Oncogene (2002) 21, 3377 ± 3390 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc

Transcriptional regulation of granulocyte and monocyte development

Alan D Friedman*,1

1Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland, MD 21231, USA

Granulocytes and monocytes develop from a common granulocyte/macrophage progenitors (GMPs), charac- myeloid progenitor. Early granulopoiesis requires the C/ terized by expression of CD34 and Fcg Receptor(II/III) EBPa, PU.1, RAR, CBF, and c-Myb transcription (Akashi et al., 2000; Traver et al., 2001). These factors, and terminal neutrophil di€erentiation is progenitors in turn give rise to granulocyte-, mono- dependent upon C/EBPe, PU.1, Sp1, CDP, and cyte-, and granulocyte/monocyte-colony forming units HoxA10. Monopoiesis can be induced by Maf-B, c- (CFU-G, CFU-M, and CFU-GM). Granulocyte-Col- Jun, or Egr-1 and is dependent upon PU.1, Sp1, and ony Stimulating Factor (G-CSF) enables the formation ICSBP. Signals eminating from cytokine receptors of CFU-G in methylcellulose cultures, Macrophage- modulate factor activities but do not determine cell Colony Stimulating Factor (M-CSF, CSF-1) enables fates. Orchestration of the myeloid developmental the growth of CFU-M, and Granulocyte/Macrophage- program is achieved via cooperative gene regulation, Colony Stimulating Factor (GM-CSF) or Interleukin-3 via synergistic and inhibitory protein ± protein interac- (IL-3) enable the proliferation of CFU-G, CFU-M, tions, via promoter auto-regulation and cross-regulation, and CFU-GM. In addition to these features of normal via regulation of factor levels, and via induction of cell cells, the ®nding that a common subset of acute cycle arrest: For example, c-Myb and C/EBPa myeloid leukemia has both a granulocytic and cooperate to activate the mim-1 and NE promoters, monocytic cellular component further underscores the PU.1, C/EBPa, and CBF, regulate the NE, MPO, and close developmental relationship of neutrophils and M-CSF Receptor genes. PU.1:GATA-1 interaction and monocytes. Fetal liver and adult marrow also contain C/EBP suppression of FOG transcription inhibits progenitors capable of producing B-lymphoid and erythroid and megakaryocyte gene expression. c-Jun:- macrophage cells in single colonies, re¯ecting both PU.1, ICSBP:PU.1, and perhaps Maf:Jun complexes the plasticity of pluripotent stem cell commitment and induce monocytic genes. PU.1 and C/EBPa activate the relatedness of these lineages (Lacaud et al., 1998; their own promoters, C/EBPa rapidly induces PU.1 and Montecino-Rodriguez et al., 2001; Traver et al., 2001). C/EBPe RNA expression, and RARa activates the C/ Monocyte progenitors not only develop into macro- EBPe promoter. Higher levels of PU.1 are required for phages but are also capable of osteoclast di€erentiation monopoiesis than for B-lymphopoiesis, and higher C/ (Roodman, 1996), and GMPs share with common EBP levels may favor granulopoiesis over monopoiesis. lymphoid progenitors (CLPs) the capacity to generate CBF and c-Myb stimulate proliferation whereas C/EBPa myeloid dendritic cells (Manz et al., 2001). induces a G1/S arrest; cell cycle arrest is required for Neutrophil maturation proceeds from the myeloblast terminal myelopoiesis, perhaps due to expression of p53 to the promyelocyte, when primary granules become or hypo-phosphorylated Rb. apparent, to the myelocyte, when cell division ceases Oncogene (2002) 21, 3377 ± 3390. DOI: 10.1038/sj/ and secondary or speci®c granules begin to appear, to onc/1205324 the metamyelocyte, band, and ®nally the neutrophil, which has a three-lobed nucleus and tertiary granules. Keywords: granulocyte; monocyte; di€erentiation; Early markers of granulopoiesis include the G-CSF C/EBP; PU.1 Receptor, the CD33 and CD13 surface markers, and primary granule components, such as myeloperoxidase (MPO), neutrophil elastase (NE), myeloblastin (MBN), Introduction lysozyme and avian mim-1. Oxidation system compo- nents, such as the gp91-phox cytochrome heavy chain, Polymorphonuclear and mononuclear phagocytes func- are expressed from the myelocyte stage onward and tion in host defense against infections and are capable later markers of neutrophil development include of recognizing, ingesting, and destroying foreign secondary granule components, such as lactoferrin materials and organisms. Pluripotent hematopoietic (LF) and neutrophil gelatinase (NG), and the Gr-1 stem cells in fetal liver or in adult marrow give rise to surface marker. Monopoiesis proceeds from the monoblast to the circulating monocyte, which matures without prolifera- tion to the tissue macrophage. Early markers of this *Correspondence: AD Friedman, Johns Hopkins University, Cancer Research Building, Room 253, 1650 Orleans Street, Baltimore, lineage include the M-CSF Receptor, lysozyme, and Maryland, MD 21231; E-mail: [email protected] the Fcg Receptor(II/III), and latter markers include Regulation of granulopoiesis and monopoiesis AD Friedman 3378 gp91-phox, and several surface proteins: Macrosialin, Table 1 Transcription factors regulating granulocytic or monocytic scavenger receptor (SR), F4/80, CD14, CD11b, and genes CD18. Fcg Receptor(II/III) is also expressed on Gene Transcription factors myeloid progenitors (Carlsson et al., 1995); the Immature granulocytes CD11b:CD18 integrin is also expressed on neutrophils, mim-1 C/EBP's, c-Myb B-lymphocytes, and some T-cell subsets (Christensen et Myeloperoxidase (MPO) C/EBPs, PU.1, CBF, c-Myb, CDP/HoxA10 al., 2001); and CD14 is also expressed in hepatocytes Neutrophil elastase (NE) C/EBPs, PU.1/GABP, CBF, c-Myb, Sp1 (Hetherington et al., 1999). Phagocytic capacity is an Myeloblastin (MBN) C/EBPs, PU.1, c-Myb G-CSF receptor C/EBPs, PU.1 additional hallmark of maturing monocytes. GM-CSF receptor C/EBPs, PU.1 CD13 c-Myb, Ets-1 or Ets, c-Maf Lysozyme C/EBPs, PU.1 Receptor signaling c-fes PU.1, Sp1 Mature granulocytes gp91phox PU.1, ICSBP, CDP, HoxA10 We have recently reviewed how G-CSF Receptor Lactoferrin (LF) C/EBPs, Sp1, CDP signals might modulate granulopoiesis (Ward et al., Immature monocytes 2000). Redundancy of receptor signaling is evident M-CSF receptor C/EBPs, CBF, PU.1, c-Jun from the ®nding that G-CSF (7/7), G-CSF; GM- Lysozyme C/EBPs, PU.1 Mature monocytes CSF (7/7; 7/7); G-CSFR (7/7), or G-CSFR; IL- Macrosialin PU.1, c-Jun 6 Receptor (7/7; 7/7) mice have only about a Scavenger receptor PU.1, c-Jun twofold reduction in marrow neutrophils (Lieschke et CD14 C/EBPs, Sp1 al., 1994; Liu et al., 1996a, 1997; Seymour et al., 1997). gp91phox PU.1, ICSBP, CDP, HoxA10 CD11b PU.1, Sp1 Bcl-2 did not replace G-CSF receptor signals during CD18 PU.1, GABP, Sp1 32D cl3 maturation-MPO and Cathepsin G induction did not occur, but nuclear morphologic changes were observed (Rodel and Link, 1996). Thus, G-CSF receptor signals provide more than a survival function insights gained from characterizing transcription factor in this setting. Similarly, G-CSF receptor signals expression patterns, the phenotypes of knockout mice, cooperated with exogenous C/EBPa to allow induction and the e€ects of factor over-expression. of MPO and NE in Ba/F3 lymphoid cells and were The promoter of the avian mim-1 promoter is required for induction of C/EBPe in 32D cl3 cells activated cooperatively by C/EBPs and c-Myb in (Wang et al., 2001; Nakajima and Ihle, 2001). Also, immature granulocytic cells (Ness et al., 1993). This introduction of exogenous GM-CSF or IL-2 receptors section will not refer to individual C/EBPs, as C/EBP allows CLPs to be redirected to the granulocyte and family members bind a common DNA motif and often monocyte lineages (Kondo et al., 2000). activate reporter constructs similarly in transient M-CSFR signaling has also recently been reviewed assays. The MPO promoter region is activated (Bourette and Rohrschneider, 2000). Transgenic ex- cooperatively by CBF and c-Myb (Suzow and Fried- pression of bcl-2 in monocytes enabled M-CSF (7/7) man, 1993; Nuchprayoon et al., 1994; Britos-Bray and mice to develop macrophages (Lagasse and Weissman, Friedman, 1997), and its distal enhancer is regulated by 1997). While this ®nding precludes an absolute C/EBPs and PU.1 (Ford et al., 1996). The NE requirement for M-CSF Receptor signals in macro- promoter is regulated cooperatively by C/EBPs, PU.1, phage development, redundant signals available from a and c-Myb; the murine but not the human NE subset of other cytokine receptors may be necessary. promoter is activated weakly by CBF; GABP can The ability of phorbol esters to induce monocytic substitute for PU.1 to cooperatively active the NE di€erentiation of hematopoietic cell lines via activation promoter; and a 76 kb NE enhancer is activated by of protein kinase Ca (PKCa) or PKCd suggests that Sp1 and an Ets family member other than PU.1 this signaling pathway is important in normal mono- (Nuchprayoon et al., 1994; 1997, 1999; Oelgeschlager et poiesis (Mischak et al., 1993). Overall, with possible al., 1996). MBN and azurocidin are serine proteases notable exceptions, signals eminating from cytokine highly related to NE. Their promoters contain receptors appear to modulate but not determine sequences elements homologous to the NE elements myeloid di€erentiation. which bind C/EBPs, PU.1, and c-Myb (Friedman, 1996a), and indeed these proteins regulate the MBN promoter (Lutz et al., 2001). The promoters of both Regulation of gene expression the G-CSF and GM ± CSF receptor genes are activated by C/EBPs and PU.1 (Hohaus et al., 1995; Smith et al., Di€erentiation is de®ned by gene expression patterns, 1996). The CD13 gene is activated directly by c-Myb and so regulatory factors must act either directly or and Ets-1 or Ets-2, and c-Maf indirectly inhibits the indirectly to induce the transcription of lineage transcription of this gene by complexing with c-Myb in markers. Therefore, studies investigating the regulation immature but not maturing granulocyte precursors of granulocyte- and monocyte-speci®c genes, as (Shapiro, 1995; Hegde et al., 1998). The avian summarized in Table 1, will be reviewed initially. The lysozyme gene 72.7 kb enhancer is regulated by C/ subsequent sections will then describe additional EBPs and PU.1, and its 76.1 kb enhancer is activated

