Characterization of the hematopoietic transcription factor NF-E2 in primary murine megakaryocytes.
Biochemical analysis of megakaryocytes, the precursors of blood platelets, is limited by their rarity in vivo, and studies on lineage-specific gene expression have been conducted exclusively in cell lines with limited megakaryocytic potential. Mice lacking the transcription factor NF-E2 display arrested megakaryocyte differentiation and profound thrombocytopenia. To study the heterodimeric NF-E2 protein in primary cells, we cultured mouse fetal livers with the c-Mpl ligand, obtained highly enriched megakaryocyte populations, and readily detected NF-E2 activity in nuclear extracts. As in erythroid cells, p45 NF-E2 is the only large subunit in primary megakaryocytes that dimerizes with distinct small Maf proteins to constitute a heterogeneous NF-E2 complex. Whereas p18/MafK is the predominant small Maf protein in erythroid cells, the related polypeptides MafG and/or MafF predominate in megakaryocytes. Although this represents the first example of differential small Maf protein expression among closely related blood lineages, the DNA-binding specificity of NF-E2 is similar in both cell types. Although the megakaryocyte protein preferentially binds an asymmetric AP-1-related motif, it also recognizes cAMP-responsive element-related sequences, albeit with lower affinity, and nucleotides outside the core sequence influence the DNA-protein interaction. These results demonstrate the feasibility of biochemical studies on primary murine megakaryocytes and provide a basis to dissect the critical functions of NF-E2 in megakaryocyte differentiation.
Lecine
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P
Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Blank
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Shivdasani
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D046508:Culture Techniques; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D008099:Liver / Q000196:embryology; D050985:MafK Transcription Factor; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; D009687:Nuclear Proteins / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013926:Thrombopoietin / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050985:MafK Transcription Factor; C494696:Mafk protein, mouse; D050983:NF-E2 Transcription Factor; D050984:NF-E2 Transcription Factor, p45 Subunit; C494693:Nfe2 protein, mouse; D009687:Nuclear Proteins; D014157:Transcription Factors; D004247:DNA; D013926:Thrombopoietin