Hematopoietic stem cells and their progenitors exhibit multilineage patterns of gene expression. Molecular mechanisms underlying the generation and refinement of these patterns during cell fate determination remain unexplored because of the absence of suitable experimental systems. Using PU.1(-/-) progenitors, we demonstrate that at subthreshold levels, this Ets transcription factor regulates a mixed pattern (macrophage/neutrophil) of gene expression within individual myeloid progenitors. Increased PU.1 levels refine the pattern and promote macrophage differentiation by modulating a novel regulatory circuit comprised of counter antagonistic repressors, Egr-1,2/Nab-2 and Gfi-1. Egr-1 and Egr-2 function redundantly to activate macrophage genes and to repress the neutrophil program. These results are used to assemble and mathematically model a gene regulatory network that exhibits both graded and bistable behaviors and accounts for the onset and resolution of mixed lineage patterns during cell fate determination.

Hematopoietic transcription factors are essential for specifying cell fates; however, the function of cytokines in such developmental decisions is unresolved. We demonstrate here that haploinsufficiency for the gene encoding the transcription factor PU.1 partially suppresses the neutropenia of mice deficient in granulocyte colony-stimulating factor. This suppression was due to an increase in granulocytic progenitors and a diminution of monocytic progenitors. With (PU.1+/-) ES cells as well as (PU.1-/-) hematopoietic progenitors, we show that higher expression of PU.1 is needed for macrophage than for neutrophil development. In a (PU.1-/-) progenitor cell line, in which graded activity of PU.1 regulates neutrophil versus macrophage development, granulocyte colony-stimulating factor signaling supported the neutrophil cell fate by increasing expression of the neutrophil transcription factor C/EBPalpha in relation to expression of PU.1. Collectively, these results indicate that cytokines can promote cell fate decisions by altering the relative concentrations of lineage-determining transcriptional regulators.

PU.1 and GATA transcription factors appear to antagonize each other's function in the development of distinct lineages of the hematopoietic system. In contrast, we demonstrate that PU.1, like GATA-2, is essential for the generation of mast cells. PU.1-/- hematopoietic progenitors can be propagated in IL-3 and differentiate into mast cells or macrophages upon restoration of PU.1 activity. Using these progenitors and a conditionally activatable PU.1 protein, we show that PU.1 can negatively regulate expression of the GATA-2 gene. In the absence of GATA-2, PU.1 promotes macrophage but not mast cell differentiation. Reexpression of GATA-2 in such progenitors enables the generation of mast cells. We propose a developmental model in which cooperative function or antagonistic crossregulation by PU.1 of GATA-2 promotes distinct myeloid cell fates.