Oncogene Regulation of granulopoiesis and monopoiesis AD Friedman 3379 by C/EBPs (Ahne and Stratling, 1994; Goethe and mature monocytes. The next section will describe Loc, 1994; Faust et al., 1999). Consistent with these additional evidence which con®rms the importance of results, we recently found that activation of exogenous these factors, as well as evidence implicating C/EBPa-ER with estradiol in the murine 32D cl3 additional transcription factors, in the regulation of myeloblast cell line rapidly induces lysozyme RNA granulocyte and monocyte development. The subse- expression in the presence of cycloheximide but not quent section will then address the question of how actinomycin D (Wang and Friedman, 2002). Finally, these factors orchestrate the myeloid developmental the c-fes tyrosine kinase promoter region is regulated program. by PU.1 and Sp1 in immature granulocytic and monocytic cells (Heydemann et al., 1996). In maturing neutrophils, the gp91-phox promoter Transcription factors region is activated by the PU.1:ICSBP dimer and is repressed by both CDP and HoxA10 ± the levels of C/EBPs the latter factors decrease during terminal neutrophil and monocyte di€erentiation (Skalnik et al., 1991; The C/EBPs homo- and hetero-dimerize via their C- Eklund and Kakar, 1999; Eklund et al., 2000). The LF terminal leucine zipper domains and bind DNA as promoter is activated by C/EBPs and Sp1 and is dimers via the adjacent basic regions (Landschulz et repressed by CDP (Khanna-Gupta et al., 1997, 2000). al., 1989). The C/EBP consensus binding site is 5'-T(T/ CDP represses both the gp91-phox and LF promoters G)NNGNAA(T/G)-3'. C/EBPa, C/EBPb, and C/EBPd via interaction with sites upstream of activator binding have N-terminal trans-activation domains, and transla- sites (Luo and Skalnik, 1996; Khanna-Gupta et al., tion initiation from internal methionines produces 1997), and CDP also represses the gp91-phox promoter truncated, dominant-inhibitory polypeptides which via a site 790 which overlaps an activator-binding site retain the bZIP domain (Friedman et al., 1989, (Catt et al., 1999). HoxA10 interacts with an AT-rich Friedman and McKnight, 1990; Descombes and element located just upstream of the 5'-CCAAT Schibler, 1991; Calkhoven et al., 2000). C/EBPe has sequence at 790, the binding site for CDP, and so both a trans-activation and a trans-repression domain like CDP may repress gp91-phox transcription, in part, (Williamson et al., 1998), and C/EBPg and CHOP are via competition with activators (Eklund et al., 2000). dominant-inhibitory due to their ability to dimerize The MPO promoter contains a potent repressor with other C/EBPs and their lack of intact basic element active in 32D cl3 cells which has been mapped regions (Habener and Ron, 1992; Cooper et al., 1995). to a 28 bp segment which includes a 5'-GAAATC Trans-activation by C/EBPa may be regulated via Ras- sequence conserved in the homologous region of the induced phosphorylation of serine 248 (Behre et al., human MPO gene (Suzow and Friedman, 1993). 1999b). Perhaps this element binds CDP or HoxA10, repressors Within hematopoiesis, full-length C/EBPa, C/EBPb, thought to be more active in immature than mature and C/EBPd are predominantly expressed in the granulocytes. granulocyte, monocyte, and eosinophil lineages (Scott In immature monocytic cells, the M-CSF Receptor et al., 1992; Muller et al., 1995; Radomska et al., 1998). promoter is activated synergistically by C/EBP, PU.1, C/EBPa is the isoform most prominently detected in and CBF (Zhang et al., 1994a,b, 1996a; Petrovick et immature granulocytes (Scott et al., 1992; Hohaus et al., 1998), and c-Jun activates the M-CSF receptor al., 1995), whereas C/EBPe is found in later-stage gene indirectly, via its ability to interact with PU.1 granulocytes and in T-cells (Antonson et al., 1996). (Bassuk and Leiden, 1995; Behre et al., 1999a). In cells CHOP is only detected in granulocytic cells subjected di€erentiating into macrophages, the macrosialin and to stress, such as DNA-damage (Friedman, 1996b), scavenger receptor promoters are activated coopera- and C/EBPg expression in this lineage is not well tively by PU.1 and c-Jun, via binding sites separated by characterized. 33 or 135 bps, respectively (Moulton et al., 1994; Li et C/EBPa (7/7) mice lack neutrophils and eosino- al., 1998). The CD14 promoter is activated by C/EBP phils, but retain monocytes, lymphocytes, erythroid and Sp1 (Zhang et al., 1994c; Pan et al., 1999). And cells, and immature myeloblasts (Zhang et al., 1997). ®nally, PU.1 and Sp1 activate both the CD11b and Fetal liver cells from these mice lack Granulocyte- CD18 promoters; optimal transcription of the latter Colony Stimulating Factor (G-CSF) Receptor RNA, gene also requires GABP, which like PU.1 is a member consistent with the ability of C/EBPa to trans-activate of the Ets family of transcription factors (Pahl et al., the G-CSF Receptor promoter (Smith et al., 1996). 1993; Chen et al., 1993; Rosmarin et al., 1995a,b, Induction of G-CSF or IL-6 Receptor cDNAs into C/ 1998). EBPa (7/7) fetal liver cells restores their ability to From these studies, C/EBPs and PU.1 emerge as generate neutrophils in vitro in response to G-CSF or the most consistent regulators of genes expressed in IL-6 (Zhang et al., 1998). C/EBPb (7/7) mice retain granulocytic and immature monocytic cells. PU.1 all of the hematopoietic lineages, and hematopoietic also activates most genes expressed in mature defects in mice lacking C/EBPd or both C/EBPb and monocytic cells, c-Myb and CBF contribute to the C/EBPd were not severe (Screpanti et al., 1995; Tanaka activation of several genes in proliferating cells, and et al., 1995, 1997). C/EBPe (7/7) mice also retain Sp1 activates several promoters in neutrophils and neutrophils, although they lack secondary granules

