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REVIEW ARTICLE
Transcription Factors, Normal Myeloid Development, and Leukemia
By Daniel G. Tenen, Robert Hromas, Jonathan D. Licht, and Dong-Er Zhang
T
RANSCRIPTION FACTORS play a major role in differentiation in a number of cell types, including the
various hematopoietic lineages.1-4 In the hematopoietic system, stem cells undergo a process of commitment to multipotential progenitors, which in turn give rise to mature blood
cells. Although a number of transcription factors have been
identified that play a role in the development of erythroid
or lymphoid lineages,5-9 only in the past few years have those
factors that influence development of myeloid cells been
identified and studied. In particular, recent studies of the
regulation of normal myeloid genes, as well as the study of
leukemias, have suggested that transcription factors play a
major role in both myeloid differentiation and leukemogenesis.10-12a To understand the process of normal myeloid differentiation, it is important to identify and characterize the transcription factors that specifically activate important genes in
the myeloid lineage. These factors may play a role in acute
myeloid leukemia (AML), in which this normal differentiation program is blocked. Finally, some of the transcription
factor genes identified at the sites of consistent chromosome
rearrangements in AML have now been shown to play a role
in normal myeloid gene regulation.
The purpose of this review will be to summarize these
recent data regarding myeloid gene regulation and the transcription factors mediating this process to understand normal
myeloid development and leukemia. Emphasis will be placed
on the identified or potential role of these factors in leukemia;
we will focus on how alteration of myeloid transcription
factors (changes in expression and structure) could lead to
alterations in normal function in myelopoiesis, leading to a
block in differentiation. In addition, we will discuss those
factors that are involved in normal human myeloid maturation or human leukemia. Although this review will of necessity discuss different classes of factors, it does not aim to
be an exhaustive compilation or listing of different factors
and does not intend to be historical. Rather, it will attempt
to highlight how recent findings can help to explain normal
myeloid development and myeloid leukemia and will focus
on certain examples to make important general points. In
addition, this review will focus on early events in myeloid
development and only briefly mention transcription factors
involved in activation of mature myeloid cells. Finally, because there have been several recent reviews on hematopoietic factors in general, GATA, AML1, and the retinoic acid
receptors, we will attempt as much as possible not to repeat
what has been presented in these reviews.3,4,10-19 Some of the
key issues that we will attempt to address in this review
are the following: (1) How is gene expression restricted to
myeloid cells? (2) How is the temporal expression of myeloid genes controlled? (3) Which transcription factors are
thought to be necessary for myeloid development based on
knockout and/or inactivation studies and which are thought
to be necessary based on their redundancy? (4) If certain
transcription factors are expressed in myeloid and nonmyeloid cells, why are certain target genes expressed in myeloid
cells specifically? (5) How does a common progenitor cell
become one type (myeloid) rather than another?
Currently, models have been advanced proposing that hematopoietic lineage determination is driven extrinsically
(through growth factors, stroma, or other external influences),20,21 intrinsically (as described in stochastic models),22
or both.23 Our working hypothesis is that, regardless of
whether the environmental or stochastic models are invoked,
transcription factors are the final common pathway driving
differentiation and that hematopoietic commitment to different lineages is driven by alternative expression of specific
combinations of transcription factors, which often induce
expression of growth factor receptors or growth factors. The
transcription factors are often activated in positive autoregulatory loops, helping to explain the irreversible differentiation process found in human hematopoiesis. Experimental
support of the hypothesis that transcription factors are the
nodal decision points for myeloid differentiation include the
observations that many genes cloned at the site of leukemic
translocation breakpoints are transcription factors,11,12,24
genes isolated by their regulation of a phenotype (such as
differentiation) are transcription factors,25 and, finally,
knockouts of these transcription factors show gross defects
in myeloid development.3,26-28
METHODS USED TO IDENTIFY AND STUDY MYELOID
TRANSCRIPTION FACTORS
Our understanding of myeloid factors has been brought about
by a number of different methods. Initially, myeloid factors were
identified by investigations of known transcription factors, particularly oncogenes; examples include myb, myc, and the homeobox
genes.12a Similarly, a number of factors have been isolated using
hybridization and/or polymerase chain reaction (PCR) methods
based on similarity to known genes, such as MZF-1, with subsequent
studies investigating the normal and forced expression of these factors in myeloid cells.29-36 A second approach is subtraction hybridization or differential screening, which identified Egr-1 as a potential
mediator of monocytic development.25 A third method has been
identification of factors through the analysis of myeloid-specific pro-
From the Hematology/Oncology Division, Department of Medicine, Beth Israel Hospital, Harvard Medical School, Boston, MA;
the Hematology/Oncology Section, Department of Medicine, Indiana
University School of Medicine, Indianapolis, IN; and the Division
of Molecular Medicine, Mt Sinai School of Medicine, New York,
NY.
Submitted November 15, 1996; accepted February 7, 1997.
Supported by National Institutes of Health Grants No. CA41456
and DK48660 (to D.G.T.), HL48914 (to R.H.), CA59936 (to. J.D.L.),
and CA59589 (to D.-E.Z.) and by American Cancer Society Grants
No. DHP-160 (to J.D.L.) and DHP-166 (to D.-E.Z.). R.H. and J.D.L.
are Scholars of the Leukemia Society of America.
Address reprint requests to Daniel G. Tenen, MD, Harvard Institutes of Medicine, Room 954, 77 Avenue Louis Pasteur, Boston, MA
02115.
q 1997 by The American Society of Hematology.
0006-4971/97/9002-0036$3.00/0
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Table 1. Myeloid Transcription Factors Involved in AML
Translocation
Gene(s)
FAB Subtype
t(3;21)
t(8;21)
t(11;17)
t(15;17)
inv(16)t(16;16)
Evi-1/AML-1
AML-1/ETO
PLZF/RARa
PML/RARa
MYH11/CBFb
M1
M2
M3
M3
M4 eo
See Mitelman and Heim455 and references cited in text.
moters. Examples are PU.1 and C/EBPa, and these studies will be
described in further detail below. Finally, a number of myeloid
transcription factors have been identified through their involvement
in leukemias, either as a result of abnormal expression, such as
PU.1,37,38 or through their involvement at the site of a consistent
chromosome translocation (examples include AML139 and PLZF40;
see Table 1).
Once identified, the role of these factors has been ascertained by
a number of inhibitory studies. These include the use of antisense
and competitor oligonucleotide techniques, which have shown the
role of myc, myb, MZF-1, and PU.1 in myeloid development.41-44
A limited number of studies have used a trans-dominant mutant
approach and have been used to demonstrate the role of the retinoic
acid receptor a in myeloid development and of PU.1 in activation
of specific gene targets.45-47 Finally, the technique of gene disruption
(knockout) has recently been used to investigate the role of specific
factors in myeloid development.3,7,26-29,48-50 A recent review has focused on the effects of these knockout studies on hematopoiesis in
general3; therefore, we will discuss them only as they relate to myeloid transcription factors.
HOW DO MYELOID PROMOTERS WORK? WHAT ARE
GENERAL RULES SO FAR?
Many myeloid promoters appear to differ from what has
been described previously for tissue-specific promoters.51 In
general, a relatively small upstream region (usually a few
hundred basepairs) is capable of directing cell-type–specific
expression in tissue culture studies, although it appears that
additional elements are necessary for position independent,
high-level expression in a chromosomal locus (Fig 1). For
example, almost all of the specificity and activity detected
in transfection of tissue culture cells by the macrophage
colony-stimulating factor (M-CSF) receptor, granulocytemacrophage colony-stimulating factor (GM-CSF) receptor
a, and granulocyte colony-stimulating factor (G-CSF) receptor promoters are contained within 80, 70, and 74 bases of the
major transcription start sites, respectively,52-54 and similar
results have been obtained with the promoters for myeloid
transcription factors such as PU.1.55,56 Although most of
these promoters direct lineage and developmental specific
expression, in general they lack a TATA box or defined
initiator sequence. Interestingly, many myeloid promoters
have a functional PU.1 binding site upstream of the transcription start site at a location corresponding to one similar to
that of a TATA box. Because PU.1 can bind to the TATA
binding protein (TBP) in vitro,57 one mechanism by which
PU.1 could activate a whole class of myeloid promoters
is by recruiting TBP, the primary component of the basal
transcription factor TFIID, and the rest of the transcriptional
apparatus. Consistent with this mechanism are studies showing that mutation of a PU.1 site in the FcgR1 promoter can
abolish myeloid-specific expression, and replacement of this
mutant site with a TATA box can restore myeloid expression.58 Other factors involved in myeloid gene regulation,
including Sp1, C/EBPa, and Oct-1, have all been shown to
interact with TBP,59-61 and these interactions could also help
mediate recruitment of the basal transcription machinery.
Finally, whereas some of these TATA-less and initiatorless myeloid promoters have multiple transcriptional start
sites,55,62 others appear to have a single strong start site.63,64
The mechanism for how the start sites are determined in
these promoters is unknown.
Consistent with their lack of a TATA box, these promoters
often are dependent on a functional Sp1 site, which can
interact not only with the TATA binding protein, TBP, as
well as a TBP-associated factor, TAF110.59 These Sp1 sites
not only mediate activity, but also specificity and inducibility
with differentiation.65-67 How the Sp1 factor mediates this
specificity is not clear, but in one case Sp1 binds to its
myeloid target in vivo in myeloid cells only,65 and in another,
increased relative expression in myeloid cells of a phosphor-
Fig 1. Specificity of the human M-CSF receptor promoter is mediated by combinatorial activity of factors in myeloid cells. The activity and
specificity of the human M-CSF receptor promoter in transient transfection studies is mediated by the 50-bp region lying between bp Ï88
and Ï40 relative to the major transcription start site in monocytic cells.52,117,215 Shown are the binding sites for C/EBP, AML1, and PU.1, all of
which are important for M-CSF receptor promoter activity. In the hematopoietic system, C/EBPa is myeloid specific, AML1 is expressed in all
white blood cells, and PU.1 is B-cell– and myeloid-specific, so therefore the major cell type in which all three are expressed is myeloid cells.
In addition, C/EBPa and AML1 can physically interact and synergize to activate this promoter.215 Others have shown using other promoters
that C/EBP proteins can synergize with PU.1.88 Also shown is CBFb, which forms a heterodimer with AML1 and augments its ability to bind
to DNA.
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TRANSCRIPTION AND MYELOID DEVELOPMENT
491
ylated form of Sp1 that shows markedly increased binding
to its DNA target site is observed.66 Although Sp1 is ubiquitous, it is preferentially expressed in hematopoietic cells.68
Differential expression in distinct hematopoietic lineages has
not been explored. In addition, Sp1 can mediate responses
to retinoic acid, thyroid hormone, and retinoblastoma protein, all of which may influence myeloid differentiation.69-72
Finally, it has been recently shown that Sp1 belongs to a
family of related factors and that at least one of these, Sp3,
may mediate repression of target genes.73,74 Sp1 can also
cooperate in repression with GATA-1,75 and this interaction
could potentially repress myeloid targets in erythroid cells
(see below).
Several of the myeloid promoters appear to have GATA sites
that can bind GATA proteins specifically in gelshift assays.
However, mutation of GATA binding sites in the CD11b76 or
PU.155 promoters does not lead to significant loss of promoter
function. Coexpression of either GATA-1 or GATA-2 leads to
a slight downregulation (2-fold) of these promoters. Interestingly, there is a functional GATA-1 site in the promoter for
the platelet-specific integrin IIb that is located at a position
almost identical to that of the nonfunctional GATA site found
in the myeloid integrin CD11b,77 suggesting evolutionary conservation of binding sites in related integrin promoters, but not
function. The differences between the ability of GATA-1 to
activate promoters such as IIb in erythroid or megakaryocytic
cells, in which GATA-1 is thought to play an important role
in lineage development and not in myeloid cells, may be due
to cell-type–specific modifications of GATA and/or interactions with other transcription factors. In addition, GATA-1 is
not expressed at significant levels in myeloid cells, and both
GATA-144 and GATA-278 are downregulated in myeloid development. These results are consistent with the preservation of
myeloid cell development in mice with targeted mutations of
GATA-1.5 In contrast to monocytic and neutrophilic cells, high
levels of GATA-1 are detected in eosinophils,79 but to date
functional GATA sites have not been detected in eosinophil
promoters. In summary, it appears that GATA proteins do not
play an important positive role in myeloid development; indeed,
downregulation may be critical for myeloid maturation.44,80,81
As noted above, almost all myeloid promoters have a functional PU.1 site close to the transcription start site. There are
a number of interesting exceptions to this rule. The CD14
promoter has a TATAA-like sequence and does not have a
functional PU.1 site in its promoter.66 This promoter region is
highly active and cell-type–specific in transient assays66 but
not in transgenic mice.82,83 This scenario is somewhat similar
to that of chicken lysozyme, which lacks a PU.1 site in its
promoter but contains one in an enhancer located 2.7 kb upstream and which is part of a genomic fragment that is capable
of high-level specific expression in mice.84,85 Whether other
myeloid promoters that lack PU.1 sites will turn out to have
PU.1-containing enhancer elements located several kilobases
from the promoter remains to be determined.
