Oncogene (2002) 21, 3377 ± 3390
ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00
Transcriptional regulation of granulocyte and monocyte development
Alan D Friedman*,1
1
Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland, MD 21231, USA
Granulocytes and monocytes develop from a common
myeloid progenitor. Early granulopoiesis requires the C/
EBPa, PU.1, RAR, CBF, and c-Myb transcription
factors, and terminal neutrophil di€erentiation is
dependent upon C/EBPe, PU.1, Sp1, CDP, and
HoxA10. Monopoiesis can be induced by Maf-B, c-
Jun, or Egr-1 and is dependent upon PU.1, Sp1, and
ICSBP. Signals eminating from cytokine receptors
modulate factor activities but do not determine cell
fates. Orchestration of the myeloid developmental
program is achieved via cooperative gene regulation,
via synergistic and inhibitory protein ± protein interac-
tions, via promoter auto-regulation and cross-regulation,
via regulation of factor levels, and via induction of cell
cycle arrest: For example, c-Myb and C/EBPa
cooperate to activate the mim-1 and NE promoters,
PU.1, C/EBPa, and CBF, regulate the NE, MPO, and
M-CSF Receptor genes. PU.1:GATA-1 interaction and
C/EBP suppression of FOG transcription inhibits
erythroid and megakaryocyte gene expression. c-Jun:-
PU.1, ICSBP:PU.1, and perhaps Maf:Jun complexes
induce monocytic genes. PU.1 and C/EBPa activate
their own promoters, C/EBPa rapidly induces PU.1 and
C/EBPe RNA expression, and RARa activates the C/
EBPe promoter. Higher levels of PU.1 are required for
monopoiesis than for B-lymphopoiesis, and higher C/
EBP levels may favor granulopoiesis over monopoiesis.
CBF and c-Myb stimulate proliferation whereas C/EBPa
induces a G1/S arrest; cell cycle arrest is required for
terminal myelopoiesis, perhaps due to expression of p53
or hypo-phosphorylated Rb.
Oncogene (2002) 21, 3377 ± 3390. DOI: 10.1038/sj/
onc/1205324
Keywords: granulocyte; monocyte; di€erentiation;
C/EBP; PU.1
Introduction
Polymorphonuclear and mononuclear phagocytes func-
tion in host defense against infections and are capable
of recognizing, ingesting, and destroying foreign
materials and organisms. Pluripotent hematopoietic
stem cells in fetal liver or in adult marrow give rise to
*Correspondence: AD Friedman, Johns Hopkins University, Cancer
Research Building, Room 253, 1650 Orleans Street, Baltimore,
Maryland, MD 21231; E-mail: adfrdman@jhmi.edu
www.nature.com/onc
granulocyte/macrophage progenitors (GMPs), charac-
terized by expression of CD34 and Fcg Receptor(II/III)
(Akashi et al., 2000; Traver et al., 2001). These
progenitors in turn give rise to granulocyte-, mono-
cyte-, and granulocyte/monocyte-colony forming units
(CFU-G, CFU-M, and CFU-GM). Granulocyte-Col-
ony Stimulating Factor (G-CSF) enables the formation
of CFU-G in methylcellulose cultures, Macrophage-
Colony Stimulating Factor (M-CSF, CSF-1) enables
the growth of CFU-M, and Granulocyte/Macrophage-
Colony Stimulating Factor (GM-CSF) or Interleukin-3
(IL-3) enable the proliferation of CFU-G, CFU-M,
and CFU-GM. In addition to these features of normal
cells, the ®nding that a common subset of acute
myeloid leukemia has both a granulocytic and
monocytic cellular component further underscores the
close developmental relationship of neutrophils and
monocytes. Fetal liver and adult marrow also contain
progenitors capable of producing B-lymphoid and
macrophage cells in single colonies, re¯ecting both
the plasticity of pluripotent stem cell commitment and
the relatedness of these lineages (Lacaud et al., 1998;
Montecino-Rodriguez et al., 2001; Traver et al., 2001).
Monocyte progenitors not only develop into macro-
phages but are also capable of osteoclast di€erentiation
(Roodman, 1996), and GMPs share with common
lymphoid progenitors (CLPs) the capacity to generate
myeloid dendritic cells (Manz et al., 2001).
Neutrophil maturation proceeds from the myeloblast
to the promyelocyte, when primary granules become
apparent, to the myelocyte, when cell division ceases
and secondary or speci®c granules begin to appear, to
the metamyelocyte, band, and ®nally the neutrophil,
which has a three-lobed nucleus and tertiary granules.
Early markers of granulopoiesis include the G-CSF
Receptor, the CD33 and CD13 surface markers, and
primary granule components, such as myeloperoxidase
(MPO), neutrophil elastase (NE), myeloblastin (MBN),
lysozyme and avian mim-1. Oxidation system compo-
nents, such as the gp91-phox cytochrome heavy chain,
are expressed from the myelocyte stage onward and
later markers of neutrophil development include
secondary granule components, such as lactoferrin
(LF) and neutrophil gelatinase (NG), and the Gr-1
surface marker.
Monopoiesis proceeds from the monoblast to the
circulating monocyte, which matures without prolifera-
tion to the tissue macrophage. Early markers of this
lineage include the M-CSF Receptor, lysozyme, and
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
Myeloblastin (MBN) C/EBPs, PU.1, c-Myb
(Hetherington et al., 1999). Phagocytic capacity is an 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
Lysozyme C/EBPs, PU.1
from the ®nding that G-CSF (7/7), G-CSF; GM- 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

by C/EBPs (Ahne and Stratling, 1994; Goethe and
Loc, 1994; Faust et al., 1999). Consistent with these
results, we recently found that activation of exogenous
C/EBPa-ER with estradiol in the murine 32D cl3
myeloblast cell line rapidly induces lysozyme RNA
expression in the presence of cycloheximide but not
actinomycin D (Wang and Friedman, 2002). Finally,
the c-fes tyrosine kinase promoter region is regulated
by PU.1 and Sp1 in immature granulocytic and
monocytic cells (Heydemann et al., 1996).