Oncogene Regulation of granulopoiesis and monopoiesis AD Friedman 3380 (Yamanaka et al., 1997; Chumakov et al., 1997; In addition to regulating di€erentiation in several Lekstrom-Himes et al., 1999; Gombart et al., 2001). lineages, C/EBPa regulates proliferation directly. Expression of C/EBPa in U937 cells led to the Decreased expression in proliferating versus quiescent development of neutrophilic cells after 2 weeks, with hepatocytes and its ability to slow 3T3-L1 preadipocyte expression of the mRNAs encoding G-CSF Receptor, proliferation provided the initial evidence that C/EBPs lactoferrin, and neutrophil collagenase (Radomska et a€ect cell cycle progression (Friedman et al., 1989; al., 1998). Introduction of C/EBPa into avian multi- Umek et al., 1991). Both C/EBPaWT-ER and KaER potent progenitors also led to expression of myeloid inhibit progression from G1 to S phase in 32D cl3 cells markers (Nerlov et al., 1998). Similarly, estradiol- (Wang et al., 1999; Friedman and Wang, 2000). These mediated activation of C/EBPa-ER in 32D cl3 cells, a proteins may inhibit the G1/S transition via interaction factor-dependent, diploid cell line, induced neutrophils with E2F (Timchenko et al., 1999; Slomiany et al., within 4 days, with induction of MPO, lactoferrin, and 2000; Johansen et al., 2001). A KaER variant with a G-CSF Receptor RNAs (Wang et al., 1999). MPO defective leucine zipper domain does not slow 32D cl3 RNA was induced within 8 h of C/EBPa-ER activa- proliferation (Wang and Friedman, 2002). Perhaps C/ tion, but not in the presence of cycloheximide, EBPa dimers are required for interaction with E2F or indicating the need for an additional C/EBP-dependent with another protein associated with the E2F com- factor. A likely candidate is PU.1, as C/EBPa-ER plexes, or perhaps C/EBPa monomers interact with one induced PU.1 RNA within 4 h, even in the presence of of these proteins via its leucine zipper. In fact, E2F cycloheximide. C/EBPa-ER also rapidly induced the contains a leucine zipper, as noted (Slomiany et al., lysozyme and C/EBPe RNAs in the presence of 2000). C/EBPe also slows 32D cl3 proliferation cycloheximide, but not actinomycin D, suggesting (Nakajima and Ihle, 2001). Inhibition of proliferation direct regulation of the murine lysozyme and C/EBPe by C/EBPa or C/EBPe may contribute to granulopoi- genes by C/EBPs (Wang and Friedman, 2002). The esis, as maneuvers which stimulate proliferation, such signi®cance of C/EBPa regulation of PU.1 and C/EBPe as over-expression of c-Myb or cdk4, prevent induction RNA expression will be discussed further below. As C/ of late markers (Bies et al., 1995; Lou et al., 2000). EBPa is expressed in non-hematopoietic cells, it is Similarly, expression of p21WAF1/CIP1 or p27Kip1 in U937 interesting to speculate that early regulators of cells induces monocytic markers (Liu et al., 1996b). On hematopoiesis, such as c-Myb, CBF, or GATA-2, the other hand, induction of p27Kip1 with mimosine prime the PU.1 and C/EBPe genes for activation by C/ induced Lysozyme RNA but inhibited MPO RNA, an EBPa. The ability of CEBPa to activate its own earlier di€erentiation marker, in 32D cl3 cells (Q Wang promoter may also assist in committing progenitors to and AD Friedman, unpublished). The potential roles the myeloid lineages (Christy et al., 1993). of p53 and hypo-phosphorylated Rb in terminal The presence of several C/EBPs in myeloid cells myeloid di€erentiation will be discussed below. suggests that family members might compensate in vivo for the lack of a single isoform, just as GATA family PU.1 and interacting proteins, ICSBP, c-Jun, and members partially compensate for the lack of GATA-1 GATA-1 (Kulessa et al., 1995; Tsai et al., 1998). Indeed, as with C/EBPa, C/EBPb, C/EBPd, or C/EBPe can induce PU.1 binds DNA as a monomer, via its C-terminal Ets granulocytic di€erentiation in myeloblastic cell lines domain, to the consensus site 5'-AAAG(A/C/ (Park et al., 1999; Nakajima and Ihle, 2001; Wang and G)GGAAG-3' (Klemsz et al., 1990). The DNA-binding Friedman, 2002), and C/EBPa, C/EBPb, or C/EBPd domain of PU.1 and its N-terminal, glutamine-rich induced monocytic genes in P388 lymphoblasts (Hu et trans-activating domain were both required to rescue al., 1998). Redundancy is also evident from the ability myelopoiesis in PU.1 (7/7) ES cells, whereas its of C/EBPb to compensate for the loss of C/EBPa in acidic trans-activating domain was dispensable (Fisher hepatocytes and granulocytes in vivo (Chen et al., 2000; et al., 1998). Jones et al., 2002). In addition, compensatory e€ects Phosphorylation of serine 148 in PU.1 allows may account for the ability of immortalized C/EBPa interaction with Pip, also known as Interferon (7/7) progenitors to express high levels of G-CSF response factor-4 (IRF-4), an essential co-factor in B Receptor in vitro, even though they could not respond cells (Eisenbeis et al., 1995). Pip or Interferon to G-CSF in vivo (Collins et al., 2001). Global consensus sequence binding protein (ICSBP, IRF-8) inhibition of C/EBP-regulated genes can be achieved can interact with PU.1 in monocytic cells, enabling by expression of KRAB-C/EBPa-ER (KaER), in which trans-activation via a hybrid DNA-element binding the C/EBPa DNA-binding domain is linked to both a element (Meraro et al., 1999; Marecki et al., 2001). KRAB trans-repression domain and the Estrogen However, an essential role for Pip or ICSBP Receptor ligand-binding domain. In 32D cl3 cells, interaction for PU.1 activity in myeloid cells has not KaER inhibits granulocytic di€erentiation despite the been established. Notably, ICSBP (7/7) mice have presence of exogenous G-CSF receptor (Wang and reduced macrophages and increased granulocytic cells, Friedman, 2002). And in marrow cells, KaER inhibits and introduction of ICSBP into a cell line derived the formation of CFU-G, CFU-M, and CFU-GM in from the knockout mice increased monocytic markers multiple cytokines, without a€ecting BFU-E produc- at the expense of granulocytic markers (Holtschke et tion (Wang and Friedman, 2002). al., 1996; Tamura et al., 2000).