Another set of myeloid genes that appear to have TATAA
boxes are the primary granule protein promoters, including
myeloperoxidase,86,87 neutrophil elastase,87,88 proteinase 3,89
and cathepsin G.90 At least three of these have been noted
to have functional PU.1 sites, and in this class of genes
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PU.1 plays an important role, but perhaps has a different
mechanism than other myeloid genes in which there is no
TATAA box. At this point it is not clear what other common
factors account for the distinct pattern of expression of these
genes, in which mRNA is dramatically upregulated at the
promyelocytic stage and then rapidly downregulated with
further myeloid development. It has also been observed that
a number of these same primary granule myeloid promoters
(cathepsin G, myeloperoxidase, neutrophil elastase, aminopeptidase N, c-fes, and MRP8) contain a common sequence
CCCCnCCC.91 Recently, a protein binding to this site has
been described, and mutations of this site in the proteinase3 promoter that abrogate binding of this factor lead to a
significant loss in promoter function.89 Therefore, identification of this protein may lead to important understanding of
the regulation of this whole family of myeloid genes.
In contrast to the primary granule protein genes, most
other myeloid-specific genes are upregulated during the
course of myeloid development and are expressed at highest
levels in mature myeloid cells. Examples include key receptors, such as the G-CSF receptor92 and CD11b,93,94 as well
as critical transcription factors, such as PU.1.44,95 In some
cases, there are emerging clues into the mechanisms of
upregulation during differentiation. For example, the upregulation of the CD14 promoter during vitamin D3-induced
monocytic differentiation is mediated by an Sp1 site,67 and
recently a novel transcription factor has been described that
mediates upregulation of CD11b promoter activity during
12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of myeloid cell lines.96 The binding site for this
factor is shared by the three leukocyte integrin promoters.
The factor identified, MS-2, like Sp1, is not specific for
myeloid cells. In other cases, the promoter elements and
transcription factors mediating upregulation during myeloid
differentiation have not been well defined. It should be noted
that the transcription factors critical for upregulation during
myeloid development versus those invoked during activation
of mature myeloid cells may well be distinct. For example,
the macrophage scavenger receptor has motifs mediating
lineage specificity (including a PU.1 site) and a distinct element mediating upregulation of stimulated macrophages
(through an AP-1/ets site).97 Similarly, distinct elements mediating lineage upregulation of the interleukin-1b (IL-1b)
promoter in macrophage lines have been described.47,98,99
Functionally important transcriptional regulatory elements
have been described in the 5* untranslated regions of a number of genes,100,101 and several myeloid promoters also contain important functional sites in their 5* untranslated region.
The human and murine PU.1 promoters are almost totally
conserved in their 5* untranslated region, and both contain
a functional PU.1 site.55 In the case of the human and murine
G-CSF receptor promoters, conservation in this region is
mainly limited to the PU.1 site found there.53
TRANSGENIC STUDIES
Often myeloid regulatory factors have been identified
through the investigation of myeloid-specific promoter elements in studies involving transient transfection studies in
tissue culture cell lines. In transgenic mice, other regulatory
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TENEN ET AL
elements, such as locus control elements, are very important.102 A number of myeloid promoters have worked to
some extent in transgenic studies involving the expression
of heterologous genes in myeloid cells. In some instances,
there is variability of levels of expression and significant
position dependence. For example, the proximal 1.5 kb of
the gp91-phox gene, normally expressed at high levels in
granulocytes, targeted reporter expression to a small subset
of monocytic cells only, and most monocytes and neutrophils
did not express the transgene.103 In other examples, the responsible regulatory elements have not yet been defined in
detail. These studies include human c-fes/fps,104 chicken lysozyme,85,105 or human cathepsin G,90 in which the investigators reported high-level, properly regulated transgene expression. In these studies, relatively large pieces of DNA (6 to
21.5 kb) were used that included the gene itself as the reporter. In some instances, the expression patterns of the
transgenes were not described in detail, but were stated to
be specific for macrophages and neutrophils, respectively.
Examples include those involving a 5-kb portion of the
monocytic M-CSF (CSF-1) receptor promoter, which was
reported to target macrophages,106,107 and the MRP8 promoter, which was used to express bcl-2 in neutrophils.108
The MRP8 promoter has also been used to induce expression
of a PML/RARa transgene in myeloid cells, resulting in a
high percentage of expressing transgenic lines, altered granulocytic differentiation, and, in some animals, an acute leukemia resembling human acute promyelocytic leukemia.109 A
recent study has shown that the human lysozyme promoter
can direct reporter gene expression in macrophages.110
A number of studies have looked at the ability of myeloid
integrin promoters to direct the expression of reporter genes
in transgenic studies.111,112 The CD11b promoter is capable
of driving high-level expression of a reporter gene in myeloid
cells at levels comparable to the endogenous CD11b promoter. This promoter construct is position-dependent, perhaps explaining why another group did not observe similar
levels of expression.112 Although in these studies the activity
of the promoter was highest in mature myeloid cells, this
promoter construct has also been used to direct expression
of the t(15:17) PML/RARa fusion protein in early myeloid
cells.113 The reason for these differences is not clear, but
may reflect a significant amount of position dependence on
promoter activity and specificity. Attempts to direct expression of reporter genes into early myeloid cells using promoters such as CD3483 and c-kit114 have not been successful.
However, recent reports suggest that upstream regions of
the cathepsin G115 and MRP8108,109 genes may be useful in
directing expression of an inserted cDNA into promyelocytes. Many of the promoters used in these studies, like most
myeloid-specific genes, lack a TATAA box,63,76 and previous
studies using transient transfections in tissue culture cells
have shown the role of PU.1 in the myeloid specificity of
the promoters (Table 2). Of the myeloid genes that have
been shown to function in transgenic studies to date, several
of them, including CD11b,116 the M-CSF receptor,117 the
chicken lysozyme enhancer,84 and c-fes,118,119 have important
functional PU.1 sites. A recent study has confirmed the importance of the PU.1 site in macrophage expression of the
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Table 2. Myeloid Gene Targets Regulated by PU.1
Gene Target
Reference
CD11b (MAC-1a)
Macrophage inflammatory protein-1a
M-CSF (CSF-1) receptor
Pahl et al116
Grove et al420
Zhang et al117
Reddy et al164
Eichbaum et al58
Perez et al448
Feinman et al449
Ahne et al84
Rosmarin et al450
Moulton et al166
Kominato et al47
Srikanth et al451
Nuchprayoon et al87
Oelgeschlager et al88
Carvalho et al452
Maury453
Smith et al53
Hohaus et al54
Kistler et al56
Chen et al55
Ray-Gallet et al118
Heydemann et al119
Himmelmann et al454
Ford et al160
Sturrock et al89
FcgR1
FcgRIIIA
Chicken lysozyme
CD18 (MAC-1 b)
Macrophage scavenger receptor
IL-1b
Neutrophil elastase
Lentivirus
G-CSF receptor
GM-CSF receptor a
PU.1
c-fes/c-fps
Bruton’s tyrosine kinase (Btk)
Myeloperoxidase (MPO)
Proteinase-3
macrophage scavenger receptor.120 These studies strongly
suggest that PU.1 will in general be a critical factor in directing myeloid expression in transgenic studies.
In summary, it appears from the transgenic studies that
myeloid promoters can drive expression in monocytic and
neutrophilic cells in transgenic mice and that the PU.1 site
is likely to be critical. However, the details of the other
critical elements necessary for myeloid-specific and position-independent expression are not defined yet, and much
work needs to be performed in this area in the future.
GENE THERAPY APPLICATIONS OF MYELOID
PROMOTERS
An exciting prospect would be to use the myeloid regulatory elements to target given genes to proper hematopoietic
compartments for gene therapy applications, particularly in
terms of directing macrophage expression of enzymes involved in glycogen storage diseases.121 Myeloid promoter
elements may be useful for viral vectors, especially adenoassociated virus (AAV), because, at least in transient assays,
very small DNA fragments of a few hundred basepairs direct
activity that is as high and as specific as much larger fragments. A note of caution is that larger elements or a combination of promoter and upstream elements (locus control regions) may be necessary, as noted above from the description
of transgenic studies. Very few reports on the use of myeloid
elements in gene therapy vectors have been reported. In
one study, the CD11b promoter failed to direct significant
reporter expression when incorporated into a retroviral vector,122 but a second study showed the same promoter directing significant amounts of glucocerebrosidase mRNA and
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TRANSCRIPTION AND MYELOID DEVELOPMENT
493
enzymatic activity in myeloid cell lines and in bone marrow.123 Again, the reason for the discrepancies observed between these two studies using a similar promoter is not clear,
and much more work needs to be performed in this area of
research.
PU.1 (Spi-1) AND OTHER ets FACTORS
PU.1: A master regulator of myeloid genes. Recent studies have focused on PU.1 as a major regulator of myeloid
development.26,44,161 PU.1 is the product of the Spi-1 oncogene identified as a consequence of its transcriptional activation in Friend virus-induced erythroleukemias.14,37,38,124 The
role of PU.1 in the malignant transformation of the proerythroblast was confirmed by subsequent studies. Overexpression of PU.1 from a retroviral construct in long-term
bone marrow cultures is able to stimulate the proliferation of
proerythroblast-like cells that differentiate at a low frequency
into hemoglobinized cells,125 and overexpression in
transgenic mice can also induce erythroleukemia.126 Moreover, PU.1 antisense oligonucleotides reduce the capacity of
Friend tumor cells to proliferate.127 Taken together, these
data suggest that deregulation of PU.1 increases the selfrenewal capacity of erythroid precursor cells and blocks their
differentiation. Furthermore, it shows the importance of
proper regulation of PU.1 in the hematopoietic system.
PU.1 is a member of the Ets transcription family.124 Ets
factors all contain a characteristic DNA binding domain of
approximately 80 amino acids.14,128,129 PU.1 and the related
Ets family member Spi-B130 form a distinct subfamily within
the larger group of Ets proteins, with very distinct structure,
patterns of expression, binding specificity, and functions that
are nonoverlapping with other members of the Ets family.
The PU.1 protein consists of 272 amino acids, with the DNA
binding domain located in the carboxyl terminal part of the
protein, whereas the amino terminus contains an activation
domain that has been implicated in interactions with other
regulatory proteins.57,124,131,132 The structure of the DNA binding domain has been recently determined and demonstrates
a winged helix-turn-helix motif.133
PU.1 shows specific patterns of hematopoietic expression.
PU.1 is expressed at highest levels in myeloid and B cells,
but not in T cells.37,95,124 During hematopoietic development,
PU.1 mRNA is expressed at low levels in murine ES cells
and human CD34/ stem cells and is specifically upregulated
with myeloid differentiation.44 The timing of this upregulation coincides with the first detection of early myeloid maturation, suggesting that this increase in expression may be an
important process in myeloid development, and inhibition of
PU.1 function at this juncture can block myeloid progenitor
formation.44 These findings were confirmed by another report
showing that PU.1 is expressed in the earliest Go CD34/
stem cells and upregulated as single CD34//CD380 cells
developed into myeloid colonies.134 Studies in both primary
CD34/ cells and human leukemic cell line models suggest
that PU.1 mRNA and DNA binding activity do not increase
further with subsequent myeloid maturation from the promyelocytic to more mature stages,44,95 but these studies do not
preclude the possibility that the high levels of mRNA observed in human monocytes and neutrophils95 are a result
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of subsequent final maturation and/or activation. Although
initially thought to be B-cell and monocyte specific,124,135
high levels of both PU.1 mRNA and DNA binding activity
have been subsequently found in neutrophils.95 PU.1 mRNA
has also been detected in human eosinophils (S.J. Ackerman
and D.G. Tenen, unpublished observations). In murine
Friend erythroleukemia cells, PU.1 is downregulated during
chemically induced erythroid differentiation.127,136,137
These expression studies suggest that regulation of PU.1
mRNA may play a significant role in the commitment of
early multipotential progenitors to the myeloid lineages, as
well as in the further differentiation and maturation of these
cells. Characterization of the murine and human PU.1 promoters shows that a highly conserved region surrounding the
transcription start sites can direct myeloid-specific activity in
transient transfection studies. The important functional sites
identified thus far in the promoter, an octamer site at bp
054, an Sp1 site at bp 039, and a site for PU.1 itself at bp
/20, are conserved in the two species. In B cells, the octamer
site plays a major role in PU.1 expression,56,138 whereas in
myeloid cells the PU.1 site is the most important for function
of the promoter.55 This study suggests the presence of a
positive feedback mechanism in the regulation of the PU.1
promoter similar to that shown for another Ets family member, Ets-1.139 Such autoregulation of transcription factors
could play a major role in commitment and differentiation
of hematopoietic multipotential progenitor cells (see below).
