report the cDNA cloning and characterization of mouse GATA-4, a new member of the family of zinc finger transcription factors that bind a core GATA motif. GATA-4 cDNA was identified by screening a 6.5-day mouse embryo library with oligonucleotide probes corresponding to a highly conserved region of the finger domains. Like other proteins of the family, GATA-4 is approximately 50 kDa in size and contains two zinc finger domains of the form C-X-N-C-(X17)-C-N-X-C. Cotransfection assays in heterologous cells demonstrate that GATA-4 trans activates reporter constructs containing GATA promoter elements. Northern (RNA) analysis and in situ hybridization show that GATA-4 mRNA is expressed in the heart, intestinal epithelium, primitive endoderm, and gonads. Retinoic acid-induced differentiation of mouse F9 cells into visceral or parietal endoderm is accompanied by increased expression of GATA-4 mRNA and protein. In vitro differentiation of embryonic stem cells into embryoid bodies is also associated with increased GATA-4 expression. We conclude that GATA-4 is a tissue-specific, retinoic acid-inducible, and developmentally regulated transcription factor. On the basis of its tissue distribution, we speculate that GATA-4 plays a role in gene expression in the heart, intestinal epithelium, primitive endoderm, and gonads.

GA Binding Protein (GABP) Is Required for Development and Maturation of Myeloid Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 776-776
Author(s):  
Zhongfa Yang ◽  
Alan G. Rosmarin
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Target Genes ◽  
Embryonic Stem ◽  
Myeloid Cells ◽  
Transactivation Domain ◽  
Wild Type ◽  
Floxed Allele

Abstract GABP is an ets transcription factor that regulates transcription of key myeloid genes, including CD18 (beta2 leukocyte integrin), neutrophil elastase, lysozyme, and other key mediators of the inflammatory response; it is also known to regulate important cell cycle control genes. GABP consists of two distinct and unrelated proteins that, together, form a functional transcription factor complex. GABPalpha (GABPa) is an ets protein that binds to DNA; it forms a tetrameric complex by recruiting its partner, GABPbeta (GABPb), which contains the transactivation domain. GABPa is a single copy gene in both the human and murine genomes and it is the only protein that can recruit GABPb to DNA. We cloned GABPa from a murine genomic BAC library and prepared a targeting vector in which exon 9 (which encodes the GABPa ets domain) was flanked by loxP (floxed) recombination sites. The targeting construct was electroporated into embryonic stem cells, homologous recombinants were implanted into pseudopregnant mice, heterozygous floxed GABPa mice were identified, and intercrossing yielded expected Mendelian ratios of wild type, heterozygous, and homozygous floxed GABPa mice. Breeding of heterozygous floxed GABPa mice to CMV-Cre mice (which express Cre recombinase in all tissues) yielded expected numbers of hemizygous mice (only one intact GABPa allele), but no nullizygous (GABPa−/−) mice among 64 pups; we conclude that homozygous deletion of GABPa causes an embryonic lethal defect. To determine the effect of GABPa deletion on myeloid cell development, we bred heterozygous and homozygous floxed mice to LysMCre mice, which express Cre only in myeloid cells. These mice had a normal complement of myeloid cells but, unexpectedly, PCR indicated that their Gr1+ myeloid cells retained an intact (undeleted) floxed GABPa allele. We detected similar numbers of in vitro myeloid colonies from bone marrow of wild type, heterozygous floxed, and homozygous floxed progeny of LysMCre matings. However, PCR of twenty individual in vitro colonies from homozygous floxed mice indicated that they all retained an intact floxed allele. Breeding of floxed GABPa/LysMCre mice with hemizygous mice indicated that retention of a floxed allele was not due to incomplete deletion by LysMCre; rather, it appears that only myeloid cells that retain an intact GABPa allele can survive to mature in vitro or in vivo. We prepared murine embryonic fibroblasts from homozygous floxed mice and efficiently deleted GABPa in vitro. We found striking abnormalities in proliferation and G1/S phase arrest. We used quantitative RT-PCR to identify mechanisms that account for the altered growth of GABPa null cells. We found dramatically reduced expression of known GABP target genes that regulate DNA synthesis and cell cycle that appear to account for the proliferative defect. We conclude that GABPa is required for growth and maturation of myeloid cells and we identified downstream targets that may account for their failure to proliferate and mature in vitro and in vivo.

scholarly journals Deficient Heme and Globin Synthesis in Embryonic Stem Cells Lacking the Erythroid-Specific δ-Aminolevulinate Synthase Gene

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 798-805
Author(s):  
Hideo Harigae ◽  
Naruyoshi Suwabe ◽  
Peter H. Weinstock ◽  
Mayumi Nagai ◽  
Hiroyoshi Fujita ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Gene Mutations ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Aminolevulinate Synthase ◽  
E Gene ◽  
In The Wild ◽  
Mutant Cells

The erythroid-specific isoform of δ-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells, and ALAS-E gene mutations are known to be responsible for x-linked sideroblastic anemia. To study the role of ALAS-E in erythroid development, we prepared mouse embryonic stem (ES) cells carrying a disrupted ALAS-E gene and examined the effect of the lack of ALAS-E gene expression on erythroid differentiation. We found that mRNAs for erythroid transcription factors and TER119-positive cells were increased similarly both in the wild-type and mutant cells. In contrast, heme content, the number of benzidine-positive cells, adult globin protein, and mRNA for β-major globin were significantly decreased in the mutant cells. These results were confirmed using another ES differentiation system in vitro and suggest that ALAS-E expression, hence heme supply, is critical for the late stage of erythroid cell differentiation, which involves hemoglobin synthesis.

