4 factors by regulating the expression of a number of myeloid- speciﬁc genes, including the cytokine receptors granulo- cyte/macrophage colony-stimulating factor receptor α (GM- CSFRα), granulocyte-CSFR (G-CSFR), macrophage-CSFR (M-CSFR), and interleukin-7 receptor α (IL-7Rα) [36, 37]. 4.1. Regulation of Macrophage versus Neutrophil Production. As a master myeloid regulator, PU.1 does not only regulate GMP development but also directs the diﬀerentiation path- ways that give rise to both neutrophils and macrophages. Again, precise control of PU.1 levels in GMPs is required to direct these distinct diﬀerentiation programs. Diﬀerent dosage of PU.1 expression in GMPs modulates distinct reg- ulatory networks which involve a number of lineage-speciﬁc transcription factors such as Egr-2 (early growth response-2), Gﬁ-1 (growth factor independent-1), and C/ebpα (CCAAT enhancer-binding protein α) [21, 38, 39]. Using myeloid cell lines with an inducible transgene allowing high or low levels of PU.1, Laslo et al. revealed that low expression of PU.1 activates a mixed lineage of macrophage and neutrophil genes [39]. When PU.1 levels exceed a certain threshold, this induces the expression of Egr-2 and the transcriptional repressor Nab-2. Together, Egr- 2 and Nab-2 repress the expression of Gﬁ-1, a transcrip- tion factor that promotes neutrophil diﬀerentiation. This results in the silencing of neutrophil genes and promotes macrophage diﬀerentiation [21, 39]. PU.1 thus indirectly represses Gﬁ-1 expression and conversely, PU.1 levels are elevated in Gﬁ-1 knockout mice [21]. Similar to Gata-1 and PU.1 antagonising each other’s function in the myeloid- erythroid decision of CMPs, it appears that Gﬁ-1 and PU.1 negatively regulate each other’s expression to determine the macrophage-neutrophil decision of GMPs [21]. Like Gﬁ-1, the neutrophil transcription factor C/ebpα is also expressed in myeloid cells and antagonises PU.1 to direct neutrophil diﬀerentiation. Dahl et al. showed that haematopoietic progenitors expressing high levels of indu- cible PU.1 predominantly develop into macrophages when cultured in IL-3 (interleukin-3) [38]. However, when these cells are pretreated with G-CSF (which promotes granulocyte development) prior to PU.1 induction, they display an upregulation of C/ebpα and instead form neutrophils. Thus, it can be deduced that with low levels of PU.1 in GMPs, the expression of Gﬁ-1 and C/ebpα are suﬃcient to antagonise PU.1 function by repressing macrophage genes and promot- ing neutrophil development. In contrast, when lineage commitment is directed towards macrophage development, PU.1 expression is upregulated to overcome the antagonism imposed by the two neutrophil regulators. 5. Regulation of Lymphopoiesis: PU.1 in B-Cell and T-Cell Maturation In addition to being a major myeloid regulator, PU.1 has also been shown to modulate lymphopoiesis, the process that gives rise to B cells and T cells. PU.1 mutant mice exhibit a loss of the B and T cell compartments and develop fatal sep- ticaemia within 2 days of birth owing to a lack of mature International Journal of Cell Biology and functional immune cells [5, 35]. Studies investigating the pattern of PU.1 expression using GFP-reporter mice have revealed that PU.1 levels increase as B-cells mature, while PU.1 is completely silenced in mature T cells [7, 8]. Despite the requirement for PU.1 for B-cell production, its expression is dispensable once progenitors are committed to the lymphoid lineage as revealed by Iwasaki et al. [12]. PU.1 CLPs generate B cells and express B-cell genes at comparable levels to wildtype CLPs in culture. In vivo, tar- geted deletion of PU.1 after the pre-B-cell stage does not disrupt immunoglobulin expression or the cells’ response to a variety of mitogenic agents. This suggests that PU.1 is not essential for B-cell maturation once it has directed progenitors to the CLP stage. One possible explanation for this is that other members of the ETS family of transcription factors, such as Spi-B, may have functional redundancy with PU.1 in B cells [40, 41]. Similar to in B cells, PU.1 is only required at the early CLP stage for T-cell generation. Once cells develop to pro-T-cells, PU.1 expression is dramatically silenced. Silencing of PU.1 is required for T cell maturation and in fact, forced overexpression of PU.1 results in growth arrest and a T-cell maturation block [42]. 6. Dosage-Dependent Regulation and Pathogenesis It is clear that precise levels of PU.1 expressed at diﬀerent stages within the haematopoietic lineage are crucial in directing proper diﬀerentiation and cell fate commitment (Figure 1). Hypomorphic mouse lines expressing varied doses of PU.1 have been particularly helpful in providing a better picture on how PU.1 activates and represses speciﬁc sets of genes. The PU.1BN and PU.1Blac mice have been esti- mated to express approximately 20% (“high” concentration) and 2% (“low” concentration) of PU.1 compared to wildtype mice, respectively, [22, 35]. By performing microarrays on PU.1BN, PU.1Blac, and PU.1−/− myeloid cell lines, it was revealed that PU.1 can regulate its targets in four distinct modes [22]. Some target genes are activated or repressed equally at both “high” and “low” concentrations of PU.1; others are exclusively activated or repressed at either “high” or “low” levels of PU.1 but not both, and the ﬁnal group of targets can be activated or repressed in a dose-dependent manner by PU.1, that is, the degree of their transcriptional regulation is dependent on the level of PU.1. Targets which are repressed in such a “gradient” manner include erythroid and T-cell-speciﬁc genes, while genes which are activated include the myeloid genes. This illustrates the dependence on PU.1 levels in the regulation of these lineage-speciﬁc genes and may in part explain why PU.1 expression varies in certain cell types during speciﬁc stages of haematopoiesis. In fact, when this delicate expression control is disrupted, the balance between diﬀerent haematopoietic regulators is upset, and in many occasions, this results in oncogenic transformation [11, 33, 43]. A notable example is the SFFV- induced transcriptional activation of PU.1 in Friend virus infected mice which develop acute erythroleukaemia [1]. In this instance, the high level of PU.1 expression disrupts