The PU.1 promoter also contains a site that can bind GATA
proteins, but no binding could be detected using nuclear
extracts from myeloid cell lines, and cotransfection of either
a GATA-1 or GATA-2 expression construct repressed the
PU.1 promoter twofold. Important unanswered questions are
whether GATA proteins affect PU.1 expression in multipotential progenitors44 or in early erythroid cells, in which PU.1
is repressed in the course of differentiation.136,137,140 Finally,
although the elements described above direct expression of
PU.1 in tissue culture cells, as much as 2 kb of upstream
murine DNA sequence failed to direct expression of a reporter cDNA in spleen or peritoneal macrophages in
transgenic studies, so other elements are required for high
level expression in the chromosomal context.83
PU.1, like other Ets factors,128 was first noted to bind to
a sequence characterized by a purine-rich core (GGAA),
hence its name.124 However, the DNA binding specificity of
PU.1 appears to be quite distinct from that of other Ets
factors. For example, PU.1 and Spi-B bind to the CD11b
PU.1 site, but not the other Ets family members Ets-1, Ets2, Elf-1, or Fli-1.44 In addition, it is not possible to predict
PU.1 binding by observation of a 5*-GAGGAA-3* sequence.
For example, there are three such sequences in the M-CSF
receptor promoter, and PU.1 does not bind to any of them
with high affinity. Instead, PU.1 binds to the nearby sequence
5*-AAAGAGGGGGAGAG-3*.117 This example is consistent
with previous work indicating that binding of PU.1 and other
Ets factors is dependent on sequences outside the GGAA
core.141 In addition, many functional PU.1 binding sites in
myeloid promoters have a string of adenosine residues immediately 5* of the GA core.54,116,117,137 These results are
consistent with a recently described consensus sequence for
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PU.1 binding.118 The availability of an x-ray crystallographic
structure for PU.1133 will assist in further studies of PU.1
binding specificity. In summary, PU.1 is distinct from other
Ets factors in DNA binding specificity. Identification of the
transactivation domain of PU.1 has been relatively difficult
because PU.1 acts as a weak transactivator; indeed, most
transactivation studies have required multiple PU.1 binding
sites and used artificial promoter constructs.53,55,124,142 Studies
using myeloid promoters have in general shown weak transactivations, on the order of twofold to fivefold.116,117 Recently, using multimerized reporter constructs, the transactivation domain of PU.1 was identified in the amino
terminus.131 These studies defined multiple relatively weak
transactivation domains; whether this indicates that each interacts with different proteins (see below) remains to be
determined.
PU.1, like other Ets proteins, interacts with other transcriptional regulators. In B cells, PU.1 recruits a second, B-cell–
specific DNA binding factor, NF-EM5 or Pip, to a site
important for Ig k 3* enhancer function.143-146 For this interaction, phosphorylation of a serine residue at position 148 is
necessary.144 However, such interactions have not been
shown previously to play a functional role in previously
described myeloid targets of PU.1, such as CD11b and the
M-CSF receptor, and mutation of Ser148 does not affect the
ability of PU.1 to activate these myeloid promoters.116,117
As noted above, PU.1 can interact with TBP in vitro.57,132
An attractive hypothesis is that PU.1 may recruit the basal
transcription machinery to myeloid promoters, which in general lack TATAA boxes, but the functional significance of
this interaction is not known at this time. Similarly, these
same studies showed that PU.1 can interact physically with
RB. It has been shown that hypophosphorylated RB can
negatively regulate the activity of another Ets factor, Elf-1,
in the course of T-cell activation.147 Because RB becomes
hypophosphorylated during myeloid differentiation,148,149 it
possibly could regulate PU.1 function in these cells. Experiments to date have failed to show an effect of RB on PU.1
function on myeloid promoters,83 although it has been shown
that RB can repress PU.1 function on an artificial promoter.132 A recent study using a protein interaction screen
has identified a number of proteins that can bind to PU.1,
although the functional consequences of these interactions
remains to be determined.142 One of these is HMG(I)Y,
which has been shown to augment the ability of the Ets
factor Elf-1 to activate the IL-2 receptor b chain.150 To date,
similar experiments with HMG(I)Y have failed to show
binding to myeloid targets such as the M-CSF receptor or
GM-CSF receptor a promoters or to augment the ability of
PU.1 to activate these promoters. Another factor cloned in
this study and shown to bind to PU.1 is NF-IL6b (same as
C/EBPd); in this case, it was shown that PU.1 and C/EBPd
could cooperatively activate an artificial reporter plasmid.
These findings are interesting because they suggest that PU.1
might also interact with C/EBPa (see below). Similarly, in
another study, PU.1 has been shown to interact with another
basic leucine zipper (BZIP) transcription factor, c-Jun.151 Finally, PU.1 has been shown to functionally interfere with
steroid hormone gene activation, and steroid hormone recep-
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tors can inhibit PU.1 transactivation function.152 Of particular
relevance to myeloid development is the fact that these interfering effects were seen with the retinoic acid receptor.152
However, the functional significance with respect to myeloid
development of all of these interactions is not known.
Ets family members have been shown to play an important
role in several signal transduction pathways.153-156 PU.1 is
phosphorylated in vitro by casein kinase II and JNK kinase,
but not by ERK1 (MAP) kinase.157 A recent study has shown
that lipopolysaccharide (LPS) can activate casein kinase II
and phosphorylation of PU.1 in LPS-stimulated macrophage
lines.158 The LPS stimulation of PU.1 transactivation of a
reporter construct was abolished by a serine to alanine mutation of residue 148, suggesting that stimulation of macrophages with LPS leads to increased phosphorylation and
activity of PU.1. The same PU.1 phosphorylation site has
been shown to be important for B-cell gene activation (see
above). A recent study implicated PU.1 in M-CSF–induced
proliferation of murine macrophages, and this effect was
abrogated by mutating two potential phosphorylation sites
(Ser41 and Ser45) in the transactivation domain of PU.1 that
are distinct from the postulated site of LPS stimulation at
Ser148.159 Finally, a recent study suggested that there may
be differences in PU.1 phosphorylation between myeloid
cells and B cells, but not during myeloid commitment of
multipotential progenitors.160
A number of studies have addressed PU.1 function in
myeloid cells by inhibiting its function. Addition of competitor oligonucleotides to human CD34/ cells specifically
blocked myeloid CFU formation.44 The inhibition was only
observed if the competitors were added very early during
GM-CSF–induced myeloid maturation, before the upregulation of PU.1 expression. Several groups have addressed the
function of PU.1 on murine development using targeted disruption of the PU.1 gene.26,161 One group reported that PU.1
0/0 embryos died in utero, usually at day E16.26 These
animals demonstrated a variable anemia, but no production
of any type of white blood cells, including monocytes, neutrophils, and B cells, as might be expected based on the
expression of PU.1 and its known target genes. The concomitant failure to produce T cells in the 0/0 animals was surprising, given that PU.1 has not been known to be expressed
in this lineage (see above). These studies suggested that the
defect was either due to a block of a very early multilineage
progenitor cell or that T-cell development is dependent on
the presence of macrophages and/or B cells.26
A second PU.1 knockout animal yielded a phenotype with
some distinct differences.161 The PU.1 0/0 animals in this
case were viable at birth and could be kept alive for days
by housing them in a sterile environment with the administration of antibiotics. These animals also lacked monocytes
and mature B cells, but were capable of producing B-cell
progenitors. Several days after birth, T cells and cells resembling neutrophils in the peripheral blood were detected. In
this case, the findings suggested a role for PU.1 in B-cell
and monocytic development, which is more in keeping with
its known pattern of expression and its known gene targets.
Although neutrophilic cells were observed, they were Mac1–negative and reduced in number, suggesting that targeting
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PU.1 results in a partial defect in neutrophil development.
The reason for these marked differences between the two
knockout phenotypes is not known at this time, but will be
important to determine in that the former knockout model
suggests that PU.1 plays an important role in multipotential
cells,26 whereas the latter suggest a more limited role in Bcell and myeloid development.161 In vitro differentiation of
PU.1 0/0 ES cells does not produce macrophages and points
to the M-CSF receptor as a major target for PU.1 in understanding its effect on myeloid differentiation.162,163
Consistent with the finding that PU.1 has a major role in
myeloid development has been the identification in the past
three years of multiple myeloid genes regulated by PU.1
(Table 2). In transfection studies, PU.1 regulates a number
of genes that appear as late markers of myeloid cells (Table
2). In addition, PU.1 sites are critical for the activity of a
number of myeloid CSF receptor promoters, including the
M-CSF receptor,117,164 the GM-CSF receptor a,54 and the GCSF receptor promoter.53 Quantitative studies will be needed
to establish whether hematopoietic cells in PU.1 knockout
animals have decreased or absent expression of these three
myeloid CSF receptors. If so, the role of expression of these
receptors in myeloid development can be tested by restoring
their expression singly or in combination in these PU.1
0/0 animals. As noted above, studies in PU.1 0/0 ES cells
have confirmed that CD11b and the M-CSF receptor are
major targets for PU.1.162,163 In addition to transactivating
the promoters of the genes cited above, it is possible that
PU.1 has other mechanisms of action. Recently, it was noted
that PU.1 (but not Spi-B or other Ets factors) can bind RNA,
suggesting that it could possibly play a role in posttranscriptional regulation.165 The biologic significance of these observations remains to be determined.
Although PU.1 was first isolated as a gene activated in
murine erythroleukemia,37 so far it has not been implicated
in the pathogenesis of human leukemias. The PU.1 gene has
been mapped to 11p11.22, not a frequent site of chromosomal translocations found in human leukemia. PU.1 expression in leukemic myeloid lines is not higher than that observed in primary myeloid cells,95 and a recent survey of
primary human leukemias found no significant discordance
between PU.1 expression in leukemic samples and that predicted by the apparent differentiation stage of the leukemic
cells (M.T. Voso, unpublished observations). However, no
studies to date have addressed whether PU.1 is mutated in
human leukemias.
Other Ets factors and myeloid development. Spi-B was
isolated by hybridization with a PU.1 cDNA probe and,
together with PU.1, forms a distinct subfamily within the
Ets factors.130 In contrast to other Ets proteins,44 Spi-B can
bind to many of the targets for PU.1 and can transactivate
many of these myeloid genes.55,95 However, the observation
that PU.1 knockout mice failed to produce certain myeloid
lineages suggested that Spi-B might be expressed quite differently from PU.1. This was confirmed by studies showing
that Spi-B is not expressed at significant levels in myeloid
cells, suggesting that its role in myeloid differentiation and
function is likely to be limited.55,95 Recently, it has been
shown that the binding specificity of Spi-B is very similar
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but not identical to that of PU.1,118,166 and perhaps this can
also contribute to the differences in function of these two
factors. In addition, Spi-B and PU.1 are phosphorylated by
different kinases.157
To date the role of other Ets factors in myeloid development has not been well defined. A number of other Ets
factors are expressed in myeloid cells, including Ets-2, Fli1, and Elf-1167,168 (D.E.Z. and D.G.T, unpublished observations). Ets-2 is capable of transactivating the murine M-CSF
receptor promoter,169 but targeted disruption of the Ets-2
gene did not affect the ability of ES cells to form macrophages in vitro,163 suggesting that Ets-2 does not play a
necessary role in myeloid development. Ets-2 may play an
important role in activation or signaling in macrophages.107,156,164 The ubiquitous Ets protein GABP is important for the activity of the CD18 protein and can functionally cooperate with PU.1.170 GABP from myeloid cells can
also bind to the neutrophil elastase promoter and cooperates
with c-myb and C/EBP to transactivate this promoter in
nonmyeloid cells (A. Rosmarin, unpublished observations).
Recently, another ubiquitously expressed Ets protein, TEL,
was isolated from a patient with chronic myelomonocytic
leukemia as a fusion protein with the platelet-derived growth
factor (PDGF) receptor.171 TEL can also form a fusion protein with AML1 in a large percentage of cases of pre-B–
cell acute lymphoblastic leukemia (ALL).172 At this time,
the precise role of TEL in myeloid development and in the
pathogenesis of myeloid leukemia is not known.173
C/EBP, A REGULATOR OF GRANULOCYTIC
DEVELOPMENT
C/EBP (CCAAT/enhancer binding protein; now known
as C/EBPa) was isolated as a rat liver protein that bound to
viral enhancer sequences174,175 and is a member of one of
several leucine zipper transcription factor families, including
the fos/Jun family and the ATF/CREB family.176-178 C/EBPa
binds as a homodimer or heterodimer with other C/EBP
proteins or other transcription factors and has been shown
to regulate a number of hepatic and adipocyte genes. C/
EBPa is upregulated and plays an important role in adipocyte
differentiation and in this cell type functions in fully differentiated, nonproliferative cells; inhibition of C/EBPa blocks
differentiation; and expression of C/EBPa induces differentiation.179,180 Indeed, several studies have indicated that C/
EBPa can act as a general inhibitor of cell proliferation and
in this respect resembles a tumor suppressor.181-183 C/EBPa
is part of a family of leucine zipper transcription factors,
including C/EBPb and C/EBPd, that heterodimerize and are
differentially regulated during adipocyte differentiation.179,184,185 Another very interesting C/EBP gene, C/EBPe,
was very recently isolated and shown to be preferentially
expressed in myeloid cells.186,187 Both C/EBPa and C/EBPb
have single mRNAs that can encode transcriptionally active
and repressive forms, depending on the usage of alternative
start AUG codons in the same reading frame.188,189 In addition, another C/EBP family member, CHOP-10, can dimerize with C/EBP proteins to inhibit transcriptional activation
and is found at a translocation breakpoint in liposarcomas.190,191 Of note is that CHOP can specifically block C/
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EBPa activity and induce increased apoptosis of myeloid
32D cells.191
In murine adipocyte cell lines, C/EBPa is transcriptionally
upregulated (and C/EBPb is downregulated) with adipocyte
differentiation and binds to its own promoter.192 In addition,
c-myc overexpression can block differentiation of adipocytes, possibly by repressing the C/EBPa promoter.193 Interestingly, although both murine and human C/EBPa promoters are autoregulated, the mechanisms of autoregulation are
different. The murine C/EBPa promoter has a binding site
for C/EBPa.192 The human C/EBPa promoter does not conserve the C/EBPa binding site found in the murine promoter,
and the autoregulation is indirect and mediated by the action
of C/EBPa on a helix-loop-helix protein, upstream stimulatory factor (USF).194 Studies of C/EBPa promoter analysis
in myeloid cells have not been reported.
