From the Departments of Microbiology and Biochemistry
and the Centre for Molecular and Cellular Biology, University of
Queensland, Brisbane 4072, Australia and the ¶ Department of
Molecular Genetics, Ohio State University, Columbus, Ohio 43210
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ABSTRACT |
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Numerous macrophage-restricted promoters lack TATA boxes or other conventional initiation motifs but contain high affinity binding sites (PU boxes) for the macrophage-restricted Ets family transcription factor PU.1. In RAW264 murine macrophages, multimerized PU boxes were not active as enhancers when placed upstream of a minimal promoter. To model their role in basal promoters, we inserted PU boxes into a promoterless luciferase reporter plasmid. Two sites, regardless of orientation, were necessary and sufficient to direct reporter gene expression in transient transfections of the RAW264 macrophage-like cell line. This activity was absent in transfected 3T3 fibroblasts but could be induced by PU.1 coexpression. Both the model promoter and the macrophage-specific mouse and human c-fms promoters were activated in RAW264 cells by other Ets family transcription factors, Ets-2 and Elf-1. In fibroblasts, the effects of PU.1 and Ets-2 were multiplicative, whereas overexpression of PU.1 in RAW264 cells reduced activation of c-fms or model promoters by the other Ets factors. The PU.1 and Ets-2 binding sites of the mouse c-fms promoter have been located by DNase footprinting. A conserved Ets-like motif at the transcription site, CAGGAAC, that bound only weakly to PU.1, was identified as an additional critical basal c-fms promoter element. Comparison of studies on the model promoter, c-fms and other myeloid promoters provides evidence for a conserved mechanism that involves three separate and functionally distinct Ets-like motifs.
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INTRODUCTION |
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Members of the large Ets family of transcription factors are characterized by a DNA binding domain (usually in the C-terminal region) that is homologous to the viral oncogene v-ets (1) and recognizes purine-rich sequences typically with a 5'-GGAA-3' core. The most divergent member of the Ets family, PU.1, is known to be restricted to macrophages, B cells, mast cells, and neutrophils (2, 3) and is necessary for normal myelopoiesis (4-7). Apart from the Ets domain, PU.1 has a glutamine-rich activation domain and a proline-, serine-, threonine-, and glutamic acid-rich (PEST) domain (8), which links the activation and Ets domains.
PU.1 recognition motifs have been shown to be important in the
expression of many macrophage-restricted genes such as
c-fms, which encodes the receptor for macrophage
colony-stimulating factor (9, 10), CD11b (11), CD18 (12-14), FcR1
(15, 16), Fc
RIIIA (17), GM-CSF receptor (18), c-fes (19),
the macrophage scavenger receptor (20), and the PU.1 gene itself (21,
22). Our own studies have concentrated upon the mouse and human
c-fms promoters. Two functional PU.1 binding sites are
present in the human c-fms promoter at
150 and
170
nucleotides from the start codon (
150H and
170H). Zhang
et al. (10) provided evidence that the more distal site has
higher PU.1 binding affinity, but deletion of either site lowers the
basal promoter activity substantially (23), indicating that both are
required. Only one of these PU.1 sites is clearly present in the mouse
promoter (Fig. 1), but at
170M there is a candidate PU.1 site on the
opposite strand.
The mouse and human c-fms genes and many of the macrophage-specific genes cited above lack proximal promoter elements that normally determine the site of initiation such as a TATA box, Inr (initiator) sequences, or the GC-rich sequences found in "housekeeping" genes (Fig. 1). The possibility that PU.1 might function in initiation in macrophage specific genes is favored by evidence that it can bind directly to both TFIID and the retinoblastoma gene product (24) and the location of PU.1 sites 30-50 bp1 upstream of the multiple sites of transcription initiation in most of the promoters cited above. This paper contains an analysis comparing artificial promoters containing only PU.1 recognition sites with the macrophage-specific mouse and human c-fms proximal promoters. As an explanation for the presence of multiple purine-rich elements in macrophage-specific promoters, we propose that PU.1 needs to interact with another member(s) of the Ets transcription factor family to promote macrophage-specific transcription initiation. In the course of this study, we provide evidence for a separate role in initiation for GGAA motifs to which PU.1 binds weakly, if at all.