Oncogene Regulation of granulopoiesis and monopoiesis AD Friedman 3381 As discussed, c-Jun also cooperates with PU.1 to apparent relaxed `stringency' re¯ects redundancy regulate several monocytic genes, either via adjacent cis among Ets family members which is more e€ective in DNA elements or via direct interaction. Notably, c- vitro. For example, GABP can substitute for PU.1 to Jun, JunB, and JunD levels increase during monocytic activate the NE promoter in 32D cl3 cells (Nuch- di€erentiation (Lord et al., 1993), and exogenous c-Fos prayoon et al., 1997). or c-Jun induced partial monocytic di€erentiation in Activation of exogenous PU.1-ER in 32D cl3 cells is M1, U937, or WEHI-B D+ cells and increased the not sucient to induce terminal granulopoiesis, responsiveness of U937 cells to phorbol esters (Lord et although increased MPO RNA is detected (Wang et al., 1993; Szabo et al., 1994; Li et al., 1994). Phorbol al., 1999). Similarly, stable expression of PU.1 in 32D esters, an agent which stimulates the monocytic cl3 cells is of no consequence in IL-3, but accelerates di€erentiation of several myeloid cell lines, directly their di€erentiation in G-CSF (Bellon et al., 1998). In induces the c-Jun promoter via binding sites for c- contrast, over-expression of PU.1 in normal B- Jun:ATF-2 dimers (van Damm et al., 1993). Phorbol lymphoid progenitors leads to the outgrowth of esters or other stimuli active N-terminal Jun Kinases macrophages at high levels of PU.1 expression and to (JNKs), which in turn phosphorylate c-Jun and ATF-2, the generation of B-cells at lower levels of PU.1 increasing their trans-activation potency (Arias et al., (DeKoter and Singh, 2000). The ability of PU.1 to 1994). Phorbol esters also induce the JunB promoter activate its own promoter may play a role in (de Groot et al., 1991). JunB (7/7) mice in which maintaining high levels of PU.1 expression in myeloid JunB expression has been rescued in tissues other than cells (Chen et al., 1995b). marrow retain normal monocyte numbers but develop granulocytic hyperplasia (Passegue et al., 2001). c-Jun Core binding factors (CBFs) (7/7) fetal liver cells reconstitute hematopoiesis in syngeneic recipients, indicating that, as with JunB, c- The role of CBFs in normal hematopoiesis and in Jun is not required for myeloid development (Eferl et leukemia was recently reviewed (Friedman, 1999). The al., 1999). These ®ndings may re¯ect both compensa- CBFs are a family of heterodimeric proteins containing tory e€ects among Jun family members during a common CBFb subunit and one of three CBFa monocytic di€erentiation and a speci®c role for JunB subunits, CBFa1, CBFa2 (commonly referred to as as an inhibitor of proliferation in the granulocyte AML1), and CBFa3 (Bae et al., 1993; Wang et al., lineage. c-Jun accelerates cell proliferation in ®bro- 1993; Ogawa et al., 1993a, Levanon et al., 1994). CBFs blasts and hepatoblasts, suggesting that c-Jun and bind the consensus site, 5'-PuACCPuCA-3' via their N- JunB are mutually antagonistic in this regard in terminal Runt homology domains (Ogawa et al., myeloid cells (Jochum et al., 2001). In fact, JunB 1993a; Meyers et al., 1993). CBFb does not contact antagonizes the trans-activation potential of c-Jun DNA, but interacts with the CBFa subunits via their (Chiu et al., 1989; Schutte et al., 1989). Runt domains and increases their anity for DNA PU.1 also interacts with GATA-1 and inhibits its (Wang et al., 1993; Ogawa et al., 1993b). AML1 can ability to activate erythroid genes, thereby likely also interact with DNA indirectly, via interaction with contributing to lineage-speci®c expression in myeloid a subset of Ets family members, including MEF, Ets-1, cells (Rekhtman et al., 1999; Zhang et al., 2000). and PU.1, and via interaction with C/EBPa (Westen- Similarly, C/EBPb, and potentially other C/EBPs, dorf et al., 1998; Mao et al., 1999). reduces FOG but not GATA-1 RNA expression, CBFa2 (AML1) expression is largely restricted to potentially directing CMPs to the GMP or to an myeloid and lymphoid cells, including CD34+ pre- eosinophil progenitor (Querfurth et al., 2000). cursors, in adult mice, whereas CBFa1 is most highly PU.1 is expressed in B lymphoid, granulocytic, and expressed in osteoblasts, and CBFb is widely expressed monocytic cells (Klemsz et al., 1990; Chen et al., (Satake et al., 1992; Wang et al., 1993; Erickson et al., 1995a). PU.1 levels increase during granulocytic and 1996; Corsetti and Calabi, 1997; Ducy et al., 1997). monocytic di€erentiation (Cheng et al., 1996). PU.1 AML1 (7/7) and CBFb (7/7) mice die as embryos (7/7) mice lack B lymphoid cells and monocytes and without developing de®nitive hematopoiesis, whereas have greatly reduced neutrophil development (Scott et CBFa1(7/7) mice have normal blood formation but al., 1994; McKercher et al., 1996). Introduction of the do not develop calci®ed bones (Okuda et al., 1996; G-CSF Receptor or M-CSF Receptor into PU.1 (7/ Wang et al., 1996a,b; Sasaki et al., 1996; Niki et al., 7) marrow cells did not enable generation of 1997; Komori et al., 1997). Adult mice chimeric for monocytes or neutrophils (DeKoter et al., 1998; CBFb-SMMHC, a dominant-inhibitor of CBFs, devel- Anderson et al., 1999). Just as C/EBPa (7/7) op erythroid but not myeloid or lymphoid cells progenitors gain the ability to generate neutrophils expressing CBFb-SMMHC, consistent with a speci®c when cultured in vitro, PU.1 (7/7) progenitors role for CBFs in the maturation of these lineages in express myeloperoxidase in IL-3 and G-CSF and adult marrow (Castilla et al., 1999). express markers of immature monocytes when cultured In addition to activating lineage-speci®c markers in the combination of IL-3, SCF, GM-CSF, and M- such as MPO and the M-CSF receptor, CBFs stimulate CSF (DeKoter et al., 1998; Henkel et al., 1999). the G1 to S transition in myeloid and lymphoid cell Similarly, PU.1 (7/7) ES cells express several early lines (Cao et al., 1997, 1998; Lou et al., 2000; Strom et myeloid RNAs (Olson et al., 1995). Perhaps this al., 2000). Exposure of 32D cl3 cells to mimosine,