Whereas the C/EBP proteins are expressed in a number
of different tissues, their expression in the hematopoietic
system may be limited to myeloid cells. It has shown that
C/EBPa is specifically expressed in human myelomonocytic
cell lines and not in human erythroid, B-cell, and T-cell
lines,195 consistent with the myeloid-specific expression previously noted for avian C/EBP.196-199 In studies of murine
32D cells and human leukemic lines, such as HL-60 and
U937 cells, the C/EBP proteins showed a pattern of expression quite different from that observed in adipocytes.195,200
In these studies, C/EBPa was observed to be highly expressed in proliferating myelomonocytic cells upon induction of differentiation and downregulated with maturation,
whereas C/EBPb and C/EBPd were upregulated. However,
recent Northern blot analysis of human primary CD34/ cells
shows that C/EBPa expression is maintained during granulocytic differentiation but is markedly downregulated with
monocytic or erythroid differentiation. In addition, Northern
blot analysis of mature peripheral blood neutrophils shows
very high levels of C/EBPa mRNA, which was completely
undetectable in adherent peripheral blood monocytes (H. Radomska and D.G.T., manuscript submitted). In addition, C/
EBPa was noted to be upregulated as single CD34//CD380
cells were differentiated into granulocytic colonies; no such
upregulation was observed during colony-forming unit-macrophage (CFU-M) colony formation.134 These studies point
to a role of C/EBPa in neutrophilic but not monocytic lineage development.
As noted above, C/EBPa was the first protein noted to
have the leucine zipper motif.176,201 The leucine zipper consists of repetitive leucine residues spaced every 7 amino
acids that results in an a helical amphipathic structure with
hydrophobic and hydrophilic faces on each side of the helix.
Just amino terminal to the zipper is a basic region that is
highly positively charged and directly interacts with DNA.
The leucine zipper is directly involved in homodimerization
and heterodimerization. C/EBPa, C/EBPb, and C/EBPd are
strongly similar in their C terminal basic region and leucine
zipper domains and diverge in the N-terminal transactivation
domain.61,179,184,202,203 Adding to the complexity, other DNA
binding proteins, including CTF/NF-1204 and NF-Y (also
known as CBF and CP1),205-207 can bind to CAAT containing
sequences. C/EBP binding sites consist of two half sites, but
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even the sequence CCAAT was not always required for
binding. Despite the high degree of similarity of the carboxyl
terminal basic-zipper DNA binding domain, C/EBPa and C/
EBPb can bind with vastly different affinities to the same
promoter site.208 The consensus binding site for C/EBPa has
been recently determined using PCR binding site selection.209
In addition to other members of the C/EBP family, the C/
EBP proteins can interact with a number of transcription
factors, including NF-kB and Rel proteins,210,211 members of
the CREB/ATF family,98,178 Sp1,212 RB,213 and members of
the fos/jun zipper family.214 The amino terminal region of
C/EBPa has been shown to physically interact with TBP.61
As noted above, C/EBPd has been shown to interact with
PU.1,142 and PU.1 can physically interact with C/EBPa83 and
C/EBPb as well (P. Auron, unpublished observations). As
noted above, C/EBPa and C/EBPb are most similar in their
carboxyl terminal basic-zipper DNA binding domain and
less so in the amino terminus, perhaps explaining why they
can also show very different abilities to interact with other
proteins.212 Another functionally important interaction of relevance to myeloid gene regulation is that between C/EBPa
and AML1, which is important for M-CSF receptor promoter
function in transfection studies (see below).215 Recently, it
has been shown that the underphosphorylated form of the
retinoblastoma (RB) protein can interact physically with C/
EBP proteins to increase DNA binding and transactivation
of target genes during adipocyte differentiation.216 As RB
becomes underphosphorylated with myeloid differentiation,148,149 it could positively regulate C/EBPa-induced granulocytic maturation.
Recently, lines of mice harboring a knockout of the C/
EBPa gene were generated.217,218 Heterozygous mice are outwardly normal, but homozygous mice die within the first
few hours after birth of impaired glucose metabolism; their
viability can be extended to about 1 day with injections of
glucose. The mice are unable to properly synthesize and
mobilize glycogen and fat. Analysis of the hematopoietic
system in embryonic and newborn mice has demonstrated a
significant defect in production of granulocytic cells.28 Newborn knockout (0/0) animals do not produce any mature
neutrophils, which comprise 90% of the peripheral blood
white blood cells of newborn heterozygote (//0) or wildtype (///) mice. Cells that appear to be immature myeloid
cells are observed in the peripheral blood. These cells stain
with Sudan black, a characteristic of myeloid cells, and have
increased myeloid CFU potential compared with those from
heterozygote blood. Interestingly, many cells in the CFU
from the knockout blood are immature in morphology as
well, similar to what is observed in CFU from leukemic
cells. Eosinophils are also not observed, but all of the other
lineages, including peripheral blood monocytes and peritoneal macrophages, red blood cells, platelets, and lymphoid
cells, appear quantitatively unaffected. Fluorescence-activated cell sorting (FACS) analysis in embryonic and newborn animals confirmed that myeloid markers (Mac-1 and
Gr-1) were greatly reduced, with normal B- and T-cell subsets. Expression of the G-CSF receptor mRNA was profoundly and selectively reduced, whereas that of M-CSF
receptor and GM-CSF receptor a, bc, and bIL3 were all com-
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TRANSCRIPTION AND MYELOID DEVELOPMENT
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parable to wild-type. Interestingly, a fourfold increase in
mRNA for Epo receptor was detected, although whether
this is due to a negative effect of C/EBPa on Epo receptor
expression in precursor cells or alternatively a reflection of
an increased number of erythroid cells in C/EBPa 0/0 livers
has not been determined. Compared with wild-type animals,
maturation of immature myeloid cells in vivo in response to
G-CSF was not observed, as assessed by both morphology
and FACS analysis of Gr-1 expression. In addition, no colony-forming units-granulocyte (CFU-G) could be obtained
using C/EBPa 0/0 newborn liver, although other types of
colonies (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM], colony-forming uniterythroid [CFU-E], colony-forming unit–granulocyte-macrophage [CFU-GM], and colony-forming unit-macrophage
[CFU-M]) were quantitatively equal to wild-type animals.
These findings suggested that much of the phenotype may be
due to decreased or absent G-CSF signaling due to markedly
reduced receptor levels. However, G-CSF receptor knockout
animals produce mature granulocytes, in contrast to C/EBPa
knockouts, strongly suggesting that there must be important
C/EBPa target genes in myeloid progenitors in addition to
the G-CSF receptor.219 Transplantation of C/EBPa fetal liver
into sublethally irradiated animals resulted in detection of T
cells but not myeloid cells of donor origin, showing that the
defect is indeed in the hematopoietic cells.28
A number of studies have pointed to the role of C/EBPb
in myeloid cells. The avian homolog of C/EBPb (NF-M) is
a myeloid-specific factor that activates the promoter for
cMGF, which is related to G-CSF and IL-6.196-199 In mammalian cells, C/EBPb has been described as having low activity
until activated by LPS, IL-1, IL-6, and other inflammatory
mediators, which induce its translocation to the nucleus,
where it can activate cytokine genes such as IL-6 and GCSF.185,220 Changes in C/EBPb phosphorylation (but not in
C/EBPa) have also been described during myeloid differentiation of a progenitor cell line.160 Human C/EBPb is also
known as NF-IL6.185 C/EBPb can also be activated in other
cell types by phosphorylation without translocation.221 In one
report, targeted disruption of C/EBPb leads to defects in
killing of Listeria and tumor cells by macrophages.222 In a
second study, disruption of C/EBPb led to an increase in
IL-6 production (previously it was thought that C/EBPb, or
NF-IL6, was essential for IL-6 production) and hyperproliferation of a number of different hematopoietic lineages,
a syndrome resembling Castleman’s disease.223 Knockout
animals from both of these studies can get this syndrome as
the animals get older, but it is not seen in young mice kept
in a sterile environment.224 In these younger mice, in the
absence of inflammation, myelopoiesis was apparently not
adversely affected, consistent with a role of C/EBPb in cytokine activation rather than lineage development.
As noted above, C/EBPb (NF-IL6) regulates a number
of cytokine genes and may contribute to normal myeloid
development as well as activation of mature myeloid cells
in response to an inflammatory stimulus. In particular, targeting of C/EBPb resulted in a reduction of G-CSF production in macrophages but not other cell types.222 C/EBPa has
been noted to regulate the promoters for several myeloid
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granule proteins, including myeloperoxidase and neutrophil
elastase.87,88,160 In addition, C/EBP sites, like PU.1 sites, are
critical for the activity of a number of myeloid CSF receptor
promoters, including the M-CSF receptor,117 the GM-CSF
receptor a,54 and the G-CSF receptor promoter.53 In the C/
EBPa knockout mice, G-CSF receptor mRNA was selectively and significantly reduced.28 Interestingly, no reduction
was noted in the levels of M-CSF receptor and GM-CSF
receptor (a and b chain) mRNA, suggesting that perhaps
other factors (such as C/EBPb) can compensate for C/EBPa
in some but not all instances.
With respect to the possible role of C/EBP proteins in
leukemia, the C/EBP proteins have not been implicated as
transforming proteins, with the exception of CHOP10.190,225
Indeed, C/EBPa has been shown to inhibit cell proliferation
in fibrosarcoma lines through the cyclin-dependent kinase
inhibitor p21183 and can act as a tumor suppressor of hepatoma lines and other cell types.181,182 The human C/EBPa
gene maps to chromosome 19q13.1 and C/EBPb maps to
20q13.1.226 Neither of these is a frequent site of chromosomal
translocations found in human myeloid leukemia. Studies of
C/EBP expression in primary human leukemias have not
been reported. However, given the phenotype of the C/EBPa
knockout mouse, in which there appears to be a block in
granulocyte maturation, it will be of interest to look for
abnormalities of C/EBP expression or mutations in myeloid
leukemias, which also represent states with a block in maturation.
In summary, the C/EBP proteins are differentially expressed and can form many interactions with themselves and
other transcription factors. These studies show that C/EBPb
(and C/EBPd) may be involved in activation of cytokines in
mature cells, whereas C/EBPa has a major role in granulocytic maturation through regulation of the G-CSF receptor
and other as yet not identified target genes.
Other factors binding to CCAAT sites and myeloid development. In addition to the C/EBP proteins, the CCAAT
displacement protein (CDP) has also been implicated in myeloid development. This protein was observed to negatively
regulate genes by binding to a CCAAT box and displacing
the binding of positively acting factors.227 It was subsequently shown to be capable of binding to the gp91phox
promoter and repressing this cytochrome component gene
expressed specifically in myeloid cells.228,229 At this time
no characterization of the positively acting factors that are
presumed to be displaced at this site has been made. CDP
is expressed at highest levels in undifferentiated myeloid cell
lines and is downregulated with differentiation, providing a
mechanism for the upregulation of gp91phox observed with
myeloid maturation. Importantly, this is one of the few examples explaining the potential mechanism of upregulation of
myeloid targets with myeloid differentiation. In most other
cases, the mechanism of upregulation is largely unknown.
Recently, it has been shown that CDP can also repress the
lactoferrin promoter.230 In these studies, overexpression of
CDP in myeloid stem cells blocked lactoferrin expression
after G-CSF–induced neutrophil maturation without
blocking phenotypic maturation. Furthermore, these investigators observed coordinate repression of multiple secondary
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granule protein genes, including lactoferrin, neutrophil collagenase, and neutrophil gelatinase, suggesting that CDP repression is a common mode of regulation of this class of
myeloid promoters (N. Berliner, unpublished observations).
Other factors might also act in a similar manner to repress
positive activators during differentiation. For example, a factor that is downregulated during adipocyte differentiation
and inhibits C/EBPa function, presumably through its similarity to carboxypeptidase, has recently been described,231
but it is not known if it inhibits C/EBPa in hematopoietic
cells.