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MATERIALS AND METHODS |
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Cell Culture-- NIH3T3 (murine fibroblast line) and RAW264 (a murine macrophage-like line) were obtained from the American Type Culture Collection (Rockville, MD) and cultivated as described by Stacey et al. (25). HeLa cells were obtained from Dr. R. Sturm and cultured in Dulbecco's modified Eagle's medium plus 5% fetal bovine serum.
Nuclear Extracts-- Nuclear extracts were prepared with a variation (9) of the method of Osborn et al. (26). Recombinant PU.1 (9) and GST-Ets-2 (25) were prepared and purified as described elsewhere.
Transient DNA Transfection Analysis and Plasmid
Construction--
The mouse pGL0.3fms, pGL3.5fms, and pGL6.7fms (27)
and human 430fms and
150H mutant human
430fms (M1) (23)
c-fms promoter-luciferase reporter plasmids are described
elsewhere. Mutation of the
103 site of the mouse promoter from CAGGAA
to CACCAA mutant was performed in the pGL0.3fms plasmid according to
the QuikchangeTM methodology (Stratagene, La Jolla, CA) and
confirmed by direct sequencing. The luciferase reporter plasmids pGL2C
(SV40 enhancer and promoter), the SV40 promoter plasmid pGL2P, and the
basal plasmid pGL2B were obtained from Promega (Madison, WI). The mouse PU.1 expression plasmid PUpECE, the mouse Ets-2 expression plasmid Ets2pECE, and the parent vector pECE used previously (9) were a gift
from Dr. Richard Maki as were the pBL-CAT series plasmids (28). The
Elf-1 expression vector, in which expression of the mouse protein is
directed by the Rous sarcoma virus long terminal repeat, was a gift
from Dr. Martine Roussel. The PU box model promoters were made by
ligating the SV40 PU box oligonucleotide into pGL2B, which had been cut
with XhoI and end-filled. The double-stranded PU box
oligonucleotide (5'-CTGAAAGAGGAACTTGGTTAGGTA-3') (28) was
phosphorylated with T4 polynucleotide kinase prior to ligation to allow
multimerization. Clones containing inserts were characterized for
number and orientation of inserts by direct sequencing. Plasmids containing 1-4 PU boxes in various orientations were isolated.
DNase I Footprinting Analysis-- Recombinant PU.1 or GST-Ets-2 was prepared as described above. To label the DNA strands, the pGL0.3fms reporter plasmid was cut with either HindIII (to 3'-label the upper strand) or MluI (to 3'-label the lower strand), and [32P]dCTP was incorporated using the Klenow fragment of DNA polymerase. The plasmid was then re-cut with MluI or HindIII, respectively, to generate the appropriate labeled fragment. The probe fragment was purified by elution from a 5% TBE acrylamide gel. Binding reactions were constituted as follows; to 25 µl of buffer D was added 2 µl of poly(dI-dC) (1 mg/ml) and nuclear extract (1, 5, or 10 µg), recombinant GST-Ets-2 (1, 5, or 10 µl), or recombinant PU.1 (1, 5, or 10 µl). Probe (2 µl, approximately 104 cpm) was added, and the volume was adjusted to 50 µl/reaction with MilliQ water. Binding reactions were left at 25 °C for 60 min before adding 51 µl of DNase I (50 ng/µl in 10 mM MgCl2, 5 mM CaCl2). After 30 s, 100 µl of stop solution (30 mM EDTA, 200 mM NaCl, 1% SDS, 100 µg/ml tRNA) was added, followed by 30 µl of ammonium acetate solution (3 M ammonium acetate, 20 mM EDTA). DNA was precipitated with 2 volumes of ethanol, washed, dried, and redissolved in 5 µl of sequencing gel loading buffer. Aliquots (2 µl) of each reaction were analyzed on a sequencing gel, using a c-fms sequencing ladder (0.3fms, pGL2 primer) for calibration.