In maturing neutrophils, the gp91-phox promoter
region is activated by the PU.1:ICSBP dimer and is
repressed by both CDP and HoxA10 ± the levels of
the latter factors decrease during terminal neutrophil
and monocyte di€erentiation (Skalnik et al., 1991;
Eklund and Kakar, 1999; Eklund et al., 2000). The LF
promoter is activated by C/EBPs and Sp1 and is
repressed by CDP (Khanna-Gupta et al., 1997, 2000).
CDP represses both the gp91-phox and LF promoters
via interaction with sites upstream of activator binding
sites (Luo and Skalnik, 1996; Khanna-Gupta et al.,
1997), and CDP also represses the gp91-phox promoter
via a site 790 which overlaps an activator-binding site
(Catt et al., 1999). HoxA10 interacts with an AT-rich
element located just upstream of the 5'-CCAAT
sequence at 790, the binding site for CDP, and so
like CDP may repress gp91-phox transcription, in part,
via competition with activators (Eklund et al., 2000).
The MPO promoter contains a potent repressor
element active in 32D cl3 cells which has been mapped
to a 28 bp segment which includes a 5'-GAAATC
sequence conserved in the homologous region of the
human MPO gene (Suzow and Friedman, 1993).
Perhaps this element binds CDP or HoxA10, repressors
thought to be more active in immature than mature
granulocytes.
In immature monocytic cells, the M-CSF Receptor
promoter is activated synergistically by C/EBP, PU.1,
and CBF (Zhang et al., 1994a,b, 1996a; Petrovick et
al., 1998), and c-Jun activates the M-CSF receptor
gene indirectly, via its ability to interact with PU.1
(Bassuk and Leiden, 1995; Behre et al., 1999a). In cells
di€erentiating into macrophages, the macrosialin and
scavenger receptor promoters are activated coopera-
tively by PU.1 and c-Jun, via binding sites separated by
33 or 135 bps, respectively (Moulton et al., 1994; Li et
al., 1998). The CD14 promoter is activated by C/EBP
and Sp1 (Zhang et al., 1994c; Pan et al., 1999). And
®nally, PU.1 and Sp1 activate both the CD11b and
CD18 promoters; optimal transcription of the latter
gene also requires GABP, which like PU.1 is a member
of the Ets family of transcription factors (Pahl et al.,
1993; Chen et al., 1993; Rosmarin et al., 1995a,b,
1998).
From these studies, C/EBPs and PU.1 emerge as
the most consistent regulators of genes expressed in
granulocytic and immature monocytic cells. PU.1
also activates most genes expressed in mature
monocytic cells, c-Myb and CBF contribute to the
activation of several genes in proliferating cells, and
Sp1 activates several promoters in neutrophils and
Regulation of granulopoiesis and monopoiesis
AD Friedman
3379
mature monocytes. The next section will describe
additional evidence which con®rms the importance of
these factors, as well as evidence implicating
additional transcription factors, in the regulation of
granulocyte and monocyte development. The subse-
quent section will then address the question of how
these factors orchestrate the myeloid developmental
program.
Transcription factors
C/EBPs
The C/EBPs homo- and hetero-dimerize via their C-
terminal leucine zipper domains and bind DNA as
dimers via the adjacent basic regions (Landschulz et
al., 1989). The C/EBP consensus binding site is 5'-T(T/
G)NNGNAA(T/G)-3'. C/EBPa, C/EBPb, and C/EBPd
have N-terminal trans-activation domains, and transla-
tion initiation from internal methionines produces
truncated, dominant-inhibitory polypeptides which
retain the bZIP domain (Friedman et al., 1989,
Friedman and McKnight, 1990; Descombes and
Schibler, 1991; Calkhoven et al., 2000). C/EBPe has
both a trans-activation and a trans-repression domain
(Williamson et al., 1998), and C/EBPg and CHOP are
dominant-inhibitory due to their ability to dimerize
with other C/EBPs and their lack of intact basic
regions (Habener and Ron, 1992; Cooper et al., 1995).
Trans-activation by C/EBPa may be regulated via Ras-
induced phosphorylation of serine 248 (Behre et al.,
1999b).
Within hematopoiesis, full-length C/EBPa, C/EBPb,
and C/EBPd are predominantly expressed in the
granulocyte, monocyte, and eosinophil lineages (Scott
et al., 1992; Muller et al., 1995; Radomska et al., 1998).
C/EBPa is the isoform most prominently detected in
immature granulocytes (Scott et al., 1992; Hohaus et
al., 1995), whereas C/EBPe is found in later-stage
granulocytes and in T-cells (Antonson et al., 1996).
CHOP is only detected in granulocytic cells subjected
to stress, such as DNA-damage (Friedman, 1996b),
and C/EBPg expression in this lineage is not well
characterized.
C/EBPa (7/7) mice lack neutrophils and eosino-
phils, but retain monocytes, lymphocytes, erythroid
cells, and immature myeloblasts (Zhang et al., 1997).
Fetal liver cells from these mice lack Granulocyte-
Colony Stimulating Factor (G-CSF) Receptor RNA,
consistent with the ability of C/EBPa to trans-activate
the G-CSF Receptor promoter (Smith et al., 1996).
Induction of G-CSF or IL-6 Receptor cDNAs into C/
EBPa (7/7) fetal liver cells restores their ability to
generate neutrophils in vitro in response to G-CSF or
IL-6 (Zhang et al., 1998). C/EBPb (7/7) mice retain
all of the hematopoietic lineages, and hematopoietic
defects in mice lacking C/EBPd or both C/EBPb and
C/EBPd were not severe (Screpanti et al., 1995; Tanaka
et al., 1995, 1997). C/EBPe (7/7) mice also retain
neutrophils, although they lack secondary granules
Oncogene
 Regulation of granulopoiesis and monopoiesis
AD Friedman
3380
(Yamanaka et al., 1997; Chumakov et al., 1997;
Lekstrom-Himes et al., 1999; Gombart et al., 2001).
