The Experts below are selected from a list of 33 Experts worldwide ranked by ideXlab platform

Elizabeth A. Eklund - One of the best experts on this subject based on the ideXlab platform.

  • HoxA10 null animals exhibit reduced platelet biogenesis.
    British Journal of Haematology, 2016
    Co-Authors: Iwona M. Konieczna, Teresa A. Deluca, Elizabeth A. Eklund, William M. Miller
    Abstract:

    Summary The Transcription Factor HoxA10 is an important regulator of myelopoiesis. Engineered over-expression of HoxA10 in mice results in a myeloproliferative disorder that progresses to acute myeloid leukaemia (AML) over time, and in humans over-expression is associated with poor outcomes in AML. Here, we report that loss of HoxA10 expression in mice results in reduced platelet count and platelet production, but does not affect clotting efficiency. About 40% fewer platelets were found in HoxA10 null animals in comparison to wild type littermates. We found a nearly 50% reduction in the percentage of reticulated platelets in HoxA10 null mice, suggesting deficient platelet production. Furthermore, HoxA10 null animals recovered less efficiently from induced thrombocytopenia, supporting our hypothesis of defective platelet production. This also correlated with reduced colony formation potential of stem and progenitor cells seeded in megakaryocyte-enhancing conditions in vitro. Together, our results indicate that HoxA10 is important for megakaryopoiesis and platelet biogenesis.

  • Constitutively Active SHP2 Cooperates with HoxA10 Overexpression to Induce Acute Myeloid Leukemia
    Journal of Biological Chemistry, 2008
    Co-Authors: Hao Wang, Iwona M. Konieczna, Stephan Lindsey, Weiqi Huang, Elizabeth Horvath, Gurveen Saberwal, Elizabeth A. Eklund
    Abstract:

    The homeodomain Transcription Factor HoxA10 is maximally expressed in myeloid progenitor cells. Sustained HoxA10 expression during differentiation has been described in poor prognosis human acute myeloid leukemia (AML). Consistent with this, engineered overexpression of HoxA10 in murine bone marrow induces a myeloproliferative disorder that progresses to AML over time. This murine model suggests that HoxA10 overexpression is sufficient for myeloproliferation but that differentiation block, and therefore AML, requires acquisition of additional mutations. In myeloid progenitor cells, HoxA10 represses Transcription of genes that encode phagocyte effector proteins such as gp91PHOX and p67PHOX. Tyrosine phosphorylation of HoxA10 during myelopoiesis decreases binding to these target genes. In immature myeloid cells, HoxA10 also activates Transcription of the DUSP4 gene that encodes Mkp2, an anti-apoptotic protein. HoxA10 binding to the DUSP4 promoter decreases during myelopoiesis. Therefore, both myeloid-specific gene repression and DUSP4 activation by HoxA10 decrease during myelopoiesis. This results in phenotypic differentiation and facilitates apoptosis as differentiation proceeds. HoxA10 is de-phosphorylated by SHP2 protein-tyrosine phosphatase in myeloid progenitors. This mechanism maintains HoxA10 in a nonphosphorylated state in immature, but not differentiating, myeloid cells. Constitutively active SHP2 mutants have been described in human AML, which dephosphorylate HoxA10 throughout myelopoiesis. In this study, we hypothesize that constitutive SHP2 activation synergizes with HoxA10 overexpression to accelerate progression to AML. Because both HoxA10 overexpression and constitutive SHP2 activation are found in poor prognosis human AML, these studies contribute to understanding biochemical aspects of disease progression in myeloid malignancy.

  • Identification of a HoxA10 Activation Domain Necessary for Transcription of the Gene Encoding β3 Integrin during Myeloid Differentiation
    Journal of Biological Chemistry, 2007
    Co-Authors: Yu Feng Lu, Elizabeth Horvath, Susan L. Bellis, Wei Zhou, Elizabeth A. Eklund
    Abstract:

    Abstract Transcription of the ITGB3 gene, which encodes β3 integrin, increases during myeloid differentiation. αvβ3 integrin mediates adhesion to fibronectin or vitronectin and regulates various aspects of the inflammatory response in mature phagocytes. In these studies, we found that the homeodomain Transcription Factor HoxA10 interacted with a specific ITGB3 cis element and activated Transcription of this gene during myeloid differentiation. We also found that increased fibronectin adhesion in differentiating myeloid cells was dependent upon this HoxA10-induced increase in β3 integrin expression. We determined that activation of ITGB3 Transcription required a HoxA10 domain that was not identical to the “hexapeptide” that mediates interaction of Hox and Pbx proteins. This activation domain was also not identical to a previously identified HoxA10 repression domain that mediates interaction with Transcriptional co-repressors. Instead, this HoxA10 activation domain had homology to “PQ” protein-protein interaction domains that have been described previously in other Transcription Factors. Consistent with this, we found that the HoxA10 PQ-like domain recruited the CREB-binding protein (CBP) to the ITGB3 promoter. This was associated with an increase in local histone acetylation in vivo. In immature myeloid cells, we previously determined that HoxA10 repressed Transcription of the CYBB and NCF2 genes, which encode the phagocyte oxidase proteins gp91PHOX and p67PHOX, respectively. Therefore, our studies indicated that HoxA10 either activates or represses gene Transcription at various points during myelopoiesis. Our studies also suggested that HoxA10 is a bifunctional protein that is involved in dynamic regulation of multiple aspects of phagocyte phenotype and function.

