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1.
Curr Opin Hematol ; 19(4): 268-72, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22517588

ABSTRACT

PURPOSE OF REVIEW: Recent work has shown that the Musashi 2 (Msi2) gene plays important roles in normal and malignant hematopoiesis. Here, we give an overview on the role of Msi2 in the regulation and function of primitive hematopoietic cells as well as in leukaemic progression. We also discuss the molecular pathways in which Msi2 acts in both normal and leukaemic blood cells. RECENT FINDINGS: Msi2 gain and loss of function experiments have shown that it plays an important role in regulating the heamatopoietic stem cell pool. Msi2 has also been found to be overexpressed in human myeloid leukaemias correlating with poor prognosis, therefore Msi2 may be considered as a prognostic marker for acute myeloid leukaemia. SUMMARY: Further studies into the molecular pathways through which Msi2 modulates primitive progenitor function will provide insight into the regulation of normal haematopoiesis and a better understanding of the mechanisms governing the leukaemic transformation process. This will be crucial for the development of effective therapies.


Subject(s)
Cell Transformation, Neoplastic/genetics , Hematopoiesis/genetics , Leukemia/genetics , RNA-Binding Proteins/physiology , Animals , Gene Expression Regulation, Leukemic , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/physiology , Humans , Leukemia/metabolism
2.
Blood ; 118(3): 554-64, 2011 Jul 21.
Article in English | MEDLINE | ID: mdl-21613258

ABSTRACT

We used a retroviral integration screen to search for novel genes that regulate HSC function. One of the genes that conferred HSC dominance when overexpressed due to an adjacent retroviral insertion was Musashi 2 (Msi2), an RNA-binding protein that can act as a translational inhibitor. A gene-trap mouse model that inactivates the gene shows that Msi2 is more highly expressed in long-term (LT) and short-term (ST) HSCs, as well as in lymphoid myeloid primed progenitors (LMPPs), but much less in intermediate progenitors and mature cells. Mice lacking Msi2 are fully viable for up to a year or more, but exhibit severe defects in primitive precursors, most significantly a reduction in the number of ST-HSCs and LMPPs and a decrease in leukocyte numbers, effects that are exacerbated with age. Cell-cycle and gene-expression analyses suggest that the main hematopoietic defect in Msi2-defective mice is the decreased proliferation capacity of ST-HSCs and LMPPs. In addition, HSCs lacking Msi2 are severely impaired in competitive repopulation experiments, being overgrown by wild-type cells even when mutant cells were provided in excess. Our data indicate that Msi2 maintains the stem cell compartment mainly by regulating the proliferation of primitive progenitors downstream of LT-HSCs.


Subject(s)
Lymphoid Progenitor Cells/physiology , Myeloid Progenitor Cells/physiology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Animals , Cell Adhesion/physiology , Cell Division/physiology , Cell Movement/physiology , Female , Genetic Testing/methods , Leukocyte Count , Lymphoid Progenitor Cells/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Mutagenesis, Insertional , Myeloid Progenitor Cells/cytology , Neomycin , Protein Synthesis Inhibitors , Retroviridae/genetics , Spleen/pathology , Thymus Gland/pathology , beta-Galactosidase/genetics
3.
Stem Cells ; 27(2): 300-6, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19008347

ABSTRACT

Several laboratories have reported the reprogramming of mouse and human fibroblasts into pluripotent cells, using retroviruses carrying the Oct4, Sox2, Klf4, and c-Myc transcription factor genes. In these experiments the frequency of reprogramming was lower than 0.1% of the infected cells, raising the possibility that additional events are required to induce reprogramming, such as activation of genes triggered by retroviral insertions. We have therefore determined by ligation-mediated polymerase chain reaction (LM-PCR) the retroviral insertion sites in six induced pluripotent stem (iPS) cell clones derived from mouse fibroblasts. Seventy-nine insertion sites were assigned to a single mouse genome location. Thirty-five of these mapped to gene transcription units, whereas 29 insertions landed within 10 kilobases of transcription start sites. No common insertion site was detected among the iPS clones studied. Moreover, bioinformatics analyses revealed no enrichment of a specific gene function, network, or pathway among genes targeted by retroviral insertions. We conclude that Oct4, Sox2, Klf4, and c-Myc are sufficient to promote fibroblast-to-iPS cell reprogramming and propose that the observed low reprogramming frequencies may have alternative explanations.


Subject(s)
Fibroblasts/cytology , Genetic Vectors/genetics , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Retroviridae/genetics , Animals , Blotting, Southern , Cell Line , Computational Biology , Fibroblasts/virology , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Mice , Octamer Transcription Factor-3/genetics , Pluripotent Stem Cells/virology , Polymerase Chain Reaction , Proto-Oncogene Proteins c-myc/genetics , SOXB1 Transcription Factors/genetics
4.
Exp Hematol ; 35(3): 490-499, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17309829