Cardiomyocyte differentiation by GATA-4-deficient embryonic stem cells

Development ◽  
1997 ◽  
Vol 124 (19) ◽  
pp. 3755-3764 ◽  
Author(s):  
N. Narita ◽  
M. Bielinska ◽  
D.B. Wilson
Keyword(s):  
Transcription Factor ◽  
In Situ Hybridization ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Cardiomyocyte Differentiation ◽  
Wild Type ◽  
Cell Stage

In situ hybridization studies, promoter analyses and antisense RNA experiments have implicated transcription factor GATA-4 in the regulation of cardiomyocyte differentiation. In this study, we utilized Gata4−/− embryonic stem (ES) cells to determine whether this transcription factor is essential for cardiomyocyte lineage commitment. First, we assessed the ability of Gata4−/− ES cells form cardiomyocytes during in vitro differentiation of embryoid bodies. Contracting cardiomyocytes were seen in both wild-type and Gata4−/− embryoid bodies, although cardiomyocytes were observed more often in wild type than in mutant embryoid bodies. Electron microscopy of cardiomyocytes in the Gata4−/− embryoid bodies revealed the presence of sarcomeres and junctional complexes, while immunofluorescence confirmed the presence of cardiac myosin. To assess the capacity of Gata4−/− ES cells to differentiate into cardiomyocytes in vivo, we prepared and analyzed chimeric mice. Gata4−/− ES cells were injected into 8-cell-stage embryos derived from ROSA26 mice, a transgenic line that expresses beta-galactosidase in all cell types. Chimeric embryos were stained with X-gal to discriminate ES cell- and host-derived tissue. Gata4−/− ES cells contributed to endocardium, myocardium and epicardium. In situ hybridization showed that myocardium derived from Gata4−/− ES cells expressed several cardiac-specific transcripts, including cardiac alpha-myosin heavy chain, troponin C, myosin light chain-2v, Nkx-2.5/Csx, dHAND, eHAND and GATA-6. Taken together these results indicate that GATA-4 is not essential for terminal differentiation of cardiomyocytes and suggest that additional GATA-binding proteins known to be in cardiac tissue, such as GATA-5 or GATA-6, may compensate for a lack of GATA-4.

Inducible Gata1 Suppression As a Novel Strategy to Expand Physiologic Megakaryocyte Production from Embryonic Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3846-3846
Author(s):  
Ji-Yoon Noh ◽  
Shilpa Gandre-Babbe ◽  
Yuhuan Wang ◽  
Vincent Hayes ◽  
Yu Yao ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hematopoietic Progenitors ◽  
Erythroid Progenitors ◽  
Transfusion Therapy ◽  
Ips Cell

Abstract Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent potential sources of megakaryocytes and platelets for transfusion therapy. However, most current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny, including platelet-releasing megakaryocytes. Mutations in the mouse and human genes encoding transcription factor GATA1 cause accumulation of proliferating, developmentally arrested megakaryocytes. Previously, we reported that in vitro differentiation of Gata1-null murine ES cells generated self-renewing hematopoietic progenitors termed G1ME cells that differentiated into erythroblasts and megakaryocytes upon restoration of Gata1 cDNA by retroviral transfer. However, terminal maturation of Gata1-rescued megakaryocytes was aberrant with immature morphology and no proplatelet formation, presumably due to non-physiological expression of GATA1. We now engineered wild type (WT) murine ES cells that express doxycycline (dox)-regulated Gata1 short hairpin (sh) RNAs to develop a strategy for Gata1-blockade that upon its release, restores physiologic GATA1 expression during megakaryopoiesis. In vitro hematopoietic differentiation of control scramble shRNA-expressing ES cells with dox and thrombopoietin (TPO) produced megakaryocytes that underwent senescence after 7 days. Under similar differentiation conditions, Gata1 shRNA-expressing ES cells produced immature hematopoietic progenitors, termed G1ME2 cells, which replicated continuously for more than 40 days, resulting in ~1013-fold expansion (N=4 separate experiments). Upon dox withdrawal with multi-lineage cytokines present (EPO, TPO, SCF, GMCSF and IL3), endogenous GATA1 expression was restored to G1ME2 cells followed by differentiation into erythroblasts and megakaryocytes, but no myeloid cells. In clonal methylcellulose assays, dox-deprived G1ME2 cells produced a mixture of erythroid, megakaryocytic and erythro-megakaryocytic colonies. In liquid culture with TPO alone, dox-deprived G1ME2 cells formed mature megakaryocytes in 5-6 days, as determined by morphology, ultrastructure, acetylcholinesterase staining, upregulated megakaryocytic gene expression (Vwf, Pf4, Gp1ba, Selp, Ppbp), CD42b surface expression, increased DNA ploidy and proplatelet production. Compared to G1ME cells rescued with Gata1 cDNA retrovirus, dox-deprived G1ME2 cells exhibited more robust megakaryocytic maturation, similar to that of megakaryocytes produced from cultured fetal liver. Importantly, G1ME2 cell-derived megakaryocytes generated proplatelets in vitro and functional platelets in vivo (~40 platelets/megakaryocyte with a circulating half life of 5-6 hours). These platelets were actively incorporated into growing arteriolar thrombi at sites of laser injury and subsequently expressed the platelet activation marker p-selectin (N=3-4 separate experiments). Our findings indicate that precise timing and magnitude of a transcription factor is required for proper terminal hematopoiesis. We illustrate this principle using a novel, readily reproducible strategy to expand ES cell-derived megakaryocyte-erythroid progenitors and direct their differentiation into megakaryocytes and then into functional platelets in clinically relevant numbers. Disclosures No relevant conflicts of interest to declare.