  • Activation of SHP2 Protein-tyrosine Phosphatase Increases HoxA10-induced Repression of the Genes Encoding gp91PHOX and p67PHOX
    Journal of Biological Chemistry, 2006
    Co-Authors: Stephan Lindsey, Weiqi Huang, Hao Wang, Elizabeth Horvath, Elizabeth A. Eklund
    Abstract:

    Abstract The CYBB and NCF2 genes encode the phagocyte oxidase proteins gp91PHOX and p67PHOX, respectively. These genes are transcribed after the promyelocyte stage of differentiation, and Transcription continues until cell death. In undifferentiated myeloid cells, homologous cis-elements in the CYBB and NCF2 genes are repressed by the homeodomain Transcription Factor HoxA10. During cytokine-induced myelopoiesis, tyrosine phosphorylation of HoxA10 decreases binding affinity for the CYBB and NCF2 cis-elements. This abrogates HoxA10-induced Transcriptional repression as differentiation proceeds. Therefore, mechanisms involved in differentiation stage-specific HoxA10 tyrosine phosphorylation are of interest because HoxA10 phosphorylation modulates myeloid-specific gene Transcription. In this study, we found that HoxA10 is a substrate for SHP2 protein-tyrosine phosphatase in undifferentiated myeloid cells. In contrast, HoxA10 is a substrate for a constitutively active mutant form of SHP2 in both undifferentiated and differentiating myeloid cells. Expression of such SHP2 mutants results in persistent HoxA10 repression of CYBB and NCF2 Transcription during myelopoiesis. Both HoxA10 overexpression and activating SHP2 mutations have been described in human myeloid malignancies. Therefore, our results suggest that these mutations could cooperate, leading to decreased myeloid-specific gene Transcription and functional differentiation block in myeloid cells with both defects.

  • HoxA10 Represses Transcription of the Gene Encoding p67phox in Phagocytic Cells
    Journal of Immunology, 2005
    Co-Authors: Stephan Lindsey, Yu Feng Lu, Elizabeth A. Eklund
    Abstract:

    p67 phox and gp91 phox are components of the phagocyte-specific respiratory burst oxidase that are encoded by the NCF2 and CYBB genes, respectively. These genes are transcribed exclusively in myeloid cells that have differentiated beyond the promyelocyte stage. In mature phagocytes, NCF2 and CYBB Transcription continues until cell death and further increases in response to IFN-γ and other inflammatory mediators. Because p67 phox and gp91 phox expression profiles are similar, we hypothesize that common Transcription Factors interact with homologous cis elements in the CYBB and NCF2 genes to coordinate Transcription. Previously, we identified a negative CYBB promoter cis element that is repressed by the homeodomain Transcription Factor HoxA10. We found that Transcriptional repression requires HoxA10-dependent recruitment of histone deacetylase activity to the CYBB cis element. In response to IFN-γ, phosphorylation of two tyrosine residues in the HoxA10 homeodomain decreases binding to CYBB promoter, thereby abrogating HoxA10-mediated repression. In the current studies, we investigate the possibility that HoxA10 similarly represses NCF2 Transcription. We identify a sequence in the NCF2 promoter that is homologous to the HoxA10-binding CYBB cis element. We find that this NCF2 promoter sequence functions as a negative cis element that is repressed by HoxA10 in a tyrosine phosphorylation and histone deacetylase-dependent manner. Our results suggest that cytokine-stimulated pathways regulate HoxA10-mediated repression of the CYBB and NCF2 genes in differentiating myeloid cells and in mature phagocytes during the inflammatory response. Because p67 phox and gp91 phox are rate-limiting components for respiratory burst activity, our studies may identify rational therapeutic targets to modulate free radical generation in pathological conditions.

Bruce H Howard - One of the best experts on this subject based on the ideXlab platform.

  • human histone deacetylase sirt2 interacts with the homeobox Transcription Factor HoxA10
    Journal of Biochemistry, 2004
    Co-Authors: Mark J Swanson, Alex Vassilev, Bruce H Howard
    Abstract:

    : Histone deacetylases are required for Transcriptional repression in eukaryotes. Saccharomyces cerevisiae has several histone deacetylases, of which ySir2p is the most conserved throughout evolution. Currently, there is no report on the interacting protein partner of a human Sir2 homolog, SIRT2. Here we show for the first time that SIRT2 interacts with the homeobox Transcription Factor, HoxA10, which was identified in a two-hybrid screen. Interactions were confirmed by co-immunoprecipitation from in vitro translations as well as in human cell-free extracts. Taken together with mouse knockout studies, our results raise the intriguing possibility that SIRT2 plays a role in mammalian development.

