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Blood, Vol. 90 No. 2 (July 15), 1997:
pp. 489-519
REVIEW ARTICLE
Transcription Factors, Normal Myeloid Development, and Leukemia
By
Daniel G. Tenen,
Robert Hromas,
Jonathan D. Licht, and
Dong-Er Zhang
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.
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INTRODUCTION |
TRANSCRIPTION 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
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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 promoters. Examples are PU.1 and C/EBP , 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 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.
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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, granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor , 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 Fc R1 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/EBP, 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 initiator-less 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.
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| 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 -88 and -40 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/EBP 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/EBP 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 CBF , which forms a heterodimer with AML1 and augments its ability to bind to DNA.
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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 phosphorylated 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 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 proteinase-3 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-1 (IL-1) 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
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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 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/RAR 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/RAR 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 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.
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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 adeno-associated 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 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.
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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 self-renewal 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+/CD38- 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 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 -54, an Sp1 site at bp -39, 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, Ets-2, 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 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  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 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 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-IL6 (same as C/EBP ); in this case, it was shown that PU.1 and C/EBP could cooperatively activate an artificial reporter plasmid. These findings are interesting because they suggest that PU.1 might also interact with C/EBP (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 receptors 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 -/- 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 -/- 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 -/- 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 Mac-1-negative and reduced in number, suggesting that targeting 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 B-cell and myeloid development.161 In vitro differentiation of PU.1 -/- 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 ,54 and the G-CSF 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 -/- animals. As noted above, studies in PU.1 -/- 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 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, Fli-1, 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
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C/EBP, A REGULATOR OF GRANULOCYTIC DEVELOPMENT |
C/EBP (CCAAT/enhancer binding protein; now known as C/EBP) 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/EBP 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/EBP is upregulated and plays an important role in adipocyte differentiation and in this cell type functions in fully differentiated, nonproliferative cells; inhibition of C/EBP blocks differentiation; and expression of C/EBP induces differentiation.179,180 Indeed, several studies have indicated that C/EBP can act as a general inhibitor of cell proliferation and in this respect resembles a tumor suppressor.181-183 C/EBP is part of a family of leucine zipper transcription factors, including C/EBP and C/EBP, that heterodimerize and are differentially regulated during adipocyte differentiation.179,184,185 Another very interesting C/EBP gene, C/EBP , was very recently isolated and shown to be preferentially expressed in myeloid cells.186,187 Both C/EBP and C/EBP 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/EBP activity and induce increased apoptosis of myeloid 32D cells.191
In murine adipocyte cell lines, C/EBP is transcriptionally upregulated (and C/EBP 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/EBP promoter.193 Interestingly, although both murine and human C/EBP promoters are autoregulated, the mechanisms of autoregulation are different. The murine C/EBP promoter has a binding site for C/EBP.192 The human C/EBP promoter does not conserve the C/EBP binding site found in the murine promoter, and the autoregulation is indirect and mediated by the action of C/EBP on a helix-loop-helix protein, upstream stimulatory factor (USF).194 Studies of C/EBP 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/EBP 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/EBP was observed to be highly expressed in proliferating myelomonocytic cells upon induction of differentiation and downregulated with maturation, whereas C/EBP and C/EBP were upregulated. However, recent Northern blot analysis of human primary CD34+ cells shows that C/EBP 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/EBP mRNA, which was completely undetectable in adherent peripheral blood monocytes (H. Radomska and D.G.T., manuscript submitted). In addition, C/EBP was noted to be upregulated as single CD34+/CD38- 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/EBP in neutrophilic but not monocytic lineage development.
As noted above, C/EBP 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 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/EBP, C/EBP, and C/EBP 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 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/EBP and C/EBP can bind with vastly different affinities to the same promoter site.208 The consensus binding site for C/EBP 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-B 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/EBP has been shown to physically interact with TBP.61 As noted above, C/EBP has been shown to interact with PU.1,142 and PU.1 can physically interact with C/EBP83 and C/EBP as well (P. Auron, unpublished observations). As noted above, C/EBP and C/EBP 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/EBP 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/EBP-induced granulocytic maturation.
Recently, lines of mice harboring a knockout of the C/EBP 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 (-/-) animals do not produce any mature neutrophils, which comprise 90% of the peripheral blood white blood cells of newborn heterozygote (+/-) or wild-type (+/+) 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 , c, and IL3 were all comparable 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/EBP on Epo receptor expression in precursor cells or alternatively a reflection of an increased number of erythroid cells in C/EBP -/- 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/EBP -/- newborn liver, although other types of colonies (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM], colony-forming unit-erythroid [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/EBP knockouts, strongly suggesting that there must be important C/EBP target genes in myeloid progenitors in addition to the G-CSF receptor.219 Transplantation of C/EBP 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/EBP in myeloid cells. The avian homolog of C/EBP (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/EBP 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 G-CSF.185,220 Changes in C/EBP phosphorylation (but not in C/EBP) have also been described during myeloid differentiation of a progenitor cell line.160 Human C/EBP is also known as NF-IL6.185 C/EBP can also be activated in other cell types by phosphorylation without translocation.221 In one report, targeted disruption of C/EBP leads to defects in killing of Listeria and tumor cells by macrophages.222 In a second study, disruption of C/EBP led to an increase in IL-6 production (previously it was thought that C/EBP, 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/EBP in cytokine activation rather than lineage development.
As noted above, C/EBP (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/EBP resulted in a reduction of G-CSF production in macrophages but not other cell types.222 C/EBP has been noted to regulate the promoters for several myeloid 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 ,54 and the G-CSF receptor promoter.53 In the C/EBP 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 ( and chain) mRNA, suggesting that perhaps other factors (such as C/EBP) can compensate for C/EBP 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/EBP 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/EBP gene maps to chromosome 19q13.1 and C/EBP 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/EBP 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/EBP (and C/EBP) may be involved in activation of cytokines in mature cells, whereas C/EBP 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 e |
Introduction
Methods
Introduction