Redirecting differentiation of hematopoietic progenitors by a transcription factor, GATA-2

Blood ◽  
2006 ◽  
Vol 107 (5) ◽  
pp. 1857-1863 ◽  
Author(s):  
Kenji Kitajima ◽  
Makoto Tanaka ◽  
Jie Zheng ◽  
Hilo Yen ◽  
Ayuko Sato ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Es Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Dependent Manner ◽  
Hematopoietic Differentiation ◽  
Macrophage Differentiation ◽  
Conditional Gene Expression

GATA-2 is a zinc finger transcription factor essential for differentiation of immature hematopoietic cells. We analyzed the function of GATA-2 by a combined method of tetracycline-dependent conditional gene expression and in vitro hematopoietic differentiation from mouse embryonic stem (ES) cells using OP9 stroma cells (OP9 system). In the presence of macrophage colony-stimulating factor (M-CSF), the OP9 system induced macrophage differentiation. GATA-2 expression in this system inhibited macrophage differentiation and redirected the fate of hematopoietic differentiation to other hematopoietic lineages. GATA-2 expression commencing at day 5 or day 6 induced megakaryocytic or erythroid differentiation, respectively. Expression levels of PU.1, a hematopoietic transcription factor that interferes with GATA-2, appeared to play a critical role in differentiation to megakaryocytic or erythroid lineages. Transcription of PU.1 was affected by histone acetylation induced by binding of GATA-2 to the PU.1 promoter region. This study demonstrates that the function of GATA-2 is modified in a context-dependent manner by expression of PU.1, which in turn is regulated by GATA-2.

scholarly journals Committing Embryonic Stem Cells to Differentiate into Thyrocyte-Like Cells in Vitro

Endocrinology ◽  
2003 ◽  
Vol 144 (6) ◽  
pp. 2644-2649 ◽  
Author(s):  
Reigh-Yi Lin ◽  
Atsushi Kubo ◽  
Gordon M. Keller ◽  
Terry F. Davies
Keyword(s):  
Transcription Factor ◽  
Genetic Manipulation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Experimental System ◽  
Embryoid Bodies ◽  
Basic Study ◽  
Thyroid Cells ◽  
Thyroid Transcription Factor

Abstract The derivation of thyrocyte-like cells in culture is of importance in the basic study of early thyroid embryogenesis and the generation of an unlimited clinical source of thyrocytes for genetic manipulation and cell transplantation. We have established an experimental system, which shows that 6-d-old embryoid bodies (EBs) differentiated from mouse embryonic stem (ES) cells expressed a set of genes traditionally associated with thyroid cells. The genes analyzed included the thyroid transcription factor PAX8, the Na+/I− symporter, thyroperoxidase, thyroglobulin, and the TSH receptor (TSHR). Immunofluorescent analysis demonstrated the presence of TSHR-positive cells as outgrowths from 8-d-old EBs cultured on chamber slides. Accordingly, this area of cells also expressed PAX8 and another thyroid transcription factor TTF2. Of importance, TSH, the main regulator of the thyroid gland, was necessary to maintain the expression of PAX8 and TSHR genes during EB differentiation. Furthermore, thyroid-specific function, such as cAMP generation by TSH, was maintained in this model. Together, these results suggested that the developmental program associated with thyrocyte development is recapitulated in the ES/EB model system. The differentiation of mouse ES cells into thyrocyte-like cells provides a powerful model for the study of thyrocyte developmental diseases associated with this lineage and contributes to the development of thyroid hormone-secreting cell lines.

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