Oncogene Regulation of granulopoiesis and monopoiesis AD Friedman 3382 which inhibits G1 progression via induction of p27Kip1, and expression of MafB in myeloblasts directed their prevented G-CSF-mediated induction of MPO, an di€erentiation to macrophages (Kelly et al., 2000). early granulocytic marker, but accelerated induction Similarly, expression of c-Maf in HL-60 or U937 cells of two later markers, LF and Lysozyme (Q Wang and led to monocytic di€erentiation (Hegde et al., 1999). c- AD Friedman, unpublished). This ®nding suggests that Maf can interact with c-Myb, interfering with its proliferation is a prerequisite for the activation of activation of early myeloid genes; however, interference proteins such as CBF and c-Myb expressed in with c-Myb activities was not sucient to induce immature cells and meant to serve a dual role by monocytic di€erentiation in HL-60 cells (Hegde et al., simultaneously stimulating proliferation and di€eren- 1998, 1999). tiation. Egr-1 and WT1 c-Myb Egr-1 is a member of a family of zinc-®nger c-Myb binds the consensus DNA site 5'-(C/T)AAC(G/ transcription factors which binds the consensus 5'- T)-3' (Biedenkapp et al., 1988). The 52 amino acid R2 GCGGGGCG-3' (Crosby et al., 1991). The DNA- and R3 segments located near the N-terminus of c- binding domain of WT1 isoforms lacking a KTS insert Myb contain tryptophans every 18 ± 19 residues which is strongly homologous, and these WT1 isoforms bind are part of the hydrophobic core of a helix ± turn ± the same consensus site (Rauscher et al., 1990). Egr-1 is helix structure (Ogata et al., 1992). c-Myb has a expressed in multiple tissue including the terminal central trans-activating domain, and its C-terminus stages of macrophage and neutrophil di€erentiation has an EVES motif which interferes with DNA- (Nguyen et al., 1993; Krishnaraju et al., 1995). Egr-1 is binding by the N-terminus (Weston and Bishop, necessary for monocytic di€erentiation of U937 or M1 1989; Dash et al., 1999). The C-terminal region of c- cells, prevents the granulocytic di€erentiation of HL-60 Myb also contains a leucine zipper, which has been or 32D cl3 cells, induces the macrophage di€erentiation shown to interact with a protein termed p160 (Tavner of M1 cells in the absence of IL-6 and endows 32D cl3 et al., 1998). c-Myb is expressed in immature myeloid, cells with the potential to di€erentiation to macro- lymphoid, and erythroid cells, and c-Myb (7/7) mice phages in response to GM-CSF (Nguyen et al., 1993; lack each of these lineages (Sheiness and Gardinier, Krishnaraju et al., 1995, 1998). In addition, ectopic 1984; Mucenski et al., 1991). Expression of exogenous expression of Egr-1 in myeloid marrow progenitors c-Myb in 32D cl3 cells allowed G-CSF induction of increased the proportion of CFU-M at the expense of MPO but prevented growth arrest and associated CFU-G and, to a lesser extent, BFU-E (Krishnaraju et induction of late markers such as LF (Bies et al., al., 2001). Mice lacking Egr-1 develop normal numbers 1995). A-Myb and B-Myb are highly homologous to of macrophages, perhaps due to compensatory e€ects c-Myb within its DNA-binding domain and bind the from other Egr family members (Lee et al., 1996). As identical consensus sequence (Trauth et al., 1994). A- Egr-1 is induced rapidly by phorbol esters even in the Myb is expressed in several tissues, including a subset absence of protein synthesis, Egr-1 may be an of B cells, while B-Myb is expressed widely in important target of M-CSF receptor signaling (Nguyen proliferating cells and may stimulate cell cycle et al., 1993). progression (Golay et al., 1991; Reiss et al., 1991; Several WT1 isoforms are expressed as a result of Trauth et al., 1994). alternative splicing. The transcriptional regulatory domain of WT1 is a€ected by the presence or absence of exon 5 and its DNA-binding domain is a€ected by MafB and c-Maf the presence or absence of a three amino acid sequence, MafB and c-Maf are members of a family of basic KTS (Haber et al., 1991). The presence of the KTS region-leucine zipper DNA-binding proteins. Mafs insert alters the DNA-binding speci®city of WT1 bind and activate transcription weakly as dimers via (Drummond et al., 1994). Lack of WT1 is lethal at a 13 bp palindrome, 5'-TGCTGACTCAGCA-3', which or before birth, and results in urogenital defects and contains a central AP-1 site, 5'-TGACTCA-3' (Katao- lack of spleen development (Kreidberg et al., 1993; ka et al., 1994a,b; Kerppola and Curran, 1994). Mafs Herzer et al., 1999). Marrow development has not been can also form heterodimers with Fos or Jun family characterized in these mice. Expression of WT1 members which bind modi®ed consensus sites (Katao- isoforms containing the KTS insert induces monocytic ka et al., 1996). Mafs interact with another bZIP di€erentiation of M1 cells, whereas isoforms lacking protein, NF-E2, in erythroid cells, enabling induction this insert, and so mimicking Egr-1 DNA-binding of globin and other erythroid-speci®c genes (Andrews speci®city, prompt a G1 arrest (Smith et al., 1998). In et al., 1993). MafB is expressed in myeloid cells, contrast, WT1(7KTS) accelerates whereas including marrow macrophages, but not in erythroid WT1(+KTS) blocks 32D cl3 granulocytic di€erentia- cells, and it inhibits erythroid gene expression via direct tion in response to G-CSF (Inoue et al., 1998; Loeb et interaction with Ets-1 (Sieweke et al., 1996). When al., 2000). Inhibition of monocyte development by avian progenitors were transformed by a Myb-Ets WT1(7KTS) may re¯ect dominant inhibition of Egr-1, fusion protein in the presence of exogenous MafB, an via competition for DNA-binding. In addition, accel- increased number of myeloid colonies were obtained, eration of granulocytic di€erentiation by WT1(7KTS)