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RESULTS |
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A PU box Multimer Does Not Function as an Enhancer in RAW264 Cells-- Some previous studies of PU.1 (8, 28) have presented the view that PU.1 can act as a conventional, albeit weak, transcriptional activator, whereas studies in B lymphocytes have emphasized the ability of PU.1 to recruit another protein to an adjacent site in an enhancer element (32, 33). To examine the enhancer activity of PU.1 in macrophages, we assessed the activity in RAW264 cells of CAT expression plasmids in which various numbers of PU box motifs are placed upstream of a minimal promoter. The plasmids are those used in the original descriptions of PU.1 (28). Preliminary experiments revealed that the presence of up to eight PU box motifs had little effect on basal transcription activity in RAW264 cells. Against this background, we reexamined the effect of a PU.1 expression plasmid on the activity of the PU box multimers in HeLa cells, which had been used previously. Table I confirms the finding of Klemsz et al. (28) that in HeLa cells, by contrast to RAW264, increasing numbers of PU box elements greatly increased the basal activity of the promoter, which could be amplified further by co-transfection with either PU.1, or another Ets family transcription factor, Ets-2. In RAW264 cells, the PU box multimer was less than twice as active as the single site control, and it could be activated by co-transfection of an Ets-2 expression plasmid but was partly repressed by PU.1.
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Two PU.1 Binding Sites Are Sufficient to Generate a Tissue-specific
Promoter--
The results in the previous section suggest that PU.1
alone functions poorly to enhance the activity of a TATA-containing promoter in RAW264 cells. In the c-fms promoters (Fig.
1) and in most of the promoters
identified in the Introduction, the PU box motifs actually occur in the
vicinity of the transcription start sites. To address directly the
alternative hypothesis that a binding site for PU.1 alone contributes
to transcription initiation in a functional macrophage-specific
promoter, we produced a series of artificial promoters in which the
high affinity PU.1 binding site from the SV40 virus was inserted into
the basal promoterless vector pGL2B. This plasmid contains a strong
polyadenylation signal and multiple in frame stop codons upstream of
the multiple cloning site, so that read through transcripts from the
plasmid backbone are almost undetectable. The plasmids were designated
pB>, pB, pB<>, and so on, where the arrows signify the
orientation and number of the PU boxes (the forward arrow signifying
the purine-rich sequence on the upper strand). Insertion of a single PU
box site in either orientation had no detectable effect on basal
promoter activity of pGL2 (data not shown). Conversely, pB
(Fig.
2) had considerable reporter activity in
RAW264 cells; the level of activity of pB
was similar to the
430-bp human fms promoter, approximately 20% of that of the
murine c-fms short promoter (0.3fms), and 80% of
that of the TATA-containing SV40 minimal promoter in the Promega plasmid, pGL2P (data not shown). In NIH3T3 fibroblasts, which lack
nuclear PU.1 or any other nuclear protein capable of binding the PU box
sequence under conditions of an electrophoretic mobility shift assay
(9, 23), the construct had the same barely detectable activity as the
basal promoterless control vector pGL2B. Co-transfection of a PU.1
expression plasmid trans-activated the pB
construct in
NIH3T3 cells but repressed basal activity in RAW264 cells (Fig. 2).
Other orientations and combinations of PU sites
(pB>>>, pB<<<, pB
, pB<>,
and pB<>>>) were examined in transfections of
RAW264 and 3T3 cells, but the level of activity and the pattern of
trans-activation by PU.1 was always similar to that of
pB
(data not shown).