AML1 AND THE CBF FAMILY
An example of a transcription factor that plays an important role in normal monocytic gene expression and in
acute myelogenous leukemia is AML1. AML1 is a member
of the core binding factor (CBF) or polyoma enhancer binding protein 2 (PEBP2) family of transcription factors. The
CBF factors consist of heterodimers between DNA binding
a subunits and a b subunit (CBFb) that does not bind DNA
directly but that enhances the binding of the a subunit.232,233
Multiple a subunit genes, including CBFA1, AML1
(CBFA2), and CBFA3, have been detected,233-237 although
the role of these other AML1 family members in myeloid
development and leukemia is not clear. In addition, alternatively spliced isoforms of the a and b subunits have been
detected. In particular, a short form and two long forms of
AML1 have been described, and it is the long form that
appears to include the transactivation function.235-238 All of
the CBFa proteins contain a runt domain, which is similar
to the Drosophila pair-rule gene, runt, an early acting segmentation protein that regulates the expression of other segmentation genes.239,240 This runt homology domain (rhd) confers the ability of AML1 to bind to its consensus sequence,
TGT/cGGT, and the ability of AML1 to interact with its
cofactor CBFb, which enhances the DNA binding ability.232,233,241
AML1, normally expressed in hematopoietic tissues and
during myeloid differentiation,134,237,242 is also expressed in
nervous tissue, skeletal muscle, and reproductive tissues as
well.243 Knockout studies of AML1 have determined that
AML1 is necessary for normal development of all hematopoietic lineages,49,50 and similar effects have been observed
with targeted disruption of the CBFb subunit.244 In these
studies, primitive hematopoiesis was apparently normal, but
severe impairment of definitive hematopoiesis was observed
both in the animals and during in vitro differentiation of
0/0 ES cells, with defects observed in all lineages examined.
AML1 may act to facilitate the action of other adjacent
transcription factors. The AML1 site in the M-CSF receptor
promoter is located between the C/EBPa and PU.1 sites,
two transcription factors that are important for myeloid gene
expression.52,215,245 Significantly, although the C/EBP site is
critical for M-CSF receptor activity in transfection experiments,52 C/EBP alone does not transactivate M-CSF receptor
promoter activity. The adjacent factor AML1, along with
CBFb, can by itself activate the M-CSF receptor promoter
sixfold, but the synergistic activation by C/EBP added to
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AML1 and CBFb is much greater (ú60-fold). All C/EBP
proteins share a highly similar bZIP domain,179,184 and AML1
interacts with the bZIP fragment of C/EBP, explaining why
C/EBPa, C/EBPb, and C/EBPd can all synergistically activate the M-CSF receptor promoter with AML1. The physical
interaction between C/EBP and AML1 could either stabilize
binding of both to the M-CSF receptor, or their interaction
might provide a surface for another transcription factor to
bind and activate the M-CSF receptor promoter. Other examples of interactions of AML1 and other transcription factors
include synergism with Ets-1 and c-myb to activate T-cell
receptor enhancers.246-249 Because Ets factors, such as PU.1,
and c-myb both play an important role in myeloid development, it is likely that these interactions with AML1 will be
important in myeloid gene expression as well.
AML1 can be phosphorylated at two serine residues
within a proline-, serine-, and threonine-rich region in its
carboxyl terminal domain after activation of the extracellular
signal regulated kinase (ERK1).250 These serine residues are
critical for AML1-dependent transformation of fibroblast cell
lines and ERK1-dependent transactivation of a T-cell receptor enhancer target gene. IL-3 treatment of the hematopoietic
cell line BaF3 results in phosphorylation of AML1, suggesting that this signalling pathway may play an important
role during hematopoietic development.
Role of AML1 in leukemogenesis. AML1 was identified
by studying one of the most frequent chromosomal translocation found in AML, t(8;21)(q22;q22).17,39,241,251,252 AML1 fusion proteins are also involved in other forms of leukemia,
including t(3;21) therapy-related acute myeloid leukemia/
myelodysplasia or chronic myelogenous leukemia in blast
crisis (AML1/MDS1, AML1/EAP, and AML1/Evi-1) and
also the t(12;21) in childhood B-cell acute lymphoblastic
leukemias (TEL/AML1).17,172,251,253-256 The b subunit of CBF,
CBFb, is also involved in a chromosomal inversion,
inv(16)(p13;q22), associated with French-American-British
(FAB) M4eo AML.257 Therefore, each of the two chains of
the CBF heterodimer is directly implicated in the pathogenesis of AML.
As noted above, AML1 is involved in the (8;21) translocation, associated with the FAB M2 form of AML, which
results in the production of the AML1/ETO fusion protein.
The mechanism by which AML1/ETO accomplishes leukemic transformation is unknown. In this case, the 5* part of
the AML1 gene, including the amino terminal DNA binding
runt domain but lacking the carboxyl terminal activation
domain, is fused to almost the entire ETO gene on chromosome 8. Recent studies have indicated that this AML1/ETO
fusion protein can act as a dominant negative inhibitor of
AML1 transactivation of such AML1 targets as the T-cell
receptor b (TCRb) enhancer.236,238,258 It has recently been
reported that the t(12;21) fusion protein, TEL/AML1, also
causes the same interference with AML1 transactivation of
the TCRb enhancer.259 Therefore, the expression of some of
the normal AML1 gene targets that play a key role in monocytic differentiation might be adversely affected by AML1/
ETO, and these include GM-CSF,236,258 IL-3,260,261 M-CSF
receptor,52 and myeloperoxidase and neutrophil elastase.87,262
Recently, it was shown that AML1/ETO and AML1 work
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synergistically to transactivate the M-CSF receptor promoter,
thus exhibiting a different activity than previously described.263
This indicates that AML1/ETO not only can repress, but also
can enhance AML1 transactivation of its target genes. Endogenous M-CSF receptor expression was examined in Kasumi-1
cells, derived from a patient with AML-M2 t(8;21) and the
promonocytic cell line, U937. Kasumi-1 cells exhibited a significantly higher level of M-CSF receptor expression than U937
cells. Bone marrow from patients with AML-M2 t(8;21) also
exhibited a higher level of expression of M-CSF receptor compared with normal controls.263 Signaling through the M-CSF
receptor promotes cell proliferation by inducing the G1/S transition because of the effect on cyclins D and E.264,265 This effect
on proliferation can be tied to the oncogenic nature of the
M-CSF receptor, which was first discovered as the cellular
counterpart to the transforming oncogene, v-fms.266 Overexpression of the M-CSF receptor causes transformation of
NIH3T3 cells and in mice leads to a myeloblastic leukemia.267,268 A premature upregulation of M-CSF receptor expression by AML1/ETO could be one of the factors contributing
to leukemogenesis AML.
To study how the fusion protein produced by the t(8:21)
translocation leads to the development of myeloid leukemia,
knock-in mice have been generated that express the AML1ETO fusion protein. Mice heterozygous for the AML1-ETO
allele die in midgestation with hemorrhaging in the central and
peripheral nervous system and exhibit a severe block in fetal
liver hematopoiesis.269 This phenotype is very similar to that
resulting from homozygous disruption of the cbfa2 (AML1)
and cbfb genes.49,50,244 However, significant differences between
AML1-ETO knock-in and cbfa2 0/0 or cbfb 0/0 mice were
observed in hematopoietic CFU assays. Yolk sac from AML1ETO knock-in mice generated solely homogenous macrophage
colonies. The total number of macrophage colonies is similar
to the sum of all type of colonies from yolk sacs of wild-type
mice. In contrast, no colonies from cbfa2 0/0 or cbfb 0/0
mice could be detected. These results, like those noted above
in which the AML1-ETO fusion protein activates the M-CSF
receptor promoter, indicate that the AML1-ETO fusion protein
affects hematopoiesis in a manner that is not simply a block
of wild-type AML1 function, and these effects could be important in leukemogenesis. Mice heterozygous for a knock-in
of the cbfb-myh11 allele, which mimics the inv(16)(p13;q22)
associated with AML-M4Eo form of AML, also die in midgestation from the same pattern of central nervous system hemorrhaging and impairment of fetal liver hematopoiesis. However,
instead of preferential macrophage differentiation from early
progenitor cells, these embryos exhibit a significant delay in
maturation of yolk sac-derived primitive erythrocytes.270 Although t(8:21) and inv(16) disrupt genes encoding different
subunits of a single transcription factor complex, the fusion
genes have different effects on hematopoiesis, consistent with
their association with distinct AML subtypes.
RARa: MYELOID DIFFERENTIATION AND ACUTE
PROMYELOCYTIC LEUKEMIA (APL)
Several lines of evidence indicate a key role for the retinoic
acid receptor (RAR) in myeloid differentiation. Vitamin A
deficiency is associated with defective hematopoiesis,271 and
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retinoids stimulate granulopoiesis.272,273 Retinoic acid (RA) can
induce differentiation of the HL-60 cell line274 and of primary
leukemic cells.275 There are three genes encoding RARs;
RARa, RARb, and RARg (reviewed previously276-278). RARa
is preferentially expressed in myeloid tissue.279-281 RARa binds
to retinoic acid response elements consisting of a direct repeat
(A/G)G(G/T)TCA separated by 2 or 5 nucleotides as a heterodimer with the retinoid X receptor RXR282-284 and stimulates
transcription in response to all-trans retinoic acid (ATRA).285
RARa was mutated in a retinoid-resistant HL-60 cell line
with this deficit corrected by re-expression of wild-type receptor.286-288 Furthermore, introduction of a dominant negative
form of RARa into a multipotent hematopoietic cell line
changed the fate of these cells from the granulocyte/monocyte
lineage to the mast cell line.289 In addition, GM-CSF–mediated
myeloid differentiation of these cells was blocked at the promyelocyte stage.45 This basic information was complimented
by clinical data indicating that patients with APL could be
induced into complete remission with ATRA (reviewed previously19,290). It was found that APL associated with t(15:17)
disrupted the RARa linking it to the PML gene, yielding the
fusion protein PML-RARa.291-296 PML-RARa is an aberrant
retinoid receptor with altered DNA binding and transcriptional
activities.292,294,296-298 PML-RARa can block the effect of wildtype retinoic acid receptor on many promoters in a dominant
negative manner. Two other APL-associated rearrangements
of the RARa gene were identified: translocation (11;17)40,299
fusing the PLZF gene to RARa and t(5;17)300 linking the
nucleophosmin (NPM) gene to RARa. All three situations
yield similar chimeric RARa proteins, yet only t(15;17) patients clearly respond to ATRA therapy.301,302 Expression of
the PML-RARa fusion protein in myeloid cells inhibits differentiation in the presence of low levels of ATRA303,304 and
actually accelerates differentiation in the presence of pharmacologic doses of RA.304 Expression of PML-RARa in
transgenic mice impairs myelopoiesis,113 and work from another group indicated that PML-RARa could induce altered
myeloid development, including increased immature and mature myeloid cells, as well as a leukemic syndrome that is
ATRA responsive.115 Another group has recently described
similar results in PML-RARa transgenic animals using another
promoter.109 Together, the experimental and clinical data indicate that RARa function is required to fully proceed through
myeloid differentiation. Disruption of RARa function likely
selects for the promyelocyte phenotype. However, the nature
of the fusion partner and its molecular mechanisms of action
apparently play a role in the ability of the leukemic cells to
differentiate in response to ATRA.
ATRA treatment drives the expression of multiple myeloid genes, including those for cell surface adhesion molecules, intrinsic host defense systems, extrinsic cytokines, colony-stimulating factor receptors, structural proteins, and
enzymes. Some of these are induced with some delay after
ATRA treatment, making them unlikely primary targets of
RARa. The few identified direct targets of RARa tend to
themselves be transcription factors, including the RARs
themselves.284 ATRA treatment of fresh APL cells upregulates RARa, correlating with the presence of a RARE in
RARa promoter.305,306 Therefore, one way that ATRA may
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induce myeloid differentiation may be to upregulate the
RARa gene, overcoming the dominant negative PML-RARa
protein. A recently identified direct target of RAR in myeloid
cells is the cyclin-dependent kinase inhibitor p21.307 Another
tantalizing set of targets for RARa in myeloid differentiation
includes hox family of homeobox-containing transcription
factors, which are expressed in myeloid cell lines in a coordinated, dynamic manner.32,33,308-313 In embryonic carcinoma
cells homozygous for a targeted disruption in the RARa
gene, ATRA treatment fails to induce the hoxb1 and CRABPII expression, indicating that these are true direct targets
of RARa.284,314 Identification of further direct target genes
of RARa during myeloid differentiation will require use of
techniques such as differential screening315 or differential
display316 of myeloid cells treated for brief periods with
ATRA.
Finally, although the RARa locus and all of the other
nuclear receptors have been disrupted by gene targeting in
mice (reviewed previously317), no major defects in hematopoiesis were described in these animals.318-320 This may reflect the ability of the receptors to compensate for each other
in a redundant manner. This is analogous to the way alternative forms of the RAR could rescue mutated HL-60 cells with
a RARa mutation.288 Multiple matings of animals deficient in
the various RAR, RXR, and other nonsteroid nuclear receptors may yield further insights as to the exact requirements
for this class of transcription factor in hematopoiesis.