Oncogene Regulation of granulopoiesis and monopoiesis AD Friedman 3383 may re¯ect limitation of G1 progression via direct regulating granulopoiesis in normal cells remains to be repression of the cyclin E promoter (Loeb et al., 2000). con®rmed. WT1 is normally expressed in immature, CD34+ HoxA10 is a homeobox protein which binds marrow cells and is down-regulated in response to preferentially to 5'-TTAT-3' as a heterodimer with stem cell factor and G-CSF (Maurer et al., 1997). The PBX (Chang et al., 1996). The ability of HoxA10 to expression of speci®c WT1 isoforms in marrow repress the gp91-phox promoter led to the suggestion progenitors remains to be determined. Perhaps that HoxA10 plays a role analogous to CDP during WT1(7KTS) serves to interfere with monocyte granulopoiesis (Eklund et al., 2000). HoxA10 is development and WT1(+KTS) serves to interfere with highly expressed in CD34+ and is only weakly granulocytic development. detected in CD347 marrow cells and is not present in mature neutrophils or monocytes (Sauvageau et al., 1994; Lawrence et al., 1995). HoxA10 is Retinoic acid receptors preferentially expressed in myeloid cell lines, HoxA10 Retinoic acid receptors a, b, g(RARa, RARb,and (7/7) mice have increased numbers of granulocytes, RARg) bind DNA via their zinc-®nger domains as and over-expression of HoxA10 inhibits monocytic heterodimers with RXR proteins. RARs are broadly colony formation in vitro and produces AML in vivo expressed, with RARa being preferentially expressed in (Lawrence et al., 1995; Zhang et al., 1996b; myeloid cells (de The et al., 1989). The DNA consensus Thorsteinsdottir et al., 1997). Overall, as HoxA10 for RAR-RXR DNA-binding is a direct repeat of 5'- expression is apparently lost prior to CDP activity, (A/G)G(G/T)TCA-3' separated by 2 ± 5 bps (Umesano it may play a role in maintaining an earlier stage in et al., 1991; Naar et al., 1991). Dominant-inhibition of myelopoiesis. While HoxA10 can activate the RARa arrests granulocytic di€erentiation at the p21WAF1/Cip1 promoter in U937 cells, it is tempting promyelocyte stage (Tsai and Collins, 1993). Mice to speculate that HoxA10 in fact represses this lacking RARa1orRARg have normal myeloid promoter in immature myeloid cells, enabling cell development, whereas while mice lacking both of these proliferation and thereby inhibiting terminal di€er- RAR isoforms have normal myeloid progenitor entiation (Bromleigh and Freedman, 2000). As we numbers, their neutrophilic di€erentiation arrests at have discussed, other Hox proteins may play a role the myelocyte stage (Labrecque et al., 1998). This in regulating myelopoiesis as well (Ward et al., phenotype is similar to that observed in C/EBPe (7/ 2000). 7) mice, and RARa directly activates the C/EBPe Like CDP, Sp1 is found in multiple tissues, but Sp1 promoter (Yamanaka et al., 1997; Chih et al., 1997). is expressed at particularly high levels in maturing While regulation of the C/EBPe promoter by RARs granulocytes (Sa€er et al., 1991). This expression may account for the requirement of RARs for terminal pattern combined with the ®nding that Sp1 regulates granulopoiesis, it remains to be established that several granulocytic and monocytic genes via its regulation of RAR activities plays a role in normal consensus-binding site, 5'-GGGCGG-3', raises the hematopoiesis. possibility that Sp1 plays a role in regulating terminal myelopoiesis. CCAAT displacement protein (CDP) and HoxA10 Myeloid zinc finger 1 (MZF-1) CDP is a widely expressed protein that represses gene expression, at least in part, via competition for MZF-1 was isolated as a zinc ®nger protein preferen- transactivator binding to DNA elements which loosely tially expressed in myeloid cell lines (Hromas et al., ®t the 5'-CCAAT-3' motif (Barberis et al., 1987; Luo 1991). MZF-1 is preferentially expressed in immature and Skalnik, 1996). Decreased CDP DNA-binding myeloid cells, and reduction of its expression inhibits during the terminal stages of granulopoiesis combined the formation of CFU-G (Bavisotto et al., 1991). The with its ability to repress the gp91-phox promoter led two zinc ®nger clusters within MZF-1 bind the to the proposal that inactivation and/or decreased consensus sites 5'-AGTGGGGA-3' and 5'- expression of CDP is required for terminal neutrophil CGGGnGAGGGGGAA-3' (Morris et al., 1994). The maturation (Skalnik et al., 1991). The mechanism MPO and LF promoters contain bindings sites capable whereby CDP DNA-binding becomes inactivated of interacting with both sets of zinc ®ngers, and the during granulopoiesis remains to be elucidated. 32D CD34 promoter contains two such sites located at cl3 cells overexpressing CDP do not express C/EBPe in about 7500 bp. MZF-1 activates the CD34 promoter response to G-CSF, and the human C/EBPe promoter via these sites in hematopoietic cell lines (Morris et al., contains a binding site for CDP at 71472 bp which 1995). MZF-1 (7/7) mice develop normal blood cells, potentially accounts for the reduced activity of an but develop a progressive increase in monocytic cells 1800 bp compared to a 726 bp promoter fragment in followed by the late formation of an in®ltrating 32Dwt18 cells (Khanna-Gupta et al., 2001). Inhibition monocytic leukemia. In addition, a much greater of C/EBPe expression may account for the combined proportion of MZF-1 (7/7) myeloid and erythroid loss of LF, neutrophil collagenase, and neutrophil progenitors are in cell cycle without increased expres- gelatinase in 32D cells expressing exogenous CDP sion of c-Myb (Gaboli et al., 2001). This phenotype (Lawson et al., 1998). However, a role for CDP in may not have been detected during antisense inhibition