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Comparative Responses of the Murine and Human c-fms Promoters to
Co-transfection of PU.1 and c-ets-2--
To ascertain whether PB
is a valid model for assessing the function of PU.1, we examined the
effects on the human and murine c-fms promoters of
coexpressing PU.1 and Ets-2 together in both macrophage and
nonmacrophage cell lines. The amounts of co-transfected plasmid (1 µg/10 µg of reporter plasmid) in these experiments were previously
determined to produce maximal activation or repression (Refs. 9 and 23;
data not shown). In RAW264 cells (Fig.
3A), co-transfection of PU.1
without Ets-2 activated the human but not the murine promoter, while
co-transfection of Ets-2 without PU.1 caused a much greater activation
of both promoters. As observed with the PB
model promoter,
co-transfection of PU.1 almost blocked the activation of the murine
c-fms promoter by Ets-2 and partly prevented the response to
Ets-2 on the human promoter. In NIH3T3 fibroblasts, which lack nuclear
PU.1, transfection of the PU.1 or Ets-2 expression plasmids each
activated both mouse and human promoters, and the two transcription
factors together resulted in a further increase in activity over that
obtainable with either factor alone (Fig. 3B). In summary,
the behavior of both fms promoters closely resembled the
artificial promoter containing only two Ets/PU.1 sites, and maximal
activity required that both PU.1 and Ets-2 be expressed.
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Elf-1 Can Substitute for c-ets-2 in Transactivating the c-fms Promoter-- In previous studies, both Ets-1 and Ets-2 were shown to activate the human c-fms promoter (23). To determine whether these are the only Ets factors able to interact with PU.1 to activate the c-fms promoter, we examined the effect of co-transfection with Elf-1, another Ets factor that is widely expressed in macrophages and other hemopoietic cells (35) and was shown by others (19) to interact with PU box motifs. As with Ets-2, co-transfection with a murine Elf-1 expression plasmid trans-activated the murine c-fms promoter in RAW264 cells (Fig. 4), and the effect was opposed by simultaneous overexpression of PU.1.
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DNase I Footprinting of the Murine c-fms Promoter--
To explain
the response of the mouse c-fms promoter to PU.1 and Ets-2,
we identified binding sites for the two factors by DNase I
footprinting. For the latter, we again employed the DNA binding domain
expressed as a glutathione S-transferase fusion protein
because the full-length protein does not bind under conditions of
electrophoretic mobility shift assay or DNase footprinting (25). Fig.
5 shows that a clear footprint was
observed in the presence of recombinant PU.1, corresponding to the
130M site predicted from the DNA sequence in Fig. 1. A second PU.1
site was observed between
160M and
170M, corresponding to the DNA sequence 5'-GAAAGGGAACT-3' (on the reverse strand; see Fig. 1). Weak
hypersensitivity on the upper strand was observed around the Ets core
at
103.
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A Possible Additional Ets Site at the Transcription Start
Region--
The more proximal of the two PU.1 binding sites at 130
in the murine c-fms promoter lies 25-30 bp upstream of the
major transcription start sites (Fig. 1). An additional GGAA core
flanks the major transcription start sites in both the murine (
103M)
and human (
105H) promoters (Fig. 1). Examination of the sequence of
the parent plasmid pGL-2B reveals that the model promoter, pB
,
also has an additional Ets core sequence (CCGGAAT, reverse strand (20 bp downstream of the XhoI site into which the PU boxes are
inserted and 35 bp 3' of the PU box in pB
); see Promega
catalogue). To assess the functional importance of an additional Ets
core, the CAGGAAC sequence at this site in the murine c-fms
promoter was mutated to CACCAAC to abolish any putative Ets factor
binding. In Fig. 6, it can be seen that
this mutation reduced the activity of the murine c-fms
promoter to base-line levels in RAW264 cells.