THE ZINC FINGER FAMILY AND MYELOID CELLS
Transcription factors belonging to the zinc finger family
are among the most numerous in vertebrate organisms. These
proteins contain cysteine and histidine residues that coordinately bind zinc ions to form a protein loop capable of specifically interacting with DNA.321 The Drosophila Krüppelrelated zinc finger proteins are distinct from other Cys/His
finger proteins because they contain a relatively invariant
amino acid sequence linking the zinc fingers of the form
HTGEKP(F/Y)XC. This H/C link is present in a number of
zinc finger transcription factor proteins of higher eukaryotes,
including Sp1,322 the EGR family of transcriptional activators,323 and the WT-1 tumor suppressor protein.324,325 Myeloid-specific zinc finger proteins were identified by their
similarity to the Krüppel sequence (MZF-1),30 by subtraction
cloning from myeloid cells (Egr-1),25 by their involvement
in chromosomal translocation involved in APL (PLZF),299,302
and by their involvement in apoptosis associated with myeloid differentiation (Requiem).326 In addition, a zinc finger
gene not normally expressed in myeloid cells was isolated
by its activation in murine models of leukemia by retroviral
insertion (Evi-1)327 and has subsequently shown to be involved in human leukemia when fused to the AML1
gene.17,328,329 Interestingly, relatively few of these proteins
were identified by traditional promoter analysis, with the
notable exception of Sp1. On the contrary, these zinc finger
proteins are in general factors in search of molecular targets
and mechanisms in myeloid differentiation.
The PLZF gene. The promyelocytic leukemia zinc finger
(PLZF) gene, potentially important for myeloid development, was identified by its rearrangement in APL with a
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variant translocation t(11;17) (q23;q21).40,299,330 The t(11;17)
APL patients were generally resistant to both chemotherapy
and differentiation therapy with ATRA,302 suggesting that
the nature of the fusion partner with RARa may determine
the clinical course of APL. PLZF on chromosome 11 encodes a zinc finger transcription factor,299 whereas the partner
protein with RARa in t(15;17) APL is PML, a RING finger
protein of unknown function (reviewed previously19). The
t(11;17) fusion yields two reciprocal transcripts expressed
in all patients tested: PLZF-RARa, predicted to encode an
aberrant retinoid receptor, and RARa-PLZF, fusing the Aactivation domain of RARa to the last 7 zinc finger motifs
of PLZF.
In the hematopoietic system, PLZF is expressed in myeloid but not in lymphoid cell lines.299 Upon treatment of
NB4 or HL-60 cells with retinoic acid, PLZF levels decline
(Chen et al299 and A. Chen, S. Waxman, and J. Licht, unpublished data). The murine homologue of PLZF was found to
be expressed at highest levels in multipotent progenitor cell
lines such as FDCP-MixA4 and at lower levels in committed
cell lines.331 When the multipotent FDCP-MixA4 cell line
was induced to differentiate, PLZF levels decreased. Murine
PLZF mRNA was also detectable in pro-B–cell lines but
not in more mature B- or T-cell lines.331 PLZF was not
expressed in undifferentiated embryonic stem cells but was
upregulated as ES cells were induced to differentiate into
embryoid bodies. In the developing mouse embryo, PLZF
is expressed in the aorta, gonadal, and mesonephros region
(AGM), a region thought to give rise to hematopoietic stem
cells. Finally, CD34/ human progenitor cells could be immunostained with PLZF antisera in a distinct nuclear speckled
pattern.331 Together, this information suggests that PLZF
may be a marker of and a critical gene for the early hematopoietic stem cell. Downregulation of PLZF may be a necessary step for the committed division and differentiation that
accompanies normal bone marrow maturation.
By immunoprecipitation of transfected cells, the PLZF
protein was identified as an 81-kD nuclear species331,332 phosphorylated on serine and threonine but not tyrosine residues
(R. Shaknovich and J. Licht, manuscript in preparation). A
biphenotypic T-cell/macrophage line, presumably representing a primitive transformed cell, expressed high levels of
PLZF mRNA and 81-kD PLZF protein when treated with the
calcium ionophore A23187, correlating with the induction of
a more monocytoid phenotype. Immunofluorescent confocal
microscopy showed that PLZF was localized to approximately 50 small nuclear subdomains, whereas the PML protein of t(15;17) APL was localized in approximately 5 to 10
larger nuclear structures (nuclear bodies or PODs333-335) and
was present before and after A23187 treatment. It is uncertain if in other cell types these two APL-associated proteins
might be localized to the same nuclear domains.
PLZF is a sequence-specific DNA binding protein recognizing a sequence with a core of TAAAT.336 This site was
cloned upstream of a reporter gene and was found to mediate
transcriptional repression by PLZF. At least one repression
domain within PLZF maps to an evolutionarily conserved
sequence known as the POZ (poxvirus-zinc finger) or BTB
(broad complex-tramtrack-bric-a-brac) domain.337,338 This
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sequence also mediates self-association by PLZF and is required for the localization of PLZF into a speckled subnuclear pattern.332,339 Whether the domains that mediate homodimerization and transcriptional repression are identical
is not known. The last 7 zinc fingers of PLZF that are retained in the RARa-PLZF fusion protein can bind to the
same site as the 9 zinc fingers of PLZF, suggesting that the
RARa-PLZF protein could act as a dominant negative protein binding and altering transcription of PLZF target genes.
The PLZF-RARa fusion protein has properties quite similar to the PML-RARa fusion protein of t(15;17)-associated
APL. Both fusion proteins can bind as homodimers to a
retinoic acid response element (RARE).297,332,339 Homodimerization by PML is mediated by a coiled-coil motif,297
whereas in PLZF the POZ domain mediates self-association.339 In an electrophoretic mobility shift assay (EMSA)
both PML-RARa and PLZF-RARa homodimers are chased
into novel complexes of higher and lower electrophoretic
mobility by addition of RXRa. This suggests that both fusion
proteins may work in a similar manner, forming multimeric
complexes in the cell that could act to sequester limiting
amounts of RXRa, the essential cofactor for RARa function,
in the cell. Both PML-RARa and PLZF-RARa can act in a
dominant negative manner to inhibit the activity of wildtype RARa294,296,298 and other nuclear receptors such as the
vitamin D3 receptor (J. Licht and M. English, unpublished
data).297 This effect can be partially relieved by overexpression of RXRa, consistent with the notion that the aberrant receptors generated in APL block myeloid differentiation at least in part through limiting the ability of RARa to
bind to its targets in combination with RXR. Dominant negative forms of RARa can block myeloid differentiation at the
promyelocyte stage.45,289,340 This correlated with the clinical
finding that RARa is rearranged in three translocations,
t(15;17), t(11;17), and t(5;17), all yielding APL.300,341 Thus,
the block in ATRA-mediated signaling may help select the
APL phenotype but may not necessarily determine whether
the disease is ATRA-responsive. Further studies of the
growth-inhibiting properties of PLZF suggest a reason why
t(11;17) APL may not respond to therapy.
Pools of murine myeloid 32DCL3 cells transduced with a
retrovirus to overexpress the PLZF protein were significantly
growth inhibited when grown in IL-3 or GM-CSF and retarded in the G1 phase of the cell cycle. In addition, the
cells exhibited a higher rate of programmed cell death and
a reduced ability to differentiate in response to G-CSF or
GM-CSF.342 The high level of expression of PLZF in the
hematopoietic precursor cell may play a role in the relative
quiescence of these cells. Downregulation of PLZF during
myeloid differentiation may be accompanied by cycles of
committed cell division. In this way, PLZF does seem similar
to PML,343-345 because both proteins can repress cell growth.
However, t(11;17) generates RARa-PLZF, a protein that
can potentially interfere with the normal transcriptional functions of PLZF by binding to and dysregulating PLZF target
genes. In contrast, the RAR-PML transcript is variably found
in t(15;17) APL19 and may not be able to affect the function
of wild-type PML. Consistent with a central role for PLZF
disruption in t(11;17) APL phenotype, RARa-PLZF may
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confer accelerated growth to 32DCL3 cells. Hence, t(11;17)
may be a resistant disease due to the presence of two oncogenes working through different mechanisms, PLZF-RARa
and RARa-PLZF. It remains to be determined what genes
are targeted by PLZF and how these play a role in the growth
and differentiation of myeloid tissue.
The myeloid zinc finger protein MZF-1. MZF-1 has multiple (13) zinc finger domains, divided in two groups.346 The
amino-terminal group has 4 fingers, and the carboxyl-terminal group has 9 fingers. There are acidic regions in the
amino-terminal nonzinc finger portion of the protein that
may be part of the transcriptional regulatory domain. Interestingly, there is a 24 amino acid glycine-proline–rich link
between the first and second zinc finger regions. Glycineproline–rich domains have also been implicated in transcriptional regulation in a large number of transcription factors,
including Oct-3, Rex-1, and CP-1.346
By Northern analysis, MZF-1 was found to be specifically
expressed in myeloid cell lines.346 By cRNA in situ hybridization of normal marrow, MZF-1 was found to be expressed
in myeloid cells,347 from myeloblasts to metamyelocytes, but
in no other marrow cell types, implying a role for MZF-1
in myeloid development. Using in situ chromosomal hybridization, MZF-1 was localized to human chromosome
19q13.4.346 It is the telomeric-most gene on 19q, with its
promoter region being less than 10 kb from the telomeric
repeats.348 The finding that MZF-1 is located at the telomere
of chromosome 19q is significant because there is evidence
that aging hematopoietic stem cells lose telomeric size with
time.349 In addition, telomeres shorten with transformation
of myelodysplasia to leukemia.350 With age, the incidence
of stem cell disorders such as myelodysplasia and leukemia
increases markedly. It is possible that the regulatory region
of MZF-1 or the transcribed sequence is disrupted with age,
which could play a role in the increased incidence of hematopoietic stem cell disorders.
The consensus DNA binding sites of MZF-1 were isolated
by mobility shift selection of degenerate oligonucleotides
and PCR amplification.351 The first finger domain, zinc fingers 1-4 (ZF1-4), bound to a consensus sequence of 5*AGTGGGGA-3*, whereas zinc fingers 5-13 (ZF5-13) bound
to a consensus of 5*-CGGGnGAGGGGGAA-3*. These guanine-rich sites resemble NF-kB binding sites. The full-length
recombinant MZF-1 could bind to either sequence. Indeed,
the two MZF-1 finger domains, ZF1-4 and ZF5-13, could
bind to each other’s consensus binding sequence. In addition,
it was discovered that ZF5-13 probably needs all 9 fingers
to bind to its consensus sequence. Deletion of as few as 3
fingers from either end of ZF5-13 abrogates DNA binding.351
Computer searches of data bases for promoter sequences
that contained these consensus MZF-1 binding sites found
several possible matches. The promoters of the myeloid
genes CD34, myeloperoxidase, c-myb, and lactoferrin all
contained consensus MZF-1 binding sites. An intriguing
finding about MZF-1 was its bifunctional transcriptional regulatory activity. In the intracellular environment of adherent,
nonhematopoietic cells it functions as a transcriptional repressor, and in hematopoietic cells it functions as an activator.352
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MZF was found to repress the CD34 promoter and the cmyb promoter in nonhematopoietic cells.352,353 However, in
one study using the hematopoietic cell lines K562 and Jurkat,
MZF-1 activated transcription from the CD34 promoter.352
In another study, using different techniques, it was found
that MZF-1 repressed the CD34 promoter in KG-1 cells.353
The presence of MZF-1 binding sites in several myeloid
promoters and the explicit regulation of the CD34 promoter
by MZF-1 point out that MZF-1 may have a general role in
the regulation of hematopoietic gene expression. Lending
evidence to the hypothesis that MZF-1 may be an important
regulator of hematopoietic development are studies showing
that blocking MZF-1 expression with antisense oligonucleotides inhibits granulopoiesis.347
MZF-1 can also disrupt the normal proliferative behavior
of embryonic fibroblasts. When MZF-1 was retrovirally
transduced and overexpressed in NIH 3T3 cells, where it is
not normally expressed, it rapidly transformed those cells.354
However, two other myeloid transcriptional regulators, PU.1
and PRH, did not transform 3T3 cells when they were similarly overexpressed. In addition, forced overexpression of
MZF-1 in IL-3–dependent FDCP1 or HL-60 cells decreased
apoptosis induced by IL-3 withdrawal or retinoic acid, respectively.355,356 FDCP1 cells transduced with MZF-1 formed
tumors in 70% of congenic hosts, whereas control cells did
not. These data imply that the aberrant expression of nodal
regulators of development can be neoplastic. It lends evidence for the hypothesis that malignancy represents normal
development gone awry.
The MZF-1 promoter was recently isolated.357 There are
MZF-1, PU.1, and retinoic acid receptor binding sites within
the MZF-1 promoter.