Expression of C/EBPa in U937 cells led to the
development of neutrophilic cells after 2 weeks, with
expression of the mRNAs encoding G-CSF Receptor,
lactoferrin, and neutrophil collagenase (Radomska et
al., 1998). Introduction of C/EBPa into avian multi-
potent progenitors also led to expression of myeloid
markers (Nerlov et al., 1998). Similarly, estradiol-
mediated activation of C/EBPa-ER in 32D cl3 cells, a
factor-dependent, diploid cell line, induced neutrophils
within 4 days, with induction of MPO, lactoferrin, and
G-CSF Receptor RNAs (Wang et al., 1999). MPO
RNA was induced within 8 h of C/EBPa-ER activa-
tion, but not in the presence of cycloheximide,
indicating the need for an additional C/EBP-dependent
factor. A likely candidate is PU.1, as C/EBPa-ER
induced PU.1 RNA within 4 h, even in the presence of
cycloheximide. C/EBPa-ER also rapidly induced the
lysozyme and C/EBPe RNAs in the presence of
cycloheximide, but not actinomycin D, suggesting
direct regulation of the murine lysozyme and C/EBPe
genes by C/EBPs (Wang and Friedman, 2002). The
signi®cance of C/EBPa regulation of PU.1 and C/EBPe
RNA expression will be discussed further below. As C/
EBPa is expressed in non-hematopoietic cells, it is
interesting to speculate that early regulators of
hematopoiesis, such as c-Myb, CBF, or GATA-2,
prime the PU.1 and C/EBPe genes for activation by C/
EBPa. The ability of CEBPa to activate its own
promoter may also assist in committing progenitors to
the myeloid lineages (Christy et al., 1993).
The presence of several C/EBPs in myeloid cells
suggests that family members might compensate in vivo
for the lack of a single isoform, just as GATA family
members partially compensate for the lack of GATA-1
(Kulessa et al., 1995; Tsai et al., 1998). Indeed, as with
C/EBPa, C/EBPb, C/EBPd, or C/EBPe can induce
granulocytic di€erentiation in myeloblastic cell lines
(Park et al., 1999; Nakajima and Ihle, 2001; Wang and
Friedman, 2002), and C/EBPa, C/EBPb, or C/EBPd
induced monocytic genes in P388 lymphoblasts (Hu et
al., 1998). Redundancy is also evident from the ability
of C/EBPb to compensate for the loss of C/EBPa in
hepatocytes and granulocytes in vivo (Chen et al., 2000;
Jones et al., 2002). In addition, compensatory e€ects
may account for the ability of immortalized C/EBPa
(7/7) progenitors to express high levels of G-CSF
Receptor in vitro, even though they could not respond
to G-CSF in vivo (Collins et al., 2001). Global
inhibition of C/EBP-regulated genes can be achieved
by expression of KRAB-C/EBPa-ER (KaER), in which
the C/EBPa DNA-binding domain is linked to both a
KRAB trans-repression domain and the Estrogen
Receptor ligand-binding domain. In 32D cl3 cells,
KaER inhibits granulocytic di€erentiation despite the
presence of exogenous G-CSF receptor (Wang and
Friedman, 2002). And in marrow cells, KaER inhibits
the formation of CFU-G, CFU-M, and CFU-GM in
multiple cytokines, without a€ecting BFU-E produc-
tion (Wang and Friedman, 2002).
Oncogene
In addition to regulating di€erentiation in several
lineages, C/EBPa regulates proliferation directly.
Decreased expression in proliferating versus quiescent
hepatocytes and its ability to slow 3T3-L1 preadipocyte
proliferation provided the initial evidence that C/EBPs
a€ect cell cycle progression (Friedman et al., 1989;
Umek et al., 1991). Both C/EBPaWT-ER and KaER
inhibit progression from G1 to S phase in 32D cl3 cells
(Wang et al., 1999; Friedman and Wang, 2000). These
proteins may inhibit the G1/S transition via interaction
with E2F (Timchenko et al., 1999; Slomiany et al.,
2000; Johansen et al., 2001). A KaER variant with a
defective leucine zipper domain does not slow 32D cl3
proliferation (Wang and Friedman, 2002). Perhaps C/
EBPa dimers are required for interaction with E2F or
with another protein associated with the E2F com-
plexes, or perhaps C/EBPa monomers interact with one
of these proteins via its leucine zipper. In fact, E2F
contains a leucine zipper, as noted (Slomiany et al.,
2000). C/EBPe also slows 32D cl3 proliferation
(Nakajima and Ihle, 2001). Inhibition of proliferation
by C/EBPa or C/EBPe may contribute to granulopoi-
esis, as maneuvers which stimulate proliferation, such
as over-expression of c-Myb or cdk4, prevent induction
of late markers (Bies et al., 1995; Lou et al., 2000).
Similarly, expression of p21WAF1/CIP1 or p27Kip1 in U937
cells induces monocytic markers (Liu et al., 1996b). On
the other hand, induction of p27Kip1 with mimosine
induced Lysozyme RNA but inhibited MPO RNA, an
earlier di€erentiation marker, in 32D cl3 cells (Q Wang
and AD Friedman, unpublished). The potential roles
of p53 and hypo-phosphorylated Rb in terminal
myeloid di€erentiation will be discussed below.
PU.1 and interacting proteins, ICSBP, c-Jun, and
GATA-1
PU.1 binds DNA as a monomer, via its C-terminal Ets
domain, to the consensus site 5'-AAAG(A/C/
G)GGAAG-3' (Klemsz et al., 1990). The DNA-binding
domain of PU.1 and its N-terminal, glutamine-rich
trans-activating domain were both required to rescue
myelopoiesis in PU.1 (7/7) ES cells, whereas its
acidic trans-activating domain was dispensable (Fisher
et al., 1998).
