The Experts below are selected from a list of 219 Experts worldwide ranked by ideXlab platform

Stuart H Orkin - One of the best experts on this subject based on the ideXlab platform.

  • gata 1 and erythropoietin cooperate to promote Erythroid Cell survival by regulating bcl xl expression
    Blood, 1999
    Co-Authors: Todd Gregory, Stuart H Orkin, Gerd A Blobel, Mitchell J Weiss
    Abstract:

    The transcription factor GATA-1 is essential for normal erythropoiesis. By examining in vitro–differentiated embryonic stem Cells, we showed previously that in the absence of GATA-1, committed Erythroid precursors fail to complete maturation and instead undergo apoptosis. The mechanisms by which GATA-1 controls Cell survival are unknown. Here we report that in Erythroid Cells, GATA-1 strongly induces the expression of the anti-apoptotic protein bcl-xL, but not the related proteins bcl-2 and mcl-1. Consistent with a role for bcl-xL in mediating GATA-1–induced Erythroid Cell survival, in vitro–differentiated bcl-xL−/− embryonic stem Cells fail to generate viable mature definitive Erythroid Cells, a phenotype resembling that of GATA-1 gene disruption. In addition, we show that erythropoietin, which is also required for Erythroid Cell survival, cooperates with GATA-1 to stimulate bcl-xL gene expression and to maintain Erythroid Cell viability during terminal maturation. Together, our data show that bcl-xL is essential for normal Erythroid development and suggest a regulatory hierarchy in which bcl-xL is a critical downstream effector of GATA-1 and erythropoietin-mediated signals.

  • a knockdown mutation created by cis element gene targeting reveals the dependence of Erythroid Cell maturation on the level of transcription factor gata 1
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Michael A Mcdevitt, Ramesh A Shivdasani, Yuko Fujiwara, Haidi Yang, Stuart H Orkin
    Abstract:

    The hematopoietic-restricted transcription factor GATA-1 is required for both mammalian Erythroid Cell and megakaryocyte differentiation. To define the mechanisms governing its transcriptional regulation, we replaced upstream sequences including a DNase I hypersensitive (HS) region with a neomycin-resistance cassette by homologous recombination in mouse embryonic stem Cells and generated mice either harboring this mutation (neoΔHS) or lacking the selection cassette (ΔneoΔHS). Studies of the consequences of these targeted mutations provide novel insights into GATA-1 function in Erythroid Cells. First, the neoΔHS mutation leads to a marked impairment in the rate or efficiency of Erythroid Cell maturation due to a modest (4- to 5-fold) decrease in GATA-1 expression. Hence, Erythroid differentiation is dose-dependent with respect to GATA-1. Second, since expression of GATA-1 from the ΔneoΔHS allele in Erythroid Cells is largely restored, transcription interference imposed by the introduced cassette must account for the “knockdown” effect of the mutation. Finally, despite the potency of the upstream sequences in conferring high-level, developmentally appropriate expression of transgenes in mice, other cis-regulatory elements within the GATA-1 compensate for its absence in Erythroid Cells. Our work illustrates the usefulness of targeted mutations to create knockdown mutations that may uncover important quantitative contributions of gene function not revealed by conventional knockouts.

  • Erythroid Cell specific properties of transcription factor gata 1 revealed by phenotypic rescue of a gene targeted Cell line
    Molecular and Cellular Biology, 1997
    Co-Authors: Mitchell J Weiss, Stuart H Orkin
    Abstract:

    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.

Michel Sadelain - One of the best experts on this subject based on the ideXlab platform.

  • stem Cell derived Erythroid Cells mediate long term systemic protein delivery
    Nature Biotechnology, 2006
    Co-Authors: Alex H Chang, Matthias Stephan, Michel Sadelain
    Abstract:

    We demonstrate here the capacity of Erythroid Cells to mediate long-term, systemic and therapeutic protein delivery in vivo. By targeting human factor IX (hFIX) expression to late-stage erythropoiesis, we achieve long-term hFIX secretion at levels significantly higher (>tenfold) than those obtained with an archetypal ubiquitous promoter in a mouse model of hemophilia B. Erythroid Cell-derived hFIX is biologically active, resulting in phenotypic correction of the bleeding disorder. In addition to achieving high expression levels and resistance to transcriptional silencing, red Cell-mediated protein delivery offers multiple advantages including immune tolerance induction, reduction of the risk of insertional oncogenesis and relative ease of application by either engrafting transduced hematopoietic stem Cells or transfusing ex vivo-generated, stem Cell-derived Erythroid Cells.