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DISCUSSION |
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This study shows that PU.1 recognition sites are weak enhancer
elements in RAW264 macrophages (Table I), but alone they are sufficient
to activate a minimal promoter in the absence of a TATA box or
consensus initiator element (Fig. 2). One PU.1 binding site was
insufficient to generate detectable promoter activity, whereas a basal
vector that contains only two PU box motifs (pB) resembled the
murine and human c-fms promoters in several important respects: (i) like the c-fms promoters, pB
displayed
promoter activity that was dependent upon endogenous or co-transfected PU.1 expression; (ii) pB
could be activated by coexpressed Ets-2 in both RAW264 macrophage-like cells and NIH3T3 fibroblasts in the same
way as the c-fms promoters; (iii)
trans-activation of both c-fms promoters and pB
in
RAW264 cells with Ets-2 was antagonized by simultaneous coexpression of
PU.1, whereas these factors produced an additive effect in NIH3T3
cells.
Based upon these observations, we hypothesize that the reason that the
c-fms promoters (Fig. 1) and all of the myeloid-specific promoters discussed in the Introduction have more than one purine-rich proximal promoter element is that maximal activity requires that one
Ets site be occupied by PU.1 and another by a second Ets factor exemplified by Ets-2. According to this model, PU.1 can act as a
repressor of the response to another co-transfected Ets factor because
it prevents binding to the second site. This occurs when PU.1 is
co-transfected with the reporter plasmid in RAW264 macrophages, where
there is already a very high basal level of nuclear PU.1 (9) but not to
the same extent in the transfected 3T3 fibroblasts when the expression
of both of the Ets factors is directed by the SV40 promoter in the pECE
vector. In the case of the murine c-fms promoter, we have
identified the 130M site as the motif to which both PU.1 and Ets-2
can bind (Fig. 5), and on this basis we hypothesize that this is the
site where PU.1 acts as a specific repressor of
trans-activation by Ets-2 in the mouse promoter. The human
promoter has the same sequence at
170H; but the
150H site can also
bind both PU.1 and Ets-2 (23), and the data in Fig. 3C
indicate that this is a major site at which the two factors can compete
with each other. The ability of PU.1 to interact with other Ets
proteins, and to act as a repressor of Ets-1 or Ets-2 actions has
several precedents. Erman and Sen (34) showed a clear instance of
context-dependent cooperation between PU.1 and Ets-1 on the
immunoglobulin heavy chain intronic enhancer, where specific domains of
each protein are required for transactivation to occur. Overexpression
of PU.1 in B cells causes repression of the immunoglobulin heavy chain
enhancer, presumably by competing with Ets-1 for occupancy of the
second site (9). Others showed that PU.1 represses activation through
an Ets motif of the class II major histocompatibility complex I-A
promoter in B cells (37). By contrast to PU.1, Ets-2, is a powerful
trans-activator (Table I). It is capable of activating
transcription in RAW264 cells through a single response element (25).
Hence, the function that Ets-2 contributes to both PB
and the
c-fms promoters may be a strong activation domain that is
not present in PU.1.
The model promoter gives insight into the possible functions of PU.1
but does not indicate how the direction of transcription and the start
sites are determined. The 103M site was considered as an additional
candidate Ets site in the c-fms promoters based upon
conservation of the Ets core between mouse and human, its position
flanked by the major transcription start sites (Fig. 1), and the
fortuitous presence of a similar sequence in the same position relative
to the PU boxes in the model promoter, pB
. If the PU box motifs
function as the equivalent of a TATA box, as hypothesized in the
introduction and supported by the transfection data, then the
103M
site is appropriately placed to be the equivalent of an initiator.