Phosphorylation of serine 148 in PU.1 allows
interaction with Pip, also known as Interferon
response factor-4 (IRF-4), an essential co-factor in B
cells (Eisenbeis et al., 1995). Pip or Interferon
consensus sequence binding protein (ICSBP, IRF-8)
can interact with PU.1 in monocytic cells, enabling
trans-activation via a hybrid DNA-element binding
element (Meraro et al., 1999; Marecki et al., 2001).
However, an essential role for Pip or ICSBP
interaction for PU.1 activity in myeloid cells has not
been established. Notably, ICSBP (7/7) mice have
reduced macrophages and increased granulocytic cells,
and introduction of ICSBP into a cell line derived
from the knockout mice increased monocytic markers
at the expense of granulocytic markers (Holtschke et
al., 1996; Tamura et al., 2000).

As discussed, c-Jun also cooperates with PU.1 to
regulate several monocytic genes, either via adjacent cis
DNA elements or via direct interaction. Notably, c-
Jun, JunB, and JunD levels increase during monocytic
di€erentiation (Lord et al., 1993), and exogenous c-Fos
or c-Jun induced partial monocytic di€erentiation in
M1, U937, or WEHI-B D+ cells and increased the
responsiveness of U937 cells to phorbol esters (Lord et
al., 1993; Szabo et al., 1994; Li et al., 1994). Phorbol
esters, an agent which stimulates the monocytic
di€erentiation of several myeloid cell lines, directly
induces the c-Jun promoter via binding sites for c-
Jun:ATF-2 dimers (van Damm et al., 1993). Phorbol
esters or other stimuli active N-terminal Jun Kinases
(JNKs), which in turn phosphorylate c-Jun and ATF-2,
increasing their trans-activation potency (Arias et al.,
1994). Phorbol esters also induce the JunB promoter
(de Groot et al., 1991). JunB (7/7) mice in which
JunB expression has been rescued in tissues other than
marrow retain normal monocyte numbers but develop
granulocytic hyperplasia (Passegue et al., 2001). c-Jun
(7/7) fetal liver cells reconstitute hematopoiesis in
syngeneic recipients, indicating that, as with JunB, c-
Jun is not required for myeloid development (Eferl et
al., 1999). These ®ndings may re¯ect both compensa-
tory e€ects among Jun family members during
monocytic di€erentiation and a speci®c role for JunB
as an inhibitor of proliferation in the granulocyte
lineage. c-Jun accelerates cell proliferation in ®bro-
blasts and hepatoblasts, suggesting that c-Jun and
JunB are mutually antagonistic in this regard in
myeloid cells (Jochum et al., 2001). In fact, JunB
antagonizes the trans-activation potential of c-Jun
(Chiu et al., 1989; Schutte et al., 1989).
PU.1 also interacts with GATA-1 and inhibits its
ability to activate erythroid genes, thereby likely
contributing to lineage-speci®c expression in myeloid
cells (Rekhtman et al., 1999; Zhang et al., 2000).
Similarly, C/EBPb, and potentially other C/EBPs,
reduces FOG but not GATA-1 RNA expression,
potentially directing CMPs to the GMP or to an
eosinophil progenitor (Querfurth et al., 2000).
PU.1 is expressed in B lymphoid, granulocytic, and
monocytic cells (Klemsz et al., 1990; Chen et al.,
1995a). PU.1 levels increase during granulocytic and
monocytic di€erentiation (Cheng et al., 1996). PU.1
(7/7) mice lack B lymphoid cells and monocytes and
have greatly reduced neutrophil development (Scott et
al., 1994; McKercher et al., 1996). Introduction of the
G-CSF Receptor or M-CSF Receptor into PU.1 (7/
7) marrow cells did not enable generation of
monocytes or neutrophils (DeKoter et al., 1998;
Anderson et al., 1999). Just as C/EBPa (7/7)
progenitors gain the ability to generate neutrophils
when cultured in vitro, PU.1 (7/7) progenitors
express myeloperoxidase in IL-3 and G-CSF and
express markers of immature monocytes when cultured
in the combination of IL-3, SCF, GM-CSF, and M-
CSF (DeKoter et al., 1998; Henkel et al., 1999).
Similarly, PU.1 (7/7) ES cells express several early
myeloid RNAs (Olson et al., 1995). Perhaps this
Regulation of granulopoiesis and monopoiesis
AD Friedman
3381
apparent relaxed `stringency' re¯ects redundancy
among Ets family members which is more e€ective in
vitro. For example, GABP can substitute for PU.1 to
activate the NE promoter in 32D cl3 cells (Nuch-
prayoon et al., 1997).
Activation of exogenous PU.1-ER in 32D cl3 cells is
not sucient to induce terminal granulopoiesis,
although increased MPO RNA is detected (Wang et
al., 1999). Similarly, stable expression of PU.1 in 32D
cl3 cells is of no consequence in IL-3, but accelerates
their di€erentiation in G-CSF (Bellon et al., 1998). In
contrast, over-expression of PU.1 in normal B-
lymphoid progenitors leads to the outgrowth of
macrophages at high levels of PU.1 expression and to
the generation of B-cells at lower levels of PU.1
(DeKoter and Singh, 2000). The ability of PU.1 to
activate its own promoter may play a role in
maintaining high levels of PU.1 expression in myeloid
cells (Chen et al., 1995b).