The early response gene Egr-1. Many groups have isolated Egr-1 (NFGI-A) as an early response gene induced by
a variety of stimuli, primarily those that induce cell proliferation.358 Egr-1 was also cloned as an early response gene
during TPA treatment of human myeloid cell lines and was
noted to be upregulated during monocytic and not granulocytic differentiation of these bipotential lines.25 Overexpression and antisense studies implicated Egr-1 as an inducer
of monocytic differentiation.25,359 An Egr-1 knockout mouse
line has been made, but no abnormalities of monocytic differentiation or function were detected.360 In this mouse
model, it is possible that other members of the Egr-1 family
can compensate for loss of the Egr-1 gene.
The Wilms’ tumor suppressor gene WT-1. An enlarging
body of literature suggests a role for the WT1 Wilms’ tumor
suppressor gene in normal and leukemic myeloid development. WT1 encodes a zinc finger transcription factor that
binds to a GC-rich sequence similar to that of the Egr-1
protein (reviewed previously361).362 WT1 can both activate
and repress transcription and in model systems inhibits cell
growth363-365 and can induce programmed cell death.361,366
WT1 is expressed in normal bone marrow, particularly in
CD34/ progenitor cells.367,368 CD43//CD330/lin0 cells, believed to be very early progenitors, express the highest levels
of WT1, whereas the CD33/ subset of CD34/ progenitors,
reflecting a later point in differentiation, express 100-fold
less WT1. WT1 is also expressed in the murine embryonic
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yolk sac and liver during definitive hematopoiesis.368 WT1
is expressed in a number of myeloid cell lines, and WT1
expression is downregulated in HL-60 and K562 cells at the
posttranscriptional level during differentiation of these cell
lines.369,370 Given that WT1 can compete for the binding site
of Egr-1 and repress transcription and that Egr-1 is an activator that promotes monocytic differentiation,25,359 it is possible
that the interplay between these two factors, which have
opposing transcriptional effects, may play a role in the tight
control of monocytic differentiation. The hypothesized role
of WT1 in hematopoiesis must be tempered by the results
of Kriedberg et al,371 who performed a targeted disruption
of the WT1 gene in the mouse. These animals exhibited
complete kidney and genitourinary agenesis, defects in the
mesothelium, and cardiac defects, but to date have demonstrated no hematopoietic deficits (J. Kreidberg, personal
communication, June 1996).
WT1 mRNA expression has been detected in up to 80%
of cases of acute myelogenous leukemia.372,373 Whereas WT1
can be found in the marrow of normal subjects, expression
in the peripheral blood is distinctly abnormal and a sign of
the presence of leukemic cells.367 In addition, there was
found to be a negative correlation between WT1 expression
and prognosis.367 Reverse transcription-PCR (RT-PCR)
assay for WT1 expression can detect minimal residual leukemic blast cells in leukemia patients and may be more sensitive than the detection of fusion gene products such as the
PML-RARa fusion found in APL.367,374 There is an inherent
paradox in these findings. WT1 is believed to be a classical
tumor-suppressor gene, and yet it is expressed in high levels
in actively proliferating leukemia. This expression may reflect the stage of differentiation of the malignant cells at the
point of transformation. Alternatively, WT1 may have an
oncogenic effect when overexpressed in hematopoietic tissue. Recent data indicate that WT1 expression may be important for leukemic cell growth. When myeloid leukemic
cell lines were treated with antisense WT1 oligonucleotides,
cellular proliferation was inhibited and programmed cell
death was induced.375,376 In addition, fresh human leukemia
sample growth was inhibited by these antisense WT1 oligonucleotides.376 Mutation of WT1 may also play a role in
leukemogenesis. Initially, a mutation was detected in the
remaining WT1 allele of a WAGR patient who had been
previously treated for Wilms’ tumor.377 This mutation affected a cysteine residue in the zinc finger region and would
be predicted to abolish DNA binding by WT1. This suggested that complete loss of WT1 function in the myeloid
lineage might be associated with the development of leukemia. More recent data from this same group indicate that
15% of cases of AML have point mutations of WT1.320 These
were heterozygous mutations leading to the formation of
truncated forms of WT1 devoid of zinc finger DNA-binding
motifs. Such truncated proteins may act in a dominant negative manner to inhibit the transcriptional effects of wild-type
WT1.378-380 This again suggests that WT1 may play a role
in the control of leukemic cell growth.
The mechanism of action of WT1 in affecting myeloid
growth is not defined, but one important candidate target
gene is c-myb, which contains an Egr-1 site within its pro-
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TRANSCRIPTION AND MYELOID DEVELOPMENT
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moter. This site acts as a negative regulatory element. WT1
binds to this site in vitro and can repress the c-myb promoter.381 This is intriguing given that forced expression of
c-myb blocks myeloid differentiation, and treatment of myeloid cells with antisense c-myb oligonucleotides blocks proliferation.42,382,383 WT1 could therefore affect myeloid growth
indirectly through action on c-myb.
HOMEOBOX PROTEINS
The homeobox genes are a group of proteins defined by
a specific helix-turn-helix DNA binding motif, the homeo
domain.313,384,385 These regulators have been shown by mutational and knockout studies to play a major role in segmental
development of many species, from Drosophila to
mouse.308,386 The genomic organization of these genes in
mammals, including humans, is arranged in four clusters that
are expressed in a temporal fashion related to their physical
position in the cluster. Recently, a number of exciting studies
have shown that this class of proteins may indeed influence
myeloid development.308,313 A number of studies have investigated the hematopoietic expression of homeobox genes,
including ones expressed in stem cells.32 One of these,
HoxA10, was noted to be expressed specifically in myeloid
cells.309 Subsequent studies showed that HoxA10 mRNA
was expressed at higher levels in human CD34/ cells than
other homeobox genes and downregulated during myeloid
development.32,33 Expression could not be detected in mature
neutrophils and monocytes. These studies suggest that
HoxA10 is upregulated and then downregulated as stem cells
differentiate into mature myeloid cells.387 Interestingly,
HoxA10 was detected in a variety of cases of myeloid leukemia, except for APL (M3), and decreased in lymphoid blast
crisis of chronic myelogenous leukemia (CML).33 HoxA10
was localized to chromosome 7p14-21.33 Its expression was
detected in several cases of monosomy 7, so complete loss
of HoxA10 in this abnormality, frequently seen in AML, is
not the mechanism of leukemogenesis. Although the targets
of HoxA10 are not known, its function has been investigated
by overexpression studies using retroviral infection of murine bone marrow.313,388 Overexpression of HoxA10 led to
abnormalities of progenitor cells, with increased numbers of
megakaryocytic and blast colonies in vitro and increased
myeloid progenitors in vivo, but peripheral blood counts
were normal. A substantial number of lethally irradiated congenic transplant recipients developed acute myelogenous
leukemia, with high blast counts, 5 to 10 months posttransplantation.
In addition to overexpression studies, the function of
HoxA10 has been investigated by knockout experiments.
Mice with targeted disruption of HoxA10 are fully viable
but have defects in both male and female fertility.389 Investigation of the hematopoietic system of these mice showed an
increase in peripheral blood neutrophils and monocytes to a
level twofold that of wild-type or heterozygote littermates.387
Interestingly, investigation of CFU in the knockout animals
showed no change in numbers of GM, M, or GEMM colonies, but these myeloid CFU were extremely large in size,
with a fivefold increase in cell number, and contained a
preponderance of immature myeloblastic cells.387 In sum-
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mary, these studies emphasize the role of HoxA10 in myeloid development and make it likely that HoxA10 plays a
critical role in control of proliferation at the myeloblastic or
promyelocytic stage. Recently, the effects of targeted disruption of the HoxA9 gene have been described.390 HoxA9 is
expressed at high levels in human CD34/ cells and myeloid
progenitors, but at lower levels in CD340 cells. It is also
expressed in lymphocytes. Disruption of HoxA9 resulted in
a 30% to 40% reduction in lymphocytes and granulocytes
and a blunted response to G-CSF, suggesting a role for
HoxA9 at the level of committed progenitors.390
A number of other homeobox genes have been studied by
overexpression and antisense studies, and many of these are
of direct relevance to myeloid development. Several of these
studies, like those of HoxA10, suggest that downregulation
of homeobox genes in progenitors may be important for
myeloid maturation. Overexpression of HoxB8, when combined with high levels of IL-3, can induce myeloid leukemia
in mice.391 These studies suggested that two events were
necessary for leukemogenesis: an increase in progenitor proliferation, mediated by IL-3, and a block in myeloid differentiation, mediated by increased HoxB8 expression.392 Consistent with the notion that downregulation of homeobox genes
may be necessary for proper differentiation, antisense studies
directed at a diverged homeobox gene expressed in CD34/
cells, HB24, was shown to block IL-3– and GM-CSF–induced proliferation, and overexpression inhibited differentiation. Interestingly, increased expression of HB24 was seen
in some cases of AML.393 Another negative regulator of
myeloid gene expression that is downregulated with myeloid
differentiation, the CDP protein discussed above, is a homeobox protein.229 The HoxB6 gene is expressed in erythroid
cells, and blocking its expression with antisense oligonucleotides can induce myeloid differentiation of erythroid lines.312
These studies suggest that HoxB6, like GATA-1, may serve
as a switch to direct multipotential progenitors from the
myeloid to the erythroid lineage (see discussion below).
In contrast to the studies cited above, in which the homeobox is either downregulated in mature myeloid cells or expressed in other lineages, several studies have looked at
homeobox genes expressed in mature myeloid cells. Hlx is
expressed in macrophages and neutrophils, and overexpression using a retroviral construct induces myeloid maturation.394 HoxB7 was shown to be induced during vitamin D3
monocytic differentiation of promyelocytic HL-60 cells.395
Overexpression of HoxB7 inhibited neutrophilic differentiation of HL-60 cells, and antisense inhibited GM-CFU.395
PRH (HEX) is an orphan homeobox gene initially identified
by three groups using PCR with degenerate primers to conserved homeodomain sequences.359,396,397 PRH is located on
human chromosome 10, where HOX 11 is also located.396
PRH is downregulated by TPA-induced monocytic differentiation, but not by retinoic acid-induced granulocytic differentiation.398 PRH is not oncogenic when transduced into 3T3
cells.354
Finally, regulators of homeobox genes may play important
roles in myeloid development and leukemia. The MLL (Hrx)
gene, a regulator of homeobox genes,399 is implicated in
mixed lineage (lymphoid/myeloid) leukemia.399-401 Another
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modulator of homeobox function is the Pbx1 gene.402 A fusion protein formed between E2A and Pbx contributes to
the t(1:19) pre-B–cell ALL, but expression of the E2A-Pbx
fusion protein in mice induces myeloid leukemia.403 Pbx1
binds cooperatively to HoxB4, B6, and B7, all of which
have been implicated in myeloid development, as described
above, but not to HoxA10.402 In summary, there are many
studies implicating homeobox genes in normal myeloid development and leukemia, and the next several years should
lead to exciting new developments.
OTHER FACTORS INVOLVED WITH MYELOID
DEVELOPMENT AND/OR LEUKEMIA
A number of other transcription factors have been implicated in myeloid development or leukemia. Because of space
limitations, they shall only be briefly mentioned.
c-myb. Myb was first isolated as a retroviral oncogene
and as an Ets fusion protein can transform chicken hematopoietic cells, resulting in a variety of different leukemias.12a,404,405 C-myb is a sequence-specific transcription activator.406 In hematopoietic cells, it is expressed in
proliferating progenitors and downregulated with differentiation, with a preferred if not specific expression pattern in
myeloid cells.42 This downregulation is important for differentiation of myeloid cell lines.382,383 Knockout of the c-myb
gene results in embryonic lethality with a failure of hematopoiesis of fetal liver,29 and c-myb has been shown to be
capable of transactivating the CD34 gene in cotransfection
studies.407 However, deletion of the c-myb binding sites does
not result in significant loss of human CD34 promoter activity,408 and murine CD34 is reportedly expressed at normal
levels in c-myb knockout mice,409 so the mechanism by
which c-myb regulates CD34 is unclear at this time. Recently, c-myb has been shown to regulate the CD13 gene in
myeloid cells410 and downregulate the M-CSF receptor.164
Finally, myb can interact with the AML1 family of transcription factors247 and can synergize with C/EBP to transactivate
a myeloid promoter, neutrophil elastase.88 Myb can also cooperate with C/EBP factors to activate target genes in myeloid cells, possibly mediated through the p300/CBP coactivator.197,411,412 All of these results point to an important role
of c-myb in myeloid development. In addition, c-myb has
been reported to regulate c-myc expression,413 and some of
its effects on myeloid development could be mediated
through this mechanism (see below).
c-myc. C-myc was isolated as an oncogene whose dysregulation plays a role in avian and human lymphomas. It is a
sequence-specific transcription factor and implicated in many
processes involving tumorigenesis and apoptosis.12a,373,414,415
Of specific relevance to this review are early studies showing
that myc is downregulated in myeloid cell lines as they are
induced to differentiate and that treatment of these lines with
antisense oligos induces myeloid differentiation.41 Activation
of an inducible myc-estrogen receptor construct in a myeloid
line can block differentiation.416 Another aspect of c-myc that
may be important in myeloid differentiation is that it has
been shown to heterodimerize with a number of partners.