Mutation of the core Ets site (GGAA to CCAA) abolished promoter
activity, demonstrating that this is an additional site that is
absolutely required for the activity of the murine fms
promoter (Fig. 6). The
103M motif is found in the basal promoters of
several other myeloid genes. Both the human GM-CSF receptor (18) and
the human CD18 (12-14) promoters have exactly the same spacing of a
distal PU.1 site and a GGAA motif adjacent to the major transcription
start sites (12-14). In the c-fes promoter the major sites
of transcription initiation also occur around the same sequence as in
murine c-fms, CAGGAAC (19). In the macrophage-specific promoter of the PU.1 gene itself, in both mouse and human, the identical sequence again lies within the transcription start region, and the same CC mutation we made in the fms promoter site
greatly reduced activity of the PU.1 promoter in myeloid cells (22). On
the basis of these many examples, a GGAA core sequence can be viewed as
a myeloid-specific initiator sequence. In none of the examples cited
does the Ets-like initiator sequence bind PU.1 with high affinity. The
CAGGAAC site in the PU.1 promoter can bind recombinant PU.1 weakly in
an electrophoretic mobility shift assay (22), an observation we have
confirmed with the c-fms sequence (data not shown).
Recombinant PU.1 caused a weak hypersensitivity on the upper strand of
the c-fms promoter in the DNase I footprinting shown in Fig.
5. It may be that the site must be weak so as to reduce competition by
other Ets factors that would not optimally fulfill the function
provided by PU.1 or to provide a sensitive relationship between the
level of PU.1 expressed and c-fms transcription. A similar
argument has been made to explain the weak binding of Elf-1 to proximal
promoter elements in T cell-specific promoters (38). DNase I
footprinting using RAW264 macrophage nuclear extract does not detect
proteins bound to this site; instead, the whole region becomes
hypersensitive to digestion.2
This observation cannot be interpreted, since some full-length Ets
factors bind weakly if at all to DNA in vitro or form
multimeric complexes with other factors, such as members of the AP1
family, bound to adjacent sites (1, 20, 25, 39, 40). Hence, it remains
possible that PU.1 itself binds alone, or cooperatively with another
factor, as in the case of B-cell-specific elements (32, 33) or that
another Ets-like factor that cannot be detected by the methods employed
in this study contributes to the function of the
103M element.
PU.1 cooperates with Ets-2 to activate both the mouse and human
c-fms promoters in 3T3 cells, but the promoters from the two species differ in basal activity and response to PU.1 co-transfection in RAW264 cells. Both species have at least three Ets core sequences: the 103M/
105H site discussed above and PU.1 binding sites at
130M/
150H and
170M/
170H. The
150H site differs from the
130M site in that it actually contains three possible GGAA or GGAT Ets cores (Fig. 1). These sites are required for the human-specific activation of the fms promoter by co-transfection of PU.1
and the maximal response of the human promoter to Ets-2 in RAW264 cells
(Ref. 23; Fig. 3C). The
150H site(s) have a lower affinity for PU.1 than the distal
170H site (23), and were not detected in
DNase I footprinting (10). Overexpression of PU.1 might permit a higher
level of occupancy of the low affinity
150H sites, leading to the
observed transcriptional activation (Fig. 3C). By contrast to the human promoter, the mouse has two strong PU.1 sites. The region
surrounding the high affinity PU.1 binding site at
170M, identified
in this study by DNase I footprinting (Fig. 5), can be clearly aligned
between mouse and human (Fig. 1), but the GGAA Ets core is not
conserved. Whereas the mouse sequence is 5'-GAAAGGGAACT-3' (reverse
strand), the human sequence is 5'-GCAAGGCAACC-3'. Functionally, therefore, the
170M site might be more accurately aligned with
170H, although the PU/Ets site is in the reverse orientation. It
remains to be seen whether the subtle differences in PU.1 dependence and relative activity of the human and mouse fms promoters
are related to different roles of c-fms and its ligand in
macrophage differentiation between the two species.