Core binding factors (CBFs)
The role of CBFs in normal hematopoiesis and in
leukemia was recently reviewed (Friedman, 1999). The
CBFs are a family of heterodimeric proteins containing
a common CBFb subunit and one of three CBFa
subunits, CBFa1, CBFa2 (commonly referred to as
AML1), and CBFa3 (Bae et al., 1993; Wang et al.,
1993; Ogawa et al., 1993a, Levanon et al., 1994). CBFs
bind the consensus site, 5'-PuACCPuCA-3' via their N-
terminal Runt homology domains (Ogawa et al.,
1993a; Meyers et al., 1993). CBFb does not contact
DNA, but interacts with the CBFa subunits via their
Runt domains and increases their anity for DNA
(Wang et al., 1993; Ogawa et al., 1993b). AML1 can
also interact with DNA indirectly, via interaction with
a subset of Ets family members, including MEF, Ets-1,
and PU.1, and via interaction with C/EBPa (Westen-
dorf et al., 1998; Mao et al., 1999).
CBFa2 (AML1) expression is largely restricted to
myeloid and lymphoid cells, including CD34+ pre-
cursors, in adult mice, whereas CBFa1 is most highly
expressed in osteoblasts, and CBFb is widely expressed
(Satake et al., 1992; Wang et al., 1993; Erickson et al.,
1996; Corsetti and Calabi, 1997; Ducy et al., 1997).
AML1 (7/7) and CBFb (7/7) mice die as embryos
without developing de®nitive hematopoiesis, whereas
CBFa1 (7/7) mice have normal blood formation but
do not develop calci®ed bones (Okuda et al., 1996;
Wang et al., 1996a,b; Sasaki et al., 1996; Niki et al.,
1997; Komori et al., 1997). Adult mice chimeric for
CBFb-SMMHC, a dominant-inhibitor of CBFs, devel-
op erythroid but not myeloid or lymphoid cells
expressing CBFb-SMMHC, consistent with a speci®c
role for CBFs in the maturation of these lineages in
adult marrow (Castilla et al., 1999).
In addition to activating lineage-speci®c markers
such as MPO and the M-CSF receptor, CBFs stimulate
the G1 to S transition in myeloid and lymphoid cell
lines (Cao et al., 1997, 1998; Lou et al., 2000; Strom et
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,
prevented G-CSF-mediated induction of MPO, an
early granulocytic marker, but accelerated induction
of two later markers, LF and Lysozyme (Q Wang and
AD Friedman, unpublished). This ®nding suggests that
proliferation is a prerequisite for the activation of
proteins such as CBF and c-Myb expressed in
immature cells and meant to serve a dual role by
simultaneously stimulating proliferation and di€eren-
tiation.
c-Myb
c-Myb binds the consensus DNA site 5'-(C/T)AAC(G/
T)-3' (Biedenkapp et al., 1988). The 52 amino acid R2
and R3 segments located near the N-terminus of c-
Myb contain tryptophans every 18 ± 19 residues which
are part of the hydrophobic core of a helix ± turn ±
helix structure (Ogata et al., 1992). c-Myb has a
central trans-activating domain, and its C-terminus
has an EVES motif which interferes with DNA-
binding by the N-terminus (Weston and Bishop,
1989; Dash et al., 1999). The C-terminal region of c-
Myb also contains a leucine zipper, which has been
shown to interact with a protein termed p160 (Tavner
et al., 1998). c-Myb is expressed in immature myeloid,
lymphoid, and erythroid cells, and c-Myb (7/7) mice
lack each of these lineages (Sheiness and Gardinier,
1984; Mucenski et al., 1991). Expression of exogenous
c-Myb in 32D cl3 cells allowed G-CSF induction of
MPO but prevented growth arrest and associated
induction of late markers such as LF (Bies et al.,
1995). A-Myb and B-Myb are highly homologous to
c-Myb within its DNA-binding domain and bind the
identical consensus sequence (Trauth et al., 1994). A-
Myb is expressed in several tissues, including a subset
of B cells, while B-Myb is expressed widely in
proliferating cells and may stimulate cell cycle
progression (Golay et al., 1991; Reiss et al., 1991;
Trauth et al., 1994).
MafB and c-Maf
MafB and c-Maf are members of a family of basic
region-leucine zipper DNA-binding proteins. Mafs
bind and activate transcription weakly as dimers via
a 13 bp palindrome, 5'-TGCTGACTCAGCA-3', which
contains a central AP-1 site, 5'-TGACTCA-3' (Katao-
ka et al., 1994a,b; Kerppola and Curran, 1994). Mafs
can also form heterodimers with Fos or Jun family
members which bind modi®ed consensus sites (Katao-
ka et al., 1996). Mafs interact with another bZIP
protein, NF-E2, in erythroid cells, enabling induction
of globin and other erythroid-speci®c genes (Andrews
et al., 1993). MafB is expressed in myeloid cells,
including marrow macrophages, but not in erythroid
cells, and it inhibits erythroid gene expression via direct
interaction with Ets-1 (Sieweke et al., 1996). When
avian progenitors were transformed by a Myb-Ets
fusion protein in the presence of exogenous MafB, an
increased number of myeloid colonies were obtained,
Oncogene
and expression of MafB in myeloblasts directed their
di€erentiation to macrophages (Kelly et al., 2000).
Similarly, expression of c-Maf in HL-60 or U937 cells
led to monocytic di€erentiation (Hegde et al., 1999). c-
Maf can interact with c-Myb, interfering with its
activation of early myeloid genes; however, interference
with c-Myb activities was not sucient to induce
monocytic di€erentiation in HL-60 cells (Hegde et al.,
1998, 1999).