Matthias Stephan - One of the best experts on this subject based on the ideXlab platform.

  • stem Cell derived Erythroid Cells mediate long term systemic protein delivery
    Nature Biotechnology, 2006
    Co-Authors: Alex H Chang, Matthias Stephan, Michel Sadelain
    Abstract:

    We demonstrate here the capacity of Erythroid Cells to mediate long-term, systemic and therapeutic protein delivery in vivo. By targeting human factor IX (hFIX) expression to late-stage erythropoiesis, we achieve long-term hFIX secretion at levels significantly higher (>tenfold) than those obtained with an archetypal ubiquitous promoter in a mouse model of hemophilia B. Erythroid Cell-derived hFIX is biologically active, resulting in phenotypic correction of the bleeding disorder. In addition to achieving high expression levels and resistance to transcriptional silencing, red Cell-mediated protein delivery offers multiple advantages including immune tolerance induction, reduction of the risk of insertional oncogenesis and relative ease of application by either engrafting transduced hematopoietic stem Cells or transfusing ex vivo-generated, stem Cell-derived Erythroid Cells.

Masaki Yasukawa - One of the best experts on this subject based on the ideXlab platform.

  • the role of zinc finger protein 521 early hematopoietic zinc finger protein in Erythroid Cell differentiation
    Journal of Biological Chemistry, 2009
    Co-Authors: Etsuko Matsubara, Ikuya Sakai, Jun Yamanouchi, Hiroshi Fujiwara, Yoshihiro Yakushijin, Takaaki Hato, Kazuhiro Shigemoto, Masaki Yasukawa
    Abstract:

    ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic Cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in Erythroid Cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL Cell lines as compared with control Cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of Erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates Erythroid Cell differentiation through direct binding with GATA-1.

James J. Bieker - One of the best experts on this subject based on the ideXlab platform.

  • Stage-Specific Repression by the EKLF Transcriptional Activator
    Molecular and cellular biology, 2004
    Co-Authors: Xiaoyong Chen, James J. Bieker
    Abstract:

    Dynamic changes in transcription factor function can be mediated by switching its interaction with coactivators and corepressors. Erythroid Kruppel-like factor (EKLF) is an Erythroid Cell-specific transcription factor that plays a critical role in beta-globin gene activation via its interactions with CBP/p300 and SWI/SNF proteins. Unexpectedly, it also interacts with Sin3A and histone deacetylase 1 (HDAC1) corepressors via its zinc finger domain. We now find that selected point mutants can uncouple activation and repression and that an intact finger structure is not required for interactions with Sin3A/HDAC1 or for transrepression. Most intriguingly, EKLF repression exhibits stage specificity, with reversible EKLF-Sin3A interactions playing a key role in this process. Finally, we have located a key lysine residue that is both a substrate for CBP acetylation and required for Sin3A interaction. These data suggest a model whereby the stage of the Erythroid Cell alters the acetylation status of EKLF and plays a critical role in directing its coactivator-corepressor interactions and downstream transcriptional effects.

  • a novel Erythroid Cell specific murine transcription factor that binds to the caccc element and is related to the kruppel family of nuclear proteins
    Molecular and Cellular Biology, 1993
    Co-Authors: I J Miller, James J. Bieker
    Abstract:

    We describe a novel Erythroid Cell-specific cDNA (EKLF [Erythroid Kruppel-like factor]) isolated by enriching for genes expressed in a mouse erythroleukemia Cell line but not expressed in a mouse monocyte-macrophage Cell line. The complete cDNA sequence is predicted to encode a protein of approximately 38,000 Da that contains a proline-rich amino domain and three TFIIIA-like zinc fingers within the carboxy domain. Additional sequence analyses reveal that the EKLF zinc fingers are most homologous to the Kruppel family of transcription factors and also allow us to predict potential DNA-binding target sites for the EKLF protein. On the basis of this prediction, we show that EKLF is able to bind the sequence CCA CAC CCT, an essential element of the beta-globin promoter. Its tissue distribution establishes that the EKLF transcript is expressed only in bone marrow and spleen, the two hematopoietic organs of the mouse, and analysis of murine Cell lines indicates that EKLF expression is limited to Erythroid and mast Cell lines. Cotransfection assays establish that EKLF transcriptionally activates a target promoter that contains its DNA-binding site. The tissue expression pattern of EKLF, in conjunction with its function as a transcriptional activator, strongly suggests that the EKLF protein may be intimately involved in establishment and/or maintenance of the Erythroid Cell phenotype.