The use of Ets-2 as a model for a second Ets factor in this study was prompted by the previous evidence of the response of the mouse and human fms promoter to this factor (9, 23) and its known ability to interact with PU box elements (Table I). It is unlikely to be the only factor that can interact with PU.1 to activate myeloid promoters. However, the relevance of the model is supported by evidence that Ets-2 can promote macrophage differentiation when overexpressed in premyeloid cells (41), and a dominant negative Ets-2 expressed from the human c-fms promoter caused significant aberrations in macrophage differentiation in vivo (42). Ets-2 protein is expressed in primary macrophages; the level of expression is regulated by CSF-1 and phorbol esters (25, 40, 42), but Ets-2 itself is not essential for the differentiation of normal primary macrophages from embryonic stem cells (4). Given the size of the Ets family, a degree of redundancy is not surprising. We showed that Elf-1 mimicked the effect of Ets-2 (Fig. 4). Elf-1 is expressed constitutively in primary murine macrophages (35). Heydemann et al. (19) described a proximal promoter element in the myeloid-specific c-fes promoter that binds PU.1 or Elf-1 with equal affinity, and both proteins were shown to be expressed at similar levels in the nuclei of human myeloid cells. Like Ets-2, Elf-1 is also not absolutely required for myeloid differentiation (19), but another Elf-1-related protein, termed MEF, recently described in murine myeloid cells (43), could also contribute. In examining the relevance of the effects of these co-transfected Ets factors, it is important to recognize that, although RAW264 is the best available transfectable murine macrophage cell line model, the level of c-fms expressed is low in comparison with bone marrow-derived macrophages (25) and lower still than in postproliferative peritoneal macrophages.3 RAW264 cells and primary macrophages have similar levels of nuclear PU.1 (9), but the cell line lacks Ets-2 mRNA and protein (25)4 or nuclear protein that binds strongly to a perfect Elf-1 consensus site.5 Increasing levels of Ets-2 or other factors during later stages of macrophage differentiation could contribute to increased expression of c-fms.
It is of some interest that Ets-2 alone can activate both the pB
model promoter and the c-fms promoter in 3T3 cells (Figs. 2
and 3), which lack PU.1 (9, 23). The c-fms promoter is active in a wide range of mouse and human tumor cell lines, and ectopic
expression of c-fms, which commonly creates a
CSF-1/c-fms autocrine loop, is a feature of malignant mouse
and human tumor cells that is correlated with invasive potential and
anchorage-independent growth (see Ref. 44 and references therein). The
c-fms proximal promoter is actually growth
factor-dependent and can be activated by both
CSF-1/c-fms (44) and GM-CSF/GM-CSF receptor (45) signaling on a normal fibroblast background. In turn, CSF-1/c-fms
signaling in 3T3 cells requires Ets-2 and can be blocked by expression
of dominant negative forms of the protein (25, 40). Expression of
c-fms mRNA, which is restricted to macrophage-like cells
in the mouse embryo (46) remains detectable in the fetal liver of PU.1
null mice (47). These findings together suggest that the fms
promoter is not absolutely PU.1-dependent, and other Ets factors such as Ets-2 are able to substitute for PU.1 activity, albeit
less effectively.
Clearly, PU/Ets sites alone do not explain the full complexity of
expression of c-fms or any other macrophage-specific gene; the PB model applies only to the basal promoter and initiation. One other factor that regulates the c-fms promoter is
c-myb, which represses promoter activity and is
down-regulated as c-fms is induced during myeloid
differentiation (23). CBF
(AML1), which is also tissue-restricted,
cooperates with CBF
and CAAT enhancer-binding protein
to
activate the human c-fms promoter in myeloid cell types (2).
Interestingly, the sites involved in this response are not conserved in
the mouse (Fig. 1). The two sequence differences that create the second
high affinity PU.1 binding site in the mouse at
170M, actually change
two key bases in the human CBF
/B site (human GTGGTTG; mouse
GTAGTTC), but despite these changes the mouse promoter is responsive to
trans-activation by another member of the CAAT
enhancer-binding protein family that has also been implicated in
myeloid differentiation, CAAT enhancer-binding protein
.6
The kinds of interactions between PU.1 and Ets-2/Elf-1 observed on the
PB and c-fms promoters are specific to those promoters. Although PU.1 is already expressed at high levels in RAW264 cells, the
ability of this factor to activate the human c-fms promoter, under the same conditions where it represses the mouse promoter, indicates that it is not saturating for weaker PU box sites. In other
studies, we have shown that co-transfection with the PU.1 expression
plasmid can activate the human immunodeficiency virus-1 long terminal
repeat in RAW264 cells. This activation is dependent upon the tandem
NF-
B sites, which bind weakly to PU.1 in vitro (48, 49).