Egr-1 and WT1
Egr-1 is a member of a family of zinc-®nger
transcription factors which binds the consensus 5'-
GCGGGGCG-3' (Crosby et al., 1991). The DNA-
binding domain of WT1 isoforms lacking a KTS insert
is strongly homologous, and these WT1 isoforms bind
the same consensus site (Rauscher et al., 1990). Egr-1 is
expressed in multiple tissue including the terminal
stages of macrophage and neutrophil di€erentiation
(Nguyen et al., 1993; Krishnaraju et al., 1995). Egr-1 is
necessary for monocytic di€erentiation of U937 or M1
cells, prevents the granulocytic di€erentiation of HL-60
or 32D cl3 cells, induces the macrophage di€erentiation
of M1 cells in the absence of IL-6 and endows 32D cl3
cells with the potential to di€erentiation to macro-
phages in response to GM-CSF (Nguyen et al., 1993;
Krishnaraju et al., 1995, 1998). In addition, ectopic
expression of Egr-1 in myeloid marrow progenitors
increased the proportion of CFU-M at the expense of
CFU-G and, to a lesser extent, BFU-E (Krishnaraju et
al., 2001). Mice lacking Egr-1 develop normal numbers
of macrophages, perhaps due to compensatory e€ects
from other Egr family members (Lee et al., 1996). As
Egr-1 is induced rapidly by phorbol esters even in the
absence of protein synthesis, Egr-1 may be an
important target of M-CSF receptor signaling (Nguyen
et al., 1993).
Several WT1 isoforms are expressed as a result of
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
the presence or absence of a three amino acid sequence,
KTS (Haber et al., 1991). The presence of the KTS
insert alters the DNA-binding speci®city of WT1
(Drummond et al., 1994). Lack of WT1 is lethal at
or before birth, and results in urogenital defects and
lack of spleen development (Kreidberg et al., 1993;
Herzer et al., 1999). Marrow development has not been
characterized in these mice. Expression of WT1
isoforms containing the KTS insert induces monocytic
di€erentiation of M1 cells, whereas isoforms lacking
this insert, and so mimicking Egr-1 DNA-binding
speci®city, prompt a G1 arrest (Smith et al., 1998). In
contrast, WT1(7KTS) accelerates whereas
WT1(+KTS) blocks 32D cl3 granulocytic di€erentia-
tion in response to G-CSF (Inoue et al., 1998; Loeb et
al., 2000). Inhibition of monocyte development by
WT1(7KTS) may re¯ect dominant inhibition of Egr-1,
via competition for DNA-binding. In addition, accel-
eration of granulocytic di€erentiation by WT1(7KTS)

may re¯ect limitation of G1 progression via direct
repression of the cyclin E promoter (Loeb et al., 2000).
WT1 is normally expressed in immature, CD34+
marrow cells and is down-regulated in response to
stem cell factor and G-CSF (Maurer et al., 1997). The
expression of speci®c WT1 isoforms in marrow
progenitors remains to be determined. Perhaps
WT1(7KTS) serves to interfere with monocyte
development and WT1(+KTS) serves to interfere with
granulocytic development.
Retinoic acid receptors
Retinoic acid receptors a, b, g(RARa, RARb, and
RARg) bind DNA via their zinc-®nger domains as
heterodimers with RXR proteins. RARs are broadly
expressed, with RARa being preferentially expressed in
myeloid cells (de The et al., 1989). The DNA consensus
for RAR-RXR DNA-binding is a direct repeat of 5'-
(A/G)G(G/T)TCA-3' separated by 2 ± 5 bps (Umesano
et al., 1991; Naar et al., 1991). Dominant-inhibition of
RARa arrests granulocytic di€erentiation at the
promyelocyte stage (Tsai and Collins, 1993). Mice
lacking RARa1 or RARg have normal myeloid
development, whereas while mice lacking both of these
RAR isoforms have normal myeloid progenitor
numbers, their neutrophilic di€erentiation arrests at
the myelocyte stage (Labrecque et al., 1998). This
phenotype is similar to that observed in C/EBPe (7/
7) mice, and RARa directly activates the C/EBPe
promoter (Yamanaka et al., 1997; Chih et al., 1997).
While regulation of the C/EBPe promoter by RARs
may account for the requirement of RARs for terminal
granulopoiesis, it remains to be established that
regulation of RAR activities plays a role in normal
hematopoiesis.
CCAAT displacement protein (CDP) and HoxA10
CDP is a widely expressed protein that represses gene
expression, at least in part, via competition for
transactivator binding to DNA elements which loosely
®t the 5'-CCAAT-3' motif (Barberis et al., 1987; Luo
and Skalnik, 1996). Decreased CDP DNA-binding
during the terminal stages of granulopoiesis combined
with its ability to repress the gp91-phox promoter led
to the proposal that inactivation and/or decreased
expression of CDP is required for terminal neutrophil
maturation (Skalnik et al., 1991). The mechanism
whereby CDP DNA-binding becomes inactivated
during granulopoiesis remains to be elucidated. 32D
cl3 cells overexpressing CDP do not express C/EBPe in
response to G-CSF, and the human C/EBPe promoter
contains a binding site for CDP at 71472 bp which
potentially accounts for the reduced activity of an
1800 bp compared to a 726 bp promoter fragment in
32Dwt18 cells (Khanna-Gupta et al., 2001). Inhibition
of C/EBPe expression may account for the combined
loss of LF, neutrophil collagenase, and neutrophil
gelatinase in 32D cells expressing exogenous CDP
(Lawson et al., 1998). However, a role for CDP in
Regulation of granulopoiesis and monopoiesis
AD Friedman
3383
regulating granulopoiesis in normal cells remains to be
con®rmed.
HoxA10 is a homeobox protein which binds
preferentially to 5'-TTAT-3' as a heterodimer with
PBX (Chang et al., 1996). The ability of HoxA10 to
repress the gp91-phox promoter led to the suggestion
that HoxA10 plays a role analogous to CDP during
granulopoiesis (Eklund et al., 2000). HoxA10 is
highly expressed in CD34+ and is only weakly
detected in CD347 marrow cells and is not present
in mature neutrophils or monocytes (Sauvageau et
al., 1994; Lawrence et al., 1995). HoxA10 is
preferentially expressed in myeloid cell lines, HoxA10
(7/7) mice have increased numbers of granulocytes,
and over-expression of HoxA10 inhibits monocytic
colony formation in vitro and produces AML in vivo
(Lawrence et al., 1995; Zhang et al., 1996b;
Thorsteinsdottir et al., 1997). Overall, as HoxA10
expression is apparently lost prior to CDP activity,
it may play a role in maintaining an earlier stage in
myelopoiesis. While HoxA10 can activate the
p21WAF1/Cip1 promoter in U937 cells, it is tempting
to speculate that HoxA10 in fact represses this
promoter in immature myeloid cells, enabling cell
proliferation and thereby inhibiting terminal di€er-
entiation (Bromleigh and Freedman, 2000). As we
have discussed, other Hox proteins may play a role
in regulating myelopoiesis as well (Ward et al.,
2000).
