Abstract
To provide insight into the mechanisms by which c-myb regulates hematopoiesis, we analyzed the expression of markers for multiple hematopoietic lineages in differentiating parental embryonic stem (ES) cells and in ES cells transfected with c-myb or with a mutant c-myb deficient in DNA binding and assessed the ability of these cells to undergo hematopoietic commitment and colony formation. Undifferentiated ES cells transfected with intact c-myb, but not cells transfected with mutant c-myb, expressed CD34, c-kit, GATA1, and flt3 mRNA as well as surface CD34, c-kit, and flt3 product. In contrast, the kinetics of GATA-2 mRNA expression was identical in parental and Myb-transfected ES cells. Transient expression assays suggested transactivation of gene expression dependent on interaction with Myb binding sites in the CD34 and GATA1 5' flanking regions. Undifferentiated parental and c-myb mutant-transfected ES cells were not clonogenic, whereas c-myb transfectants formed erythromyeloid colonies in methylcellulose cultures in the absence of added hematopoietic growth factors and, at higher frequency, in the presence of kit and flt-3 ligands. Colony formation was suppressed by treatment with antisense oligodeoxynucleotides specifically downregulating c-kit and flt-3 expression. These findings indicate that c-myb regulates hematopoietic commitment and progenitor cell proliferation and differentiation through the activation of certain genes that define the stem/progenitor cell compartment.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Antigens, CD34 / biosynthesis
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Antigens, CD34 / genetics
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Antigens, Differentiation / biosynthesis*
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Antigens, Differentiation / genetics
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Base Sequence
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Biomarkers
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Cell Differentiation / drug effects
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Cell Division
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Cell Line
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Cell Lineage
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Clone Cells
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DNA-Binding Proteins / biosynthesis
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / physiology*
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Erythroid-Specific DNA-Binding Factors
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GATA1 Transcription Factor
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Gene Expression Regulation* / drug effects
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Growth Substances / pharmacology
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Hematopoiesis / drug effects
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Hematopoiesis / physiology*
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Hematopoietic Stem Cells / drug effects
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Humans
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Mice
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Molecular Sequence Data
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Oligonucleotides, Antisense / pharmacology
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Promoter Regions, Genetic
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Proto-Oncogene Proteins / biosynthesis
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / physiology*
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Proto-Oncogene Proteins c-myb
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Recombinant Fusion Proteins / metabolism
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Stem Cells / classification
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Stem Cells / cytology*
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Stem Cells / drug effects
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Stem Cells / metabolism
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Trans-Activators / biosynthesis
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Trans-Activators / genetics
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Trans-Activators / physiology*
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Transcription Factors / biosynthesis
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Transcription Factors / genetics
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Transcriptional Activation
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Transfection
Substances
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Antigens, CD34
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Antigens, Differentiation
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Biomarkers
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DNA-Binding Proteins
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Erythroid-Specific DNA-Binding Factors
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GATA1 Transcription Factor
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GATA1 protein, human
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Gata1 protein, mouse
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Growth Substances
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Oligonucleotides, Antisense
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-myb
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Recombinant Fusion Proteins
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Trans-Activators
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Transcription Factors