The same sites also function as Ets-2-responsive elements, but specific
mutations that prevent PU.1 (and NF-
B) binding and activation
do not block Ets-2 action (49). Furthermore, PU.1 does not prevent
activation by Ets-2,7 further
indicating that the ability of PU.1 to block Ets-2 action on the mouse
and human c-fms promoters is not due to nonspecific effects
on Ets-2 expression or function under the conditions of transfection.
Another promoter that responds to both factors is the
tartrate-resistant acid phosphatase promoter, a TATA
box-containing promoter functional in osteoclasts. In another study, we
have shown that PU.1 co-transfection activates this promoter 5-10-fold in RAW264 cells via a weak PU box site in the proximal
promoter.8 In contrast to
these promoters, the CSF-1-inducible promoter of the urokinase
plasminogen activator gene is responsive only to Ets-2, which acts via
a conserved Ets/AP1 element in a distal enhancer that does not bind
PU.1 (25). It is important that such elements do not bind PU.1, since
Ets-2 is a key component of the ras/raf/mitogen-activated
protein kinase signaling pathway (40) and competition by PU.1 could
prevent the operation of this pathway in macrophages. These
observations highlight the fact that the interactions between PU.1 and
other Ets family factors depend upon promoter context, the relative
affinity of the binding sites for different Ets factors, and the level
of expression. An additional complexity may arise based upon recent
observations that PU.1 can be inducibly phosphorylated in macrophages
(49) and that a significant proportion of macrophage PU.1 is found in
the cytoplasm.7
In this study, we have provided evidence that the multiple purine-rich
motifs found in many macrophage-specific proximal promoters contribute
different functions to transcription initiation. We have shown that
PU.1 is not the only factor that binds them and that PU.1 is not
sufficient, and may not be necessary, for maximal promoter activity. In
essence, the archetypal basic myeloid promoter probably has three
factors bound to purine-rich motifs: PU.1, a second Ets factor with a
powerful activation domain, and a third factor/complex that interacts
with the GGAA initiator sequence. The hypothesis derived from our
studies of the PB and fms promoters should have general
relevance to understanding macrophage differentiation and the functions
contributed by PU.1 and other Ets factors to the activity of
myeloid-specific TATA-less promoters.
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ACKNOWLEDGEMENTS |
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Technical assistance was provided by C. J. K. Barnett. We thank Dr. Michael Rehli for criticism of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the National Health and Medical Research Council of Australia and the Queensland Cancer Fund. The Center for Molecular and Cellular Biology is a Commonwealth Special Research Center of the Australian Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dana Farber Cancer Institute, Boston, MA 02115.
To whom correspondence should be addressed: Dept. of
Microbiology, University of Queensland, Brisbane 4072, Australia. Tel.: 61-7-3365 4493; Fax: 61-7-3365 4388; E-mail:
D.Hume{at}cmcb.uq.edu.au.
1 The abbreviations used are: bp, base pair(s); CSF, colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; RLU, relative light units; CAT, chloramphenicol acetyltransferase.
2 X. Yue and D. A. Hume, unpublished observations.
3 D. A. Hume, unpublished observations.
4 D. A. Hume and M. C. Ostrowski, unpublished observations.
5 I. L. Ross and D. A. Hume, unpublished observations.
6 X. Yue, C. Chen, M. A. Stevenson, D. A. Hume, P. E. Auron, and S. K. Calderwood, submitted for publication.
7 M. Sweet and D. A. Hume, unpublished observations.
8 N. Angel, M. Cahill, A. King, M. C. Ostrowski, D. A. Hume, and A. I. Cassady, submitted for publication.
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REFERENCES |
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