Dimerization with max leads to transcriptional activation, and
dimerization with mad leads to loss of transcriptional activity.
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It has been shown that mad can be induced with maturation
of myeloid cell lines, consistent with the loss of proliferation
in these cells.417,418 Finally, a third interesting aspect of myc
is that it has been shown to repress the C/EBPa promoter in
adipocytes.193 C/EBPa expression increases with adipocyte
differentiation as c-myc decreases, and blocking myc function can increase C/EBPa expression and differentiation.
Whether c-myc plays a similar role in myeloid expression of
C/EBPa is not clear at this time.
NF-kB and rel proteins. In addition to being a major
regulator of lymphoid activation, NF-kB has been shown to
be activated in myeloid cell lines induced toward monocytic
differentiation with TPA. It is thought that this activation
may play an important role in human immunodeficiency
virus (HIV) replication and production of cytokines, such as
macrophage inflammatory protein-1a.419,420 The NF-kB and
related rel proteins can interact with C/EBP proteins and as
such may modulate the role of C/EBP factors in myeloid
cells.210,211,421 Targeted disruption of the relB gene leads to
myeloid hyperplasia, suggesting a role for this family in
myeloid development.422
Mixed lineage leukemia (MLL) protein. The MLL protein (also known as Hrx or All-1) has been shown to be
involved in translocations of 11q23 associated with mixed
(myeloid and lymphoid) leukemia.12a,399-401,423,424 MLL is a
very large transcription factor with a number of different
motifs, including a SET domain found in the trithorax and
polycomb genes, which regulate homeobox gene expression
in Drosophila. MLL has activation and repression domains,
and the translocations of the MLL gene suggest that loss of
the activation and retention of the repression domains could
alter the expression of as yet unidentified target genes as a
mechanism of leukemogenesis.425 The knockout of the MLL
gene caused altered Hox expression and segmental identity,
with myeloid defects in heterozygote animals.399 In addition,
MLL or All-1 0/0 ES cell show a block in maturation during
CFU formation in vitro, suggesting that normal function of
this gene is important for hematopoietic differentiation.426
p53. The p53 protein is a tumor-suppressor gene involved in many types of cancer and thought to play a normal
role in elimination of cells that have damaged genomes. p53
has been showed to be mutated in many types of human
leukemia, including myeloid leukemias.427,428 Other studies
have implicated mutations of p53 in CML, particularly in
blast crisis.429 p53 has also been shown to be a sequence
specific transcriptional activator that can activate target
genes, such as the cyclins.430 Wild-type p53 has been shown
to induce differentiation of myeloid cell lines.431 Therefore,
it is likely that loss of p53 function plays an important role
in leukemogenesis.
c-Jun. The c-jun proto-oncogene forms a heterodimer
with c-fos to form the AP-1 transcription factor, which has
been shown to be involved in cell proliferation and activation.432 C-jun is an early response gene in differentiating
myeloid cells, and its expression is upregulated in myeloid
cell lines during differentiation.433,434 One known myeloid
target is the macrophage scavenger receptor.166 AP-1 is antagonized by the retinoic acid receptor,435 but the PML/RAR
fusion protein found in APL is an activator of c-jun, and
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TRANSCRIPTION AND MYELOID DEVELOPMENT
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Fig 2. Structure of the human myeloid CSF receptor promoters. As with many myeloid promoters, relatively small regions direct activity and specificity in
transient transfection studies; in addition, there is
no well-defined TATA box. The major transcription
start site is designated by the horizontal arrow.
Shown are the locations of binding sites for PU.1, C/
EBP, and AML1. The PU.1 site in the G-CSF receptor
promoter is located in the 5* untranslated region, at
bp "3653; a functional PU.1 site is also found in the
5* UT region of the PU.1 promoter.55 In unstimulated
myeloid cell lines, the major C/EBP gene product is
C/EBPa.53,54,215 In newborn livers from C/EBPa Ï/Ï
animals, only expression of the G-CSF receptor is
significantly reduced.28 In PU.1 Ï/Ï animals and ES
cells, expression of the M-CSF receptor is significantly reduced or undetectable.162,163
this may contribute to the proliferative status of the leukemic
cells.436 C-jun can also physically interact with PU.1151; this
interaction may be important during myeloid differentiation.
MODELS OF HOW MYELOID TRANSCRIPTION FACTORS
WORK ON PROMOTERS TO DIRECT MYELOID
DIFFERENTIATION
The study of myeloid promoter elements has suggested
several models of how myeloid genes are regulated. In addition, these studies suggest a model of how myeloid differentiation might be directed by transcription factors in normal
cells and how abnormal function of transcription factors may
play a role in leukemia. There is a strikingly similar promoter
geography of three myeloid CSF receptor promoters, all of
which use C/EBP and PU.1 sites (Fig 2). There is likely
significant redundancy of some CSF pathways, in that different myeloid CSFs can direct myeloid CFU formation in vitro
and that knockout studies of some CSFs, such as GM-CSF,
did not show significant hematopoietic abnormalities. However, single transcription factors, such as C/EBPa and/or
PU.1, could affect the myeloid lineage by regulating expression of multiple CSF receptors. Alternatively, a factor such
as AML1, which is expressed at high levels in T cells in
addition to myeloid cells,437 could possibly regulate not only
myeloid CSF receptors, such as the M-CSF receptor, but
also CSFs produced in T cells that bind to those receptors,
such as GM-CSF258 and IL-3.260
The M-CSF receptor promoter itself is a good example
of how combinations of factors, not exclusively myeloidspecific, can act together to generate a myeloid-specific promoter (Fig 1). In the case of the M-CSF receptor promoter,
there are adjacent binding sites for PU.1,117 which is expressed in myeloid and B cells95,124; AML1, expressed in all
white blood cells234,437; and C/EBP, expressed in myeloid
cells and not lymphocytic cells.52,195,199 The common cell
type in which all three of these factors are expressed is
myeloid and not lymphocytic cells. In addition to a shared
specificity of expression in myeloid cells, AML1 and C/
EBP215 can interact physically in a specific manner and can
synergize to activate the M-CSF receptor.215 C/EBP and PU.1
can also physically interact in a specific way.88,142 Therefore,
the combinatorial effect of these factors is enhanced.
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The findings described in this review of how these transcription factors function suggest a model of how they may
direct myeloid development. This model suggests that the
apparently irreversible pattern of hematopoietic lineage development may proceed in three steps (Fig 3). (1) In stem
cells or more likely early multipotential progenitors, processes that are not defined yet result in the activation of a
specific transcription factor, such as GATA-1 or PU.1. (2)
Increased activation of expression and/or function of this
factor leads to increased expression of a specific growth
factor receptor and at the same time through an autoregulatory mechanism increases expression of the specific transcription factor. (3) Further differentiation is mediated by
the influence of the specific growth factor, augmented by
the upregulation or downregulation of other differentiation
genes regulated by the transcription factor.
Both GATA-1 and PU.1 activate important growth factor
receptors for the erythroid and myeloid lineages, respectively. For example, during erythroid development from
multipotential progenitors, GATA-1 is activated (and PU.1
is repressed).44,438 Previous studies have shown that GATA1, which plays a key role in erythroid development,5 can
transactivate its own promoter.439 Among its many gene targets, GATA-1 can stimulate expression of the erythropoietin
receptor.440 It is possible that GATA-1 activation initiates an
irreversible differentiation process in which GATA-1 stimulates its own expression and that of the erythropoietin receptor, allowing that progenitor to proliferate and survive in the
presence of erythropoietin.
Conversely, activation of PU.1 (and repression of GATA144,438) in a progenitor may lead to stimulation of PU.1 expression through an autoregulatory loop,55 as well as the MCSF receptor,117 allowing that progenitor to proceed along
the path of myeloid development. This model would also
suggest that downregulation of GATA-1 during myeloid differentiation and of PU.1 during erythroid commitment might
also be important. Consistent with this concept are results
suggesting that overexpression of GATA-1 in multipotential
cell lines result in a block of myeloid differentiation81,441 and
the finding that overexpression of PU.1 due to Friend virus
insertion near the Spi-1 locus in early erythroblasts leads to
a block in erythroid differentiation and murine erythroleuke-
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Fig 3. Model of induction of hematopoietic differentiation by specific transcription factors. In this model, transcription factors are expressed
at low levels in CD34" stem cells,134 as are specific growth factor receptors. Under direction of signals that are as yet not defined, such as the
influence of stromal interactions or growth factor signalling, specific transcription factors, such as GATA-1 or PU.1,44 are upregulated. Upregulation of specific transcription factors leads to their autoregulation and upregulation of specific growth factor receptors, resulting in increases
in proliferation, differentiation, and suppression of apoptosis of specific lineages. Downregulation of specific factors (such as GATA-1 during
myeloid development) may also play an important role.
mia.37,38,125,126 A final addition to this model would be findings showing that GATA-1 and PU.1 could downregulate
each other. Although there is a GA-rich sequence in the
proximal GATA-1 promoter, PU.1 reportedly does not bind
to this region.439 There is a site in the proximal PU.1 promoter that binds to GATA-1 and GATA-2, and coexpression
of either GATA protein results in a twofold repression in
PU.1 expression in myeloid and nonmyeloid lines.55 The role
of this site in downregulation of PU.1 in early myeloid cells
remains to be determined. Finally, this model is consistent
with findings indicating that GATA-1 does not play a major
role in myeloid development5 and that GATA-2 is either
not expressed in or downregulated in myeloid cells.78 Other
myeloid promoters, such as CD11b,76 also have sites in their
promoters that bind GATA-1 and GATA-2, but are not functional as assessed either by transient analysis of specific
point mutations or by transactivation studies.116
Recent findings suggest that C/EBP factors, notably C/
EBPa, may also contribute to myeloid development using
mechanisms similar to the model invoking GATA and PU.1.
It is not yet known how C/EBP proteins are regulated during
multilineage development of CD34/ cells, but it does appear
to be selectively expressed at high levels in neutrophilic and
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not monocytic or erythroid cells (H.S. Radomska and D.G.
Tenen, manuscript submitted). If it is upregulated selectively
during neutrophilic development, it may also be autoregulated in these cells as it is in adipocytes.192,194 It is known
to augment expression of the G-CSF receptor promoter53
and by this mechanism could promote the development of
neutrophilic cells in a manner similar to that described above
for PU.1 and GATA-1 for the monocytic and erythroid lineages, respectively.
QUESTIONS FOR THE FUTURE
In this review, we have attempted to summarize what is
known about myeloid transcription factors, lineage development, and differentiation. Clearly many advances have been
made in the last few years, but many very important questions remain unanswered.
How do the combinations of these myeloid transcription
factors make a specific lineage? Although we have provided
some concepts based on studies of the M-CSF receptor,
clearly other genes are regulated by other combinations of
factors.
What differences among myeloid subtypes (monocytes,
neutrophils, and eosinophils) are determined by transcription
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TRANSCRIPTION AND MYELOID DEVELOPMENT
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factors? An example is the role of C/EBPa in neutrophilic
development.
How are the regulators (PU.1, C/EBPa, etc) themselves
regulated in stem cells and multipotential progenitors? Are
they activated by cytokines or other extrinsic signals, or is
the process stochastic and autoregulatory? Are they in turn
regulated by other regulators, such as homeobox genes?
Are there truly myeloid locus control regions, scaffold
and matrix attachment regions, and other elements that are
important for expression of myeloid genes in the proper
chromosomal context?
What are the negative regulatory loops? Do repressors
like CDP228 or AEBP1231 play a role? What is the role of
GATA-1?
Why is it that factors expressed in other lineages (such as
PU.1 in B cells) do not induce expression of myeloid genes
in those lineages? Is it because combinatorial effects of factors are needed for myeloid expression or because of negative regulation in nonmyeloid cells?
What is the role of myeloid transcription factors in
apoptosis? Do they promote myeloid development by inhibition of apoptosis?
How does signalling affect myeloid development? What
is the role of Janus kinases (JAKs) and STATs?13,442,443 To
date, knockout studies have not shown these factors to be
essential for myeloid development. What is the role of hematopoietic cell phosphatase, whose mutation results in increased macrophage production?444,445 What is the role of
NF1?446,447
What are the mechanisms of leukemic development? The
studies summarized in this review, in which we have characterized targets of myeloid transcription factors and models
of how they could induce myeloid development, suggest a
number of mechanisms in which abnormalities of myeloid
transcription factor function could play a role in myeloid
leukemias, which represent a block in early myeloid differentiation. Translating the knowledge of what we understand
about myeloid gene regulation to understanding the pathogenesis of leukemia and perhaps developing new therapies
will be the major challenge for future studies.
ACKNOWLEDGMENT
The authors apologize in advance to the many investigators whose
work we were unable to cite due to space limitations. We also thank
David Hume, Hanna Radomska, Laura Smith, Milton Datta, and
Gerhard Behre for their careful reading of the manuscript and
thoughtful suggestions and Jim Griffin for his patience.
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1997 90: 489-519
Transcription Factors, Normal Myeloid Development, and Leukemia
Daniel G. Tenen, Robert Hromas, Jonathan D. Licht and Dong-Er Zhang
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