Like CDP, Sp1 is found in multiple tissues, but Sp1
is expressed at particularly high levels in maturing
granulocytes (Sa€er et al., 1991). This expression
pattern combined with the ®nding that Sp1 regulates
several granulocytic and monocytic genes via its
consensus-binding site, 5'-GGGCGG-3', raises the
possibility that Sp1 plays a role in regulating terminal
myelopoiesis.
Myeloid zinc finger 1 (MZF-1)
MZF-1 was isolated as a zinc ®nger protein preferen-
tially expressed in myeloid cell lines (Hromas et al.,
1991). MZF-1 is preferentially expressed in immature
myeloid cells, and reduction of its expression inhibits
the formation of CFU-G (Bavisotto et al., 1991). The
two zinc ®nger clusters within MZF-1 bind the
consensus sites 5'-AGTGGGGA-3' and 5'-
CGGGnGAGGGGGAA-3' (Morris et al., 1994). The
MPO and LF promoters contain bindings sites capable
of interacting with both sets of zinc ®ngers, and the
CD34 promoter contains two such sites located at
about 7500 bp. MZF-1 activates the CD34 promoter
via these sites in hematopoietic cell lines (Morris et al.,
1995). MZF-1 (7/7) mice develop normal blood cells,
but develop a progressive increase in monocytic cells
followed by the late formation of an in®ltrating
monocytic leukemia. In addition, a much greater
proportion of MZF-1 (7/7) myeloid and erythroid
progenitors are in cell cycle without increased expres-
sion of c-Myb (Gaboli et al., 2001). This phenotype
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
(Bavisotto et al., 1991).
p53, retinoblastoma protein (Rb), STAT3, and BLIMP-1
When induced by DNA strand breaks, p53 activates
pathways leading to apoptosis or cell cycle arrest
(Canman et al., 1995). p53 is detected at low levels in
mature myeloid and lymphoid cells and p53 levels
increase in ML-1 cells as they di€erentiate to
monocytes in response to phorbol esters (Kastan et
al., 1991). As c-Jun represses the p53 gene, this e€ect of
phorbol esters is likely indirect, perhaps via PKC
activation or cell cycle arrest (Schreiber et al., 1999).
Exogenous p53 prompts monocytic di€erentiation in
U937 cells or in 32D cl3 cells also expressing v-src or
an activated M-CSF receptor, without inducing G1
arrest or apoptosis (Soddu et al., 1996; Martinelli et
al., 1997; Ehinger et al., 1998). The induction of
monocytic di€erentiation in 32D cl3 cells is striking,
given their propensity for granulocytic maturation.
Induction of U937 cell di€erentiation by p53 depended
upon the integrity of its trans-activating domain and
was not inhibited by bcl-2 (Chylicki et al., 2000).
Although mice lacking p53 develop normally, the p53-
related protein, p73, potentially compensates for p53
de®ciency as p73 is expressed in immature but not
terminally di€erentiated myeloid cells (Lowe et al.,
1993; Peters et al., 1999).
Rb cooperates with E2F family members to repress
genes required for S phase entry (Weinberg, 1995). Rb
is down-regulated during granulopoiesis but is up-
regulated during monocyte development (Bergh et al.,
1999). Consistent with this expression pattern, anti-
sense inhibition of Rb expression in marrow progeni-
tors reduced CFU-M in favor of CFU-G (Bergh et al.,
1999). Perhaps complexes which form between hypo-
phosphorylated Rb and either C/EBPb or PU.1 are
essential for monocyte maturation (Hagemeier et al.,
1993; Chen et al., 1996). Rb (7/7) mice are
embryonic lethal and have increased erythroblasts,
whereas lack of the Rb-related protein p107 leads to
myeloid hyperplasia (Lee et al., 1992; Lecouter et al.,
1998). As with p53, Rb family members may
compensate for each other in knockout mice to help
mediate myelopoiesis.
Dominant inhibition of STAT3 in 32D cl3 cells
allows expression of early markers but prevents cell
cycle arrest and induction of late markers, although C/
EBPe induction was not prevented (Shimozaki et al.,
1997; Nakajima and Ihle, 2001). These ®ndings suggest
that STAT3 is required for induction of G1 cell cycle
arrest during granulopoiesis. Consistent with this
model, dominant inhibition of STAT3 prevents
p27Kip1 induction by G-CSF in 32D cl3 cells, and the
p27Kip1 promoter is activated directly by STAT3 (De
Koning et al., 2000).
BLIMP-1 is a zinc-®nger DNA-binding protein
which may contribute to terminal myeloid and B-
lymphoid di€erentiation at least in part by repressing
transcription of the c-myc gene (Chang et al., 2000).
Oncogene
Lineage commitment and progression
The widespread role of C/EBPs and PU.1 in regulating
myeloid genes combined with the defects evident in C/
EBPa (7/7) and PU.1 (7/7) mice make these
transcription factors leading candidates for roles in
determining the myeloid lineages. As PU.1 (7/7) mice
lack B cells and monocytes and have greatly reduced
numbers of neutrophils, whereas C/EBPa (7/7) mice
only lack maturing granulocytes, it would seem
reasonable to place PU.1 `upstream' of C/EBPs in this
regard. Further support for this idea comes from the
®nding that PU.1 and GATA-1 inhibit each others
activities, suggesting their relative importance in the
myeloid versus the erythrod/megakaryocyte lineages.