Mark J Swanson - One of the best experts on this subject based on the ideXlab platform.

  • human histone deacetylase sirt2 interacts with the homeobox Transcription Factor HoxA10
    Journal of Biochemistry, 2004
    Co-Authors: Mark J Swanson, Alex Vassilev, Bruce H Howard
    Abstract:

    : Histone deacetylases are required for Transcriptional repression in eukaryotes. Saccharomyces cerevisiae has several histone deacetylases, of which ySir2p is the most conserved throughout evolution. Currently, there is no report on the interacting protein partner of a human Sir2 homolog, SIRT2. Here we show for the first time that SIRT2 interacts with the homeobox Transcription Factor, HoxA10, which was identified in a two-hybrid screen. Interactions were confirmed by co-immunoprecipitation from in vitro translations as well as in human cell-free extracts. Taken together with mouse knockout studies, our results raise the intriguing possibility that SIRT2 plays a role in mammalian development.

Keith Humphries - One of the best experts on this subject based on the ideXlab platform.

  • High level in vitro expansion of murine hematopoietic stem cells.
    Current protocols in stem cell biology, 2020
    Co-Authors: Sanja Sekulovic, Suzan Imren, Keith Humphries
    Abstract:

    Development of strategies to extensively expand hematopoietic stem cells (HSCs) in vitro will be a major Factor in enhancing the success of a range of transplant-based therapies for malignant and genetic disorders. In addition to potential clinical applications, the ability to increase the number of HSCs in culture will facilitate investigations into the mechanisms underlying self-renewal. In this unit, we describe a robust strategy for consistently achieving over 1000-fold net expansion of HSCs in short-term in vitro culture by using novel engineered fusions of the N-terminal domain of nucleoporin 98 (NUP98) and the homeodomain of the hox Transcription Factor, HoxA10 (so called NUP98-HoxA10hd fusion). We also provide a detailed protocol for monitoring the magnitude of HSC expansion in culture by limiting dilution assay of competitive lympho-myeloid repopulating units (CRU Assay). These procedures provide new possibilities for achieving significant numbers of HSCs in culture, as well as for studying HSCs biochemically and genetically.

  • Current Protocols in Stem Cell Biology - High Level In Vitro Expansion of Murine Hematopoietic Stem Cells
    Current protocols in stem cell biology, 2008
    Co-Authors: Sanja Sekulovic, Suzan Imren, Keith Humphries
    Abstract:

    Development of strategies to extensively expand hematopoietic stem cells (HSCs) in vitro will be a major Factor in enhancing the success of a range of transplant-based therapies for malignant and genetic disorders. In addition to potential clinical applications, the ability to increase the number of HSCs in culture will facilitate investigations into the mechanisms underlying self-renewal. In this unit, we describe a robust strategy for consistently achieving over 1000-fold net expansion of HSCs in short-term in vitro culture by using novel engineered fusions of the N-terminal domain of nucleoporin 98 (NUP98) and the homeodomain of the hox Transcription Factor, HoxA10 (so called NUP98-HoxA10hd fusion). We also provide a detailed protocol for monitoring the magnitude of HSC expansion in culture by limiting dilution assay of competitive lympho-myeloid repopulating units (CRU Assay). These procedures provide new possibilities for achieving significant numbers of HSCs in culture, as well as for studying HSCs biochemically and genetically. Curr. Protoc. Stem Cell Biol. 4:2A.7.1-2A.7.14. © 2008 by John Wiley & Sons, Inc. Keywords: NUP98-HOX fusion; HSC expansion; CRU assay; multilineage reconstitution

Alex Vassilev - One of the best experts on this subject based on the ideXlab platform.

  • human histone deacetylase sirt2 interacts with the homeobox Transcription Factor HoxA10
    Journal of Biochemistry, 2004
    Co-Authors: Mark J Swanson, Alex Vassilev, Bruce H Howard
    Abstract:

    : Histone deacetylases are required for Transcriptional repression in eukaryotes. Saccharomyces cerevisiae has several histone deacetylases, of which ySir2p is the most conserved throughout evolution. Currently, there is no report on the interacting protein partner of a human Sir2 homolog, SIRT2. Here we show for the first time that SIRT2 interacts with the homeobox Transcription Factor, HoxA10, which was identified in a two-hybrid screen. Interactions were confirmed by co-immunoprecipitation from in vitro translations as well as in human cell-free extracts. Taken together with mouse knockout studies, our results raise the intriguing possibility that SIRT2 plays a role in mammalian development.