Oncogene Regulation of granulopoiesis and monopoiesis AD Friedman 3384 of MZF-1 in CFU-G due to residual expression Lineage commitment and progression (Bavisotto et al., 1991). The widespread role of C/EBPs and PU.1 in regulating myeloid genes combined with the defects evident in C/ p53, retinoblastoma protein (Rb), STAT3, and BLIMP-1 EBPa (7/7) and PU.1 (7/7) mice make these When induced by DNA strand breaks, p53 activates transcription factors leading candidates for roles in pathways leading to apoptosis or cell cycle arrest determining the myeloid lineages. As PU.1 (7/7) mice (Canman et al., 1995). p53 is detected at low levels in lack B cells and monocytes and have greatly reduced mature myeloid and lymphoid cells and p53 levels numbers of neutrophils, whereas C/EBPa (7/7) mice increase in ML-1 cells as they di€erentiate to only lack maturing granulocytes, it would seem monocytes in response to phorbol esters (Kastan et reasonable to place PU.1 `upstream' of C/EBPs in this al., 1991). As c-Jun represses the p53 gene, this e€ect of regard. Further support for this idea comes from the phorbol esters is likely indirect, perhaps via PKC ®nding that PU.1 and GATA-1 inhibit each others activation or cell cycle arrest (Schreiber et al., 1999). activities, suggesting their relative importance in the Exogenous p53 prompts monocytic di€erentiation in myeloid versus the erythrod/megakaryocyte lineages. U937 cells or in 32D cl3 cells also expressing v-src or However, several lines of evidence suggest that C/EBPs an activated M-CSF receptor, without inducing G1 might play a role in specifying the bipotent granulo- arrest or apoptosis (Soddu et al., 1996; Martinelli et cyte-monocyte progenitor (GMP): C/EBP-mediated al., 1997; Ehinger et al., 1998). The induction of down-regulation of FOG RNA is essential for monocytic di€erentiation in 32D cl3 cells is striking, eosinophil development, and perhaps is also required given their propensity for granulocytic maturation. for GMP formation (Querfurth et al., 2000). Second, Induction of U937 cell di€erentiation by p53 depended fractionation studies suggest that pluripotent stem cells upon the integrity of its trans-activating domain and ®rst commit to a common lymphoid progenitor (CLP) was not inhibited by bcl-2 (Chylicki et al., 2000). and a common myeloid progenitor (CMP) and that B- Although mice lacking p53 develop normally, the p53- cells and macrophages develop from the CLP and the related protein, p73, potentially compensates for p53 CMP respectively (Akashi et al., 2000); in this model, it de®ciency as p73 is expressed in immature but not is dicult to draw a 1 : 1 correspondence between PU.1 terminally di€erentiated myeloid cells (Lowe et al., and a speci®c progenitor. Third, the CMP gives rise to 1993; Peters et al., 1999). the GMP, the megakaryocyte-erythroid progenitor Rb cooperates with E2F family members to repress (MEP), and perhaps an eosinophilic progenitor genes required for S phase entry (Weinberg, 1995). Rb (EoP), and evidence has been presented indicating that is down-regulated during granulopoiesis but is up- high-level GATA-1 speci®es the MEP whereas the regulated during monocyte development (Bergh et al., combination of a C/EBP and lower levels of GATA-1 1999). Consistent with this expression pattern, anti- speci®c to the eosinophil lineage (Kulessa et al., 1995; sense inhibition of Rb expression in marrow progeni- McDevitt et al., 1997; Yamaguchi et al., 1999). tors reduced CFU-M in favor of CFU-G (Bergh et al., Therefore, if the third branch eminating from the 1999). Perhaps complexes which form between hypo- CMP, the GMP, were determined by the expression of phosphorylated Rb and either C/EBPb or PU.1 are C/EBP without GATA-1, then the tripartite decision of essential for monocyte maturation (Hagemeier et al., the CMP would depend on only two factors ± such 1993; Chen et al., 1996). Rb (7/7) mice are simplicity is attractive. And fourth, higher levels of embryonic lethal and have increased erythroblasts, PU.1 are present in and are required for the monocyte whereas lack of the Rb-related protein p107 leads to lineage, compared to the B-lineage (DeKoter and myeloid hyperplasia (Lee et al., 1992; Lecouter et al., Singh, 2000), and as C/EBPa rapidly induces PU.1 1998). As with p53, Rb family members may RNA expression in 32D cl3 and Ba/F3 cells, it might compensate for each other in knockout mice to help do so in the GMP as well (Wang et al., 1999). One mediate myelopoiesis. dicult with this alternative is that C/EBP would be Dominant inhibition of STAT3 in 32D cl3 cells expected to induce PU.1 in eosinophils, which would allows expression of early markers but prevents cell inhibit GATA-1 activity. PU.1 is present in mature cycle arrest and induction of late markers, although C/ eosinophils, and PU.1 RNA is strongly upregulated EBPe induction was not prevented (Shimozaki et al., when human CD34+ cells di€erentiate to eosinophils 1997; Nakajima and Ihle, 2001). These ®ndings suggest in response to IL-5 (J Du and SJ Ackerman, personal that STAT3 is required for induction of G1 cell cycle communication). PU.1 7/7 mice have greatly reduced arrest during granulopoiesis. Consistent with this numbers of mature eosinophils, and direct evidence for model, dominant inhibition of STAT3 prevents the regulation of an eosinophil-speci®c gene by PU.1 p27Kip1 induction by G-CSF in 32D cl3 cells, and the has been presented (Du et al., 1998; van Dijk et al., p27Kip1 promoter is activated directly by STAT3 (De 1998). Perhaps, partial inhibition of GATA-1 by PU.1 Koning et al., 2000). in fact serves to direct CMPs to the EoP, as opposed to BLIMP-1 is a zinc-®nger DNA-binding protein the MEP. Figure 1 illustrates two alternatives for the which may contribute to terminal myeloid and B- speci®cation of the GMP, by high levels of PU.1 alone lymphoid di€erentiation at least in part by repressing or by high levels of PU.1 achieved via expression of C/ transcription of the c-myc gene (Chang et al., 2000). EBPs, likely including C/EBPa as this isoform is