ABSTRACT

OBJECTIVE: Development of a mouse line permitting live imaging of cells expressing CD41/GpIIb as a means to study megakaryopoiesis. MATERIALS AND METHODS: The gene encoding yellow fluorescent protein (eyfp) was inserted by homologous recombination into embryonic stem cells at the start site of the gpIIb locus. A knockin mouse line, designated CD41-yellow fluorescent protein (YFP), was developed and was characterized by fluorescence microscopy and flow cytometry. Activity of YFP(+) platelets was determined by induction of P-selectin expression in response to thrombin stimulation. RESULTS: CD41-YFP mice contained YFP-labeled megakaryocytes and platelets, the proportions of which varied, depending on the genotype and individual animal, while lymphoid, myelomonocytic, and erythroid lineages were negative. In addition, a fraction of hematopoietic stem cells and intermediate progenitors expressed YFP at low levels. Crossing CD41-YFP mice with lysozyme green fluorescent protein and globin cyan fluorescent protein mice, followed by in vivo imaging of fetal liver, revealed megakaryocytic cells as a subset distinct from myeloid and erythroid cells. This experiment is also the first to show the distribution of three hematopoietic lineages in a minimally perturbed organ. Surprisingly, analysis of CD41-YFP platelets showed that the YFP(+) subset is more responsive to thrombin stimulation than the YFP(-) subset. Experiments aimed at determining the stability of the YFP(+) platelets showed that after lethal irradiation of CD41-YFP mice, the proportion of labeled platelets in the blood declines more rapidly than the bulk of the platelets. CONCLUSION: The newly developed mouse line should become useful not only for in vivo imaging experiments of megakaryocytes and platelets, but also for studies on platelet aging and function. Our irradiation experiments suggest that the YFP(+) platelets are enriched for newly made cells because YFP has a shorter half-life than platelets. Therefore, the finding that YFP(+) platelets are more responsive to thrombin stimulation raises the possibility that platelet activity decreases rapidly during physiological aging.


Subject(s)
Blood Platelets/drug effects , Luminescent Proteins/genetics , Megakaryocytes/immunology , Platelet Membrane Glycoprotein IIb/genetics , Recombinant Fusion Proteins/biosynthesis , Thrombin/pharmacology , Animals , Blood Platelets/immunology , Blood Platelets/radiation effects , Cell Line , Dose-Response Relationship, Drug , Genotype , Megakaryocytes/cytology , Mice , Mice, Transgenic , P-Selectin/biosynthesis , P-Selectin/drug effects , P-Selectin/immunology , Recombinant Fusion Proteins/genetics , Staining and Labeling , Time Factors , Whole-Body Irradiation
5.
Proc Natl Acad Sci U S A ; 103(5): 1400-5, 2006 Jan 31.
Article in English | MEDLINE | ID: mdl-16432238

ABSTRACT

PML-RARalpha induces a block of hematopoietic differentiation and acute promyelocytic leukemia. This block is based on its capacity to inactivate target genes by recruiting histone deacetylase (HDAC) and DNA methyltransferase activities. Here we report that MBD1, a member of a conserved family of proteins able to bind methylated DNA, cooperates with PML-RARalpha in transcriptional repression and cellular transformation. PML-RARalpha recruits MBD1 to its target promoter through an HDAC3-mediated mechanism. Binding of HDAC3 and MBD1 is not confined to the promoter region but instead is spread over the locus. Knock-down of HDAC3 expression by RNA interference in acute promyelocytic leukemia cells alleviates PML-RAR-induced promoter silencing. We further demonstrate that retroviral expression of dominant-negative mutants of MBD1 in hematopoietic precursors compromises the ability of PML-RARalpha to block their differentiation and thus restored cell differentiation. Our results demonstrate that PML-RARalpha functions by recruiting an HDAC3-MBD1 complex that contributes to the establishment and maintenance of the silenced chromatin state.


Subject(s)
DNA-Binding Proteins/physiology , Neoplasm Proteins/metabolism , Neoplasm Proteins/physiology , Oncogene Proteins, Fusion/metabolism , Oncogene Proteins, Fusion/physiology , Transcription Factors/physiology , Blotting, Western , Cell Differentiation , Cell Line , Cell Line, Tumor , Cell Transformation, Neoplastic , Chromatin/chemistry , Chromatin/metabolism , Chromatin Immunoprecipitation , DNA, Complementary/metabolism , DNA-Binding Proteins/chemistry , Epigenesis, Genetic , Gene Silencing , Genes, Dominant , Genetic Vectors , HeLa Cells , Hematopoietic Stem Cells/cytology , Histone Deacetylases/metabolism , Humans , Immunoprecipitation , Leukemia/metabolism , Luciferases/metabolism , Models, Biological , Oligonucleotides/chemistry , Plasmids/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Transcription Factors/chemistry
6.
Methods Mol Med ; 105: 395-412, 2005.
Article in English | MEDLINE | ID: mdl-15492410

ABSTRACT

Here, we present a computer-controlled time-lapse system for imaging of cultured hematopoietic cells labeled by the expression of different fluorescent proteins. First, we describe experiments to optimize the visualization of three green fluorescent protein variants (cyan-, green-, and yellow-enhanced fluorescent protein) and the red-fluorescent protein (DsRed) by standard wide-field fluorescence microscopy. Then, we describe procedures to best distinguish combinations of cells expressing these proteins using seven commercially available filter sets, based on the relative fluorescence intensities of the individual fluorescent proteins. Finally, we make recommendations about which of these filters to choose when working with specific fluorescent proteins.


Subject(s)
Cell Differentiation/physiology , Green Fluorescent Proteins , Hematopoietic Stem Cells/physiology , Microscopy, Fluorescence/methods , Animals , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Green Fluorescent Proteins/genetics , Humans , Microscopy, Fluorescence/instrumentation
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