The transcription factor PU.1 plays a pivotal role in normal myeloid differentiation. PU.1 ؊/؊ mice exhibit a complete block in myeloid differentiation. Heterozygous PU.1 mutations were reported in some patients with acute myeloid leukemia (AML), but not in AML with translocation t(8;21), which gives rise to the fusion gene AML1-ETO.

IntroductionHematopoiesis is the regulated development of distinct cellular lineages from a common precursor, the hematopoietic stem cell. Fundamental changes in gene expression result in each cell type expressing a characteristic complement of genes necessary for its function. This is achieved through the action of transcriptional regulators with general and restricted expression patterns in the hematopoietic system. 1 The ets domain transcription factor PU.1 is preferentially expressed in myeloid and B cells. 2,3 Inactivation of the PU.1 gene in mice causes defects in the development of multiple hematopoietic lineages, including B and T lymphocytes, monocytes, and granulocytes. 4,5 PU.1 regulates the expression of almost all characterized myeloid genes, including growth factor receptors. In particular, it directs the monocyte-specific expression of the macrophage colony-stimulating factor receptor. 6,7 PU.1 probably plays an important role at several stages in the differentiation process, and there is evidence that it is active at an early stage, mediating commitment of multipotential progenitor cells to the myeloid lineage. 8 CCAAT/enhancer-binding protein alpha (C/EBP␣) was initially identified in liver and adipose tissue, where it was found to be important for terminal differentiation. 9-14 C/EBP␣ expression is prominent in immature myeloid cells. 15-17 C/EBP␣-null mice lack the entire granulocyte lineage but develop normal monocytes. 18 Recently, we identified dominant-negative mutations of C/EBP␣ in acute myeloid leukemia 19 and a down-regulation of C/EBP␣ expression by the leukemic fusion protein AML1/ETO, 20 suggesting an important role of C/EBP␣ in leukemogenesis. Ectopic expression of C/EBP␣ in U937 monocyte leukemia cells induces granulocytic differentiation over a 2-week period and inhibits monocyte differentiation. 16 These hematopoietic progenitors require PU.1 to initiate monocyte differentiation and C/EBP␣ to initiate granulopoiesis. PU.1 has been shown to interact with a C/EBP family member, C/EBP␦. 21 However, the interaction of these transcription factors in differentiating to a specific lineage is still unclear.We propose here that the granulocyte factor C/EBP␣ interacts with the myeloid master regulator PU.1 and inactivates PU.1. c-Jun belongs to the b-ZIP group of DNA-binding proteins and is a component of AP-1 transcription complexes. 22 c-Jun has been shown to be a coactivator of PU.1, resulting in increased macrophage-colony-stimulating factor (M-CSF) receptor expression, and it is involved in the development of the monocyte lineage. 23 Here we show that C/EBP␣ blocks PU.1 function by displacing c-Jun, the coactivator of PU.1. Furthermore, C/EBP␣ specifies the fate of myeloid progenitor cells to the granulocyte lineage by inactivating PU.1 through protein-protein interactions. Materials and methods Cell lines and cell cultureFibroblast F9 and 293T cells were cultured in Dulbecco Modified Eagle Medium (PAN Biotech GmbH, Karlsruhe, Germany) containing 10% fetal bovine serum (FBS; Gibco BR...

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