International Journal of Cell Biology this, PU.1 levels have been estimated to be approximately eightfold higher in macrophages compared to B cells [2, 8]. PU.1 not only inﬂuences the myeloid-lymphoid decision of MPPs but also regulates the potential diﬀerentiation pathways to which CMPs and CLPs can commit. For instance, ﬁne tuning of PU.1 levels in CMPs is important in directing diﬀerentiation towards MEPs or GMPs. PU.1 is expressed at high levels in GMPs and directs commitment towards the neutrophil and monocyte lineages. In contrast, PU.1 expression is downregulated in MEPs, and this is essential for the development of megakaryocytes and RBCs [7, 8, 20]. Within the lymphoid pathway, diﬀerentiation of CLPs to form B cells or T cells is accompanied by a decrease in PU.1 levels, and PU.1 is further silenced as T cells develop. Conversely, PU.1 expression in B cells gradually increases as they mature, although not to the extent observed in macrophages [2, 7, 8]. Together, these data collectively illustrate the importance of PU.1 in maintaining the HSC pool and in directing diﬀerentiation towards the myeloid and lymphoid pathways. The role of PU.1 in speciﬁc lineage diﬀerentiation programs will now be discussed in more detail. 3. PU.1 Is a Negative Regulator of Erythropoiesis Downregulation of PU.1 expression in committed MEPs is important for erythroid diﬀerentiation. Low levels of PU.1 in early erythroid precursors are essential for them to undergo proliferation before terminally diﬀerentiating into RBCs. Using a PU.1 mouse model, Back et al. showed that ery-throid cells from these mice diﬀerentiate prematurely and are susceptible to apoptosis [6]. Another interesting transgenic model generated by Tavitian’s group overexpresses PU.1 in all haematopoietic lineages except T cells [11]. This results in splenic hyperplasia and anaemia in approximately half of the transgenic mice, characterised by high num- bers of immature blast cells and poorly haemoglobinised erythroblasts. The respective phenotypes from these two transgenic lines complement each other well. Whereas under normal physiological conditions PU.1 expression is diminished to allow terminal diﬀerentiation of erythroblasts, the persistent expression of PU.1 in Tavitian’s mouse line results in a maturation block with an overrepresentation of immature erythroblasts [11, 20, 23]. Conversely, elimi- nation of PU.1 results in premature diﬀerentiation of these cells. In fact, several groups have clearly demonstrated this point utilising the widely employed MEL (murine ery- throleukemia) cell line. This cell line is derived from mice infected with Friend virus where the SFFV component has integrated into the Spi-1/PU.1 locus and transcriptionally activates the gene, resulting in erythroblast transformation [1, 24]. MEL cells, which exhibit ectopic PU.1 expression, are arrested at the erythroblast stage and undergo continual proliferation without diﬀerentiation [25, 26]. Treatment with chemical agents such as DMSO (dimethyl sulfoxide) 3 or HMBA (hexamethylene bisacetamide) results in growth arrest, erythroid diﬀerentiation, and haemoglobin produc- tion, along with a concomitant decrease in PU.1 expression [27, 28]. The reduction in PU.1 expression is crucial for the diﬀerentiation of these cells. Forced expression of PU.1 in MELs inhibits DMSO- and HMBA-induced diﬀerentiation, while silencing PU.1 in these cells has been shown to be suﬃcient to drive terminal diﬀerentiation in the absence of any chemical inducers [25, 27–29]. PU.1 thus promotes erythroid cell proliferation and prevents diﬀerentiation. It has been proposed that one mech- anism by which PU.1 does this is by regulating controllers of the cell cycle [25]. PU.1 directly activates expression of Cdk6 (cyclin-dependent kinase 6), a G1 phase-speciﬁc cell cycle kinase known to inhibit MEL cell diﬀerentiation [25, 30]. Cdk6 associates with D-cyclins to regulate cell cycle progression through the G1 phase, and like PU.1, Cdk6 is constitutively expressed in proliferating MELs [30]. After chemical induction, downregulation of PU.1 results in decreased Cdk6 levels, thus arresting proliferation and permitting diﬀerentiation. Another established mechanism by which PU.1 inhibits erythroid diﬀerentiation is by antagonising the erythroid regulator Gata-1. These two transcription factors are known to exert opposing eﬀects on each other, and the interplay between these two master regulators is instrumental in the decision for CMPs to commit towards either the myeloid or erythroid lineage [31, 32]. PU.1 interacts with Gata-1 and prevents its transcriptional activity while conversely, Gata- 1 inhibits PU.1 function by disrupting its interaction with the coactivator c-Jun [32–34]. Hence, these data collectively suggest that PU.1 inhibits erythroid diﬀerentiation by upreg- ulating Cdk6 to promote proliferation and by antagonising the master erythroid regulator Gata-1. This may also explain why PU.1 expression must be downregulated during erythro- poiesis to allow normal production of RBCs. 4. PU.1 Is a Master Myeloid Regulator Both neutrophils and monocytes are generated from GMPs. Diﬀerentiation towards either of these cell fates is highly dependent on PU.1 [7, 8]. Multiple knockout models have demonstrated the importance of PU.1 in myelopoiesis; abrogation of PU.1 results in a marked lack of CMPs, defec- tive granulocytic neutrophil production, and an absence of mature macrophages [4, 5, 12, 35]. Because PU.1 is absolutely required early during myelopoiesis at the CMP stage, questions have been asked as to whether it is dis- pensable once cells have committed to the CMP pathway. In answering this, Iwasaki et al. puriﬁed PU.1 CMPs and GMPs from a conditional knockout mouse and assessed their capacity to form myeloid colonies in culture [12]. Both CMPs and GMPs were found to be unable to contribute to the mature myeloid fraction, indicating that PU.1 is further required following commitment to the myeloid lineage to promote diﬀerentiation to generate granulocytes and macrophages. PU.1 has since been shown to enable committed cells to respond to a variety of myeloid growth