Oncogene Regulation of granulopoiesis and monopoiesis AD Friedman 3385

Figure 1 A model for transcriptional regulation of granulocyte and monocyte lineage commitment and progression. Detailed discussions of lymphoid and erythroid lineage determination are presented elsewhere in this issue. Pluripotent hematopoietic stem cells (HSC) are directed to the CLP in part by Ikaros, T cell development required GATA-3, and B-cell development requires Pax5 and low levels of PU.1. The speci®cation of the CMP from the HSC remains obscure ± perhaps this is a default pathway in the absence of Ikaros. Development of the MEP requires high-level GATA-1, whereas the development of the eosinophil lineage requires lower level (or lower activity) of GATA-1 along with a C/EBP. PU.1 induced by C/EBPs may in fact serve to reduce GATA-1 activity in this lineage. High-level GATA-1 may further direct the MEP along the erythroid lineage (E), NF-E2, FOG, and GATA-1 may specify megakaryocyte progenitors (Meg), and MITF in combination with GATA-1 or GATA-2 may allow basophilic progenitors to form (Baso). Development of the GMP requires high level PU.1 expression, which may develop through both C/EBP-dependent and C/EBP-independent mechanisms. Redundancy with respect to DNA-binding and trans-activation potency might allow one or more C/EBP family members to play a role in this regard. Maf and/or Jun family members may then direct the GMP towards a committed monocyte progenitor, and increased levels of C/EBPs may specify the neutrophil progenitor. Monocyte lineage progression depends upon the continued presence of PU.1 and perhaps C/EBPs, and may require Egr-1, which could also serve to repress the granulocytic program. ICSBP may be an important PU.1 co-factor during monocyte maturation. Terminal maturation may in addition depend upon increased expression of hypo-phosphorylated Rb and/or p53 as a consequence of cell cycle arrest. Granulocyte maturation requires PU.1, C/EBPs, RARs, C/EBPe, and Sp1 and may also require inactivation of CDP DNA-binding. RARs, C/EBPa, and/or decreasing CDP activities may in fact serve to induce C/EBPe

expressed most prominently in immature hematopoietic speculation that Maf:Maf, Maf:Jun, Maf:Fos, Jun:Jun, cells (Scott et al., 1992; Radomska et al., 1998). or Jun:Fos dimers initiate monopoiesis in conjunction Detailed discussions of erythroid and lymphoid lineage with PU.1. The continued presence of C/EBPs, to development are presented elsewhere in this issue ± of maintain elevated PU.1 levels, may be required as well. note however is the ®nding that Pax5 suppresses GM- In the absence of Maf or Jun proteins, C/EBPs and CSF Receptor RNA expression, again demonstrating PU.1 may specify the granulocyte lineage. The ®nding the role of negative cross-talk between hematopoietic that increased levels of C/EBPa favor granulocytic over lineages (Chiang and Monroe, 2001). Both C/EBP- monocytic di€erentiation in U937 cells suggests that, as dependent and -independent commitment from the with PU.1 and GATA-1, C/EBP levels may play a role CMP to the GMP might occur, with requisite elevation in lineage commitment decisions (Radomska et al., of PU.1 levels. Our ®nding that either C/EBPa-ER or 1998). A model for granulocyte versus monocyte G-CSF receptor signals, acting in the presence of a lineage determination is illustrated in Figure 1. dominant-inhibitory C/EBP, elevate PU.1 RNA in 32D Egr-1 may serve to simultaneously enable monocyte cl3 cells is consistent with this idea (Wang et al., 1999; lineage progression and to prevent cross-over to Wang and Friedman, 2002). neutrophil development. Granulocyte lineage progres- The issue of granulocyte versus monocytic lineage sion requires C/EBPe, which may be induced by C/ choice also must be resolved. The ®ndings that Jun and EBPa and/or RARs. Loss of CDP DNA-binding is Maf family members can each induce the monocyte also required for terminal granulopoiesis. In addition, lineage in cell lines and that these proteins both progression to neutrophils and macrophages is asso- interact with AP-1 consensus sites leads to the ciated with cell cycle arrest, elevating hypo-phosphory-

Oncogene Regulation of granulopoiesis and monopoiesis AD Friedman 3386 lated Rb and p53. These proteins may participate in co-repressors which participate in lineage speci®cation? terminal di€erentiation, as best shown for monopoiesis. With respect to key transcription factors, further And cell cycle arrest may inactivate factors such as clari®cation of the regulatory network illustrated in CBF and c-Myb which participate in early stages of Figure 1 is needed: Which factor or factors specify di€erentiation. A model of myeloid lineage progression each lineage and at what levels of expression; can is shown in Figure 1. family members act redundantly in this regard; what additional cooperative mechanisms operate among transcriptional regulators; what roles do cytokine Perspectives for the future receptor signaling and transcription factor modi®ca- tions play in each commitment decision and in each Many questions remain unanswered regarding the step of lineage progression? The answers to these transcriptional regulation of granulocyte and monocyte questions will provide general lessons in developmental development. With respect to the cellular basis for biology and insights into leukemogenesis and will initiating these lineages: What are the relative con- enable applications in clinical hematology, oncology, tributions of granulocyte/monocyte and B-cell/mono- and gene therapy. cyte progenitors to mature blood elements; do some granulocyte or monocyte progenitors develop directly from pluripotent stem cells; how irreversible are commitment decisions? With respect to gene regula- tion: Are there additional important transcriptional Acknowledgments regulators of myeloid genes remaining to be uncovered AD Friedman is a Scholar of the Leukemia and Lympho- via detailed investigation of promoters and distal ma Society and his research is supported by the Children's enhancers; are there lineage-restricted co-activators or Cancer Foundation.

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