However, several lines of evidence suggest that C/EBPs
might play a role in specifying the bipotent granulo-
cyte-monocyte progenitor (GMP): C/EBP-mediated
down-regulation of FOG RNA is essential for
eosinophil development, and perhaps is also required
for GMP formation (Querfurth et al., 2000). Second,
fractionation studies suggest that pluripotent stem cells
®rst commit to a common lymphoid progenitor (CLP)
and a common myeloid progenitor (CMP) and that B-
cells and macrophages develop from the CLP and the
CMP respectively (Akashi et al., 2000); in this model, it
is dicult to draw a 1 : 1 correspondence between PU.1
and a speci®c progenitor. Third, the CMP gives rise to
the GMP, the megakaryocyte-erythroid progenitor
(MEP), and perhaps an eosinophilic progenitor
(EoP), and evidence has been presented indicating that
high-level GATA-1 speci®es the MEP whereas the
combination of a C/EBP and lower levels of GATA-1
speci®c to the eosinophil lineage (Kulessa et al., 1995;
McDevitt et al., 1997; Yamaguchi et al., 1999).
Therefore, if the third branch eminating from the
CMP, the GMP, were determined by the expression of
C/EBP without GATA-1, then the tripartite decision of
the CMP would depend on only two factors ± such
simplicity is attractive. And fourth, higher levels of
PU.1 are present in and are required for the monocyte
lineage, compared to the B-lineage (DeKoter and
Singh, 2000), and as C/EBPa rapidly induces PU.1
RNA expression in 32D cl3 and Ba/F3 cells, it might
do so in the GMP as well (Wang et al., 1999). One
dicult with this alternative is that C/EBP would be
expected to induce PU.1 in eosinophils, which would
inhibit GATA-1 activity. PU.1 is present in mature
eosinophils, and PU.1 RNA is strongly upregulated
when human CD34+ cells di€erentiate to eosinophils
in response to IL-5 (J Du and SJ Ackerman, personal
communication). PU.1 7/7 mice have greatly reduced
numbers of mature eosinophils, and direct evidence for
the regulation of an eosinophil-speci®c gene by PU.1
has been presented (Du et al., 1998; van Dijk et al.,
1998). Perhaps, partial inhibition of GATA-1 by PU.1
in fact serves to direct CMPs to the EoP, as opposed to
the MEP. Figure 1 illustrates two alternatives for the
speci®cation of the GMP, by high levels of PU.1 alone
or by high levels of PU.1 achieved via expression of C/
EBPs, likely including C/EBPa as this isoform is
 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
cells (Scott et al., 1992; Radomska et al., 1998).
Detailed discussions of erythroid and lymphoid lineage
development are presented elsewhere in this issue ± of
note however is the ®nding that Pax5 suppresses GM-
CSF Receptor RNA expression, again demonstrating
the role of negative cross-talk between hematopoietic
lineages (Chiang and Monroe, 2001). Both C/EBP-
dependent and -independent commitment from the
CMP to the GMP might occur, with requisite elevation
of PU.1 levels. Our ®nding that either C/EBPa-ER or
G-CSF receptor signals, acting in the presence of a
dominant-inhibitory C/EBP, elevate PU.1 RNA in 32D
cl3 cells is consistent with this idea (Wang et al., 1999;
Wang and Friedman, 2002).
The issue of granulocyte versus monocytic lineage
choice also must be resolved. The ®ndings that Jun and
Maf family members can each induce the monocyte
lineage in cell lines and that these proteins both
interact with AP-1 consensus sites leads to the
speculation that Maf:Maf, Maf:Jun, Maf:Fos, Jun:Jun,
or Jun:Fos dimers initiate monopoiesis in conjunction
with PU.1. The continued presence of C/EBPs, to
maintain elevated PU.1 levels, may be required as well.
In the absence of Maf or Jun proteins, C/EBPs and
PU.1 may specify the granulocyte lineage. The ®nding
that increased levels of C/EBPa favor granulocytic over
monocytic di€erentiation in U937 cells suggests that, as
with PU.1 and GATA-1, C/EBP levels may play a role
in lineage commitment decisions (Radomska et al.,
1998). A model for granulocyte versus monocyte
lineage determination is illustrated in Figure 1.
Egr-1 may serve to simultaneously enable monocyte
lineage progression and to prevent cross-over to
neutrophil development. Granulocyte lineage progres-
sion requires C/EBPe, which may be induced by C/
EBPa and/or RARs. Loss of CDP DNA-binding is
also required for terminal granulopoiesis. In addition,
progression to neutrophils and macrophages is asso-
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
terminal di€erentiation, as best shown for monopoiesis.
And cell cycle arrest may inactivate factors such as
CBF and c-Myb which participate in early stages of
di€erentiation. A model of myeloid lineage progression
is shown in Figure 1.
Perspectives for the future
Many questions remain unanswered regarding the
transcriptional regulation of granulocyte and monocyte
development. With respect to the cellular basis for
initiating these lineages: What are the relative con-
tributions of granulocyte/monocyte and B-cell/mono-
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
regulators of myeloid genes remaining to be uncovered
via detailed investigation of promoters and distal
enhancers; are there lineage-restricted co-activators or
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Oncogene
co-repressors which participate in lineage speci®cation?
With respect to key transcription factors, further
clari®cation of the regulatory network illustrated in
Figure 1 is needed: Which factor or factors specify
each lineage and at what levels of expression; can
family members act redundantly in this regard; what
additional cooperative mechanisms operate among
transcriptional regulators; what roles do cytokine
receptor signaling and transcription factor modi®ca-
tions play in each commitment decision and in each
step of lineage progression? The answers to these
questions will provide general lessons in developmental
biology and insights into leukemogenesis and will
enable applications in clinical hematology, oncology,
and gene therapy.
Acknowledgments
AD Friedman is a Scholar of the Leukemia and Lympho-
ma Society and his research is supported by the Children's
Cancer Foundation.
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