Previous observations suggested that functional antagonism between FLI-1 and EKLF might be involved in the commitment toward erythrocytic or megakaryocytic differentiation. We show here, using inducible shRNA expression, that EKLF knockdown in mouse erythroleukemia (MEL) cells decreases erythrocytic and increases megakaryocytic as well as Fli-1 gene expression. Chromatin immunoprecipitation analyses revealed that the increase in megakaryocytic gene expression is associated with a marked increase in RNA pol II and FLI-1 occupancy at their promoters, albeit FLI-1 protein levels are only minimally affected. Similarly, we show that human CD34 ؉ progenitors infected with shRNA lentivirus allowing EKLF knockdown generate an increased number of differentiated megakaryocytic cells associated with increased levels of megakaryocytic and Fli-1 gene transcripts. Single-cell progeny analysis of a cell population enriched in bipotent progenitors revealed that EKLF knockdown increases the number of megakaryocytic at the expense of erythrocytic colonies. Taken together, these data indicate that EKLF restricts megakaryocytic differentiation to the benefit of erythrocytic differentiation and suggest that this might be at least partially mediated by the inhibition of FLI-1 recruitment to megakaryocytic and Fli-1 gene promoters. (Blood. 2008; 112:576-584)
IntroductionErythrocytic and megakaryocytic lineages derive from a common bipotent progenitor (megakaryocyte/erythroid progenitor, MEP) able to generate only erythrocytic or megakaryocytic progenitors. 1,2 A common bipotent precursor, precursor for erythroid and megakaryocytic cells (PEM), has also been characterized in the spleen of anemic mice. 3,4 Despite this very close proximity, the molecular mechanisms controlling commitment toward either one of these 2 lineages remain poorly understood.Two main models have been proposed to explain the commitment of multipotent hematopoietic progenitors. 5,6 According to the "instructive model," lineage commitment is dictated by specific extracellular signals such as cytokines. Although erythropoietin and thrombopoietin enhance the proliferation, survival, and differentiation of already committed erythrocytic and megakaryocytic progenitors expressing erythropoietin-or thrombopoietin-specific receptors, it is now well established that they have no instructive role in the commitment. 7,8 Available data are more compatible with a "stochastic model," suggesting that commitment is dictated by the spontaneous formation of specific and mutually exclusive combinations of transcription factors. 5,6 At least 10 different transcription factors involved in erythrocytic and/or megakaryocytic differentiation have been identified. [9][10][11][12] Most of them, such as GATA-1, NF-E2, TAL-1, LMO2, FOG1, and GFI-1B, are involved in the regulation of both erythrocytic and megakaryocytic differentiation. However, there is no indication that differential expression of either one of these 6 factors might be involved in the commitment decision. Several transgenic...
