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Departments of Microbiology and Biochemistry and Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Australia; and
Department of Molecular Genetics, Ohio State University, Columbus, OH 43210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In recent years, the targeted disruption of several genes (e.g., PU.1, c-fos, c-src) and the molecular characterization of natural mutations (op/op, mi/mi) have led to new insights into developmental processes and functions of macrophages and osteoclasts (6, 7, 8, 9, 10). In op/op and PU.1-/- mice, both cell types are severely affected, probably at the stage of a common precursor. The osteopetrotic op/op mouse carries an inactivating mutation of CSF-1 (macrophage CSF), an important survival and differentiation signal for macrophages and osteoclasts normally provided from the surrounding tissue (9). The receptor for CSF-1, c-Fms, is itself macrophage/osteoclast specific (11), and in the absence of CSF-1, macrophage and osteoclast numbers are severely reduced (12). PU.1 is an Ets family transcription factor (13) that binds key elements of macrophage-specific promoters, including that of c-fms (14), and the targeted disruption of the transcription factor PU.1 demonstrated its importance for the development of both cell types (6, 15, 16).
Three mutations of the transcription factor Mitf selectively affect osteoclast development or function and result in osteopetrosis (17, 18). Mitf, which is encoded by the microphthalmia gene (10), is a member of the large family of basic helix-loop-helix leucine zipper (bHLH-ZIP)3 transcription factors and, like other bHLH-ZIP proteins, forms homo- or heterodimers that bind a cognate DNA sequence termed the E box (CANNTG) (19). Sequence analysis and biochemical studies demonstrated a close relationship between Mitf and the three transcription factors, TFE3 (20), TFEB (21), and TFEC (22, 23), which are less well understood in terms of expression pattern and function. Together, these four transcription factors form the MiT subfamily of bHLH-ZIP transcription factors, and they have been shown to form heterodimers with each other, but not with known bHLH-ZIP factors of other subfamilies (19). Mitf itself is known to be expressed in melanocytes and to bind various E boxes with an extended site, termed M boxes, in promoters of several melanocyte-specific genes, e.g., tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2 (24, 25, 26). Numerous mutant alleles of the murine microphthalmia gene have been characterized that all affect pigment-producing melanocytes (17). By contrast, only a subset of Mitf mutations affects cells of hemopoietic origin (mast cells, osteoclasts, NK cells). These mutations are subtle variants that produce dominant-negative proteins that retain their ability to homo- and heterodimerize, but cannot bind to DNA (27). An obvious corollary is that osteoclasts express dimerization partners for Mitf.
Macrophages with mutations in Mitf have not been studied very thoroughly, and dramatic functional or developmental defects in macrophages have not been reported, with the exception of a reduction of superoxide dismutase activity (28). Hence, the most obvious explanation for the absence of an overt phenotype in the macrophage lineage of mi/mi is that the level or pattern of expression of MiT dimerization partners renders them resistant to the dominant-negative gene. Of course, the other alternative is that MiT transcription factors have no function in the macrophage lineage.
To address this issue and to investigate a possible role of MiT subfamily transcription factors in macrophages, we aimed to determine which MiT members are expressed during differentiation of the monocyte-macrophage lineage and how their expression is controlled.
In this study, we show that all four known members of the MiT family are expressed in macrophages, including Mitf, TFE3, TFEB, and the murine homologue of TFEC, which we cloned during this study. Interestingly, the expression of murine TFEC appeared restricted to cells of the monocyte-macrophage lineage. We show that the tissue-restricted expression of mTFEC is controlled by a typical TATA-less, macrophage-type promoter that contains several Ets motifs that bind the myeloid and B cell-restricted transcription factor PU.1. These findings establish mTFEC as a macrophage-specific transcription factor that might itself, individually or in concert with Mitf, TFE3, and TFEB, play a role in macrophage-specific gene regulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All chemical reagents used were purchased from Ajax Chemicals (Auburn, Australia), unless otherwise noted. Oligonucleotides were synthesized by Pacific Oligo (Lismore, Australia).
Cell culture
All cell lines used in this study were obtained from the American Type Culture Collection (Manassas, VA). The cell lines J774, WR19 M.1, WR19L, EL-4, MPC-11, MOPC31C, B16, and NIH3T3 and L929 were maintained in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (Life Technologies). RAW264.7 and M1 cell lines were cultivated in RPMI 1640 medium (BioWhittaker, Walkersville, MD) plus 10% FCS. Murine bone marrow-derived macrophages (BMMs) and thioglycolate-elicited peritoneal macrophages (TGE-PMs) were obtained and cultured as described previously (29). Osteoclast-like cells (OLCs) were generated in a coculture system of primary mouse calvarial osteoblasts with spleen cells in the presence of 1,25-dihydroxyvitamin D3, as described by Takahashi et al. (5).
RNA preparation and Northern blot analysis
Total RNA was isolated from different cell lines by the guanidine thiocyanate/acid phenol method (30). Electrophoresis, Northern blotting, and cDNA hybridization were conducted as described previously (31). The probes used were: a 1200-bp cDNA of the mTFEC coding region, an 816-bp PU.1 cDNA, and an oligonucleotide complementary to mouse 18S rRNA.
PCR amplifications
Total RNA from BMMs and in vitro differentiated osteoclasts was
reverse transcribed and, together with degenerate primers (bHLHZIP-S,
-AS; Fig. 2
), used as a template to amplify the conserved bHLH-ZIP
region of MiT subfamily members. Conditions were 94°C for 1 min,
52°C for 1 min, and 72°C for 2 min, repeated for 35 cycles.
Amplified cDNA fragments were subcloned, and several independent
colonies were used for restriction and sequencing analysis.
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To amplify genomic fragments of the proximal promoter region of
mTFEC, we used nested gene-specific primers (IN, OUT) and the
Promoter Finder DNA Walking Kit (Clontech), following manufacturer’s
instructions. Positions and sequence of oligonucleotides used for
cloning purposes are indicated in Fig. 2
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DNA sequence analysis
The cDNA sequencing was done by Dye Deoxy Terminator Cycle Sequencing (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions, and sequences were analyzed on the Applied Biosystems DNA Sequencing System (model 373A) by the Australian Genome Research Facility.
Plasmid construction and purification
PCR fragments were inserted into the plasmid vector pCR2.1 (TA Cloning Kit; Invitrogen, San Diego, CA) for sequencing and subcloning purposes. To generate the pGL2-TFEC reporter plasmid, a 615-bp fragment of the TFEC proximal promoter was subcloned into pGL2-B (Promega, Madison, WI) cut with XhoI and HindIII. The mouse PU.1 expression plasmid pECE-PU.1 and mouse Ets-2 expression plasmid pECE-Ets-2 were a gift from Dr. Richard Maki, (Burnham Institute, La Jolla, CA). For transient transfections, plasmids were isolated by the alkaline-SDS method and purified by two subsequent CsCl density-gradient centrifugations (32).
Transient DNA transfections
NIH3T3 fibