Induction of hematopoietic commitment and erythromyeloid differentiation in embryonal stem cells constitutively expressing c-myb.
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.
Melotti
P
P
Departments of Microbiology and Immunology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA USA.
Calabretta
B
B
D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis / Q000235:genetics; D000943:Antigens, Differentiation / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D015415:Biomarkers; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division; D002460:Cell Line; D019070:Cell Lineage; D002999:Clone Cells; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D005786:Gene Expression Regulation / Q000187:drug effects; D006133:Growth Substances / Q000494:pharmacology; D006410:Hematopoiesis / Q000187:drug effects / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000187:drug effects; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013234:Stem Cells / Q000145:classification / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D015533:Transcriptional Activation; D014162:Transfection
D018952:Antigens, CD34; D000943:Antigens, Differentiation; D015415:Biomarkers; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; C494689:Gata1 protein, mouse; D006133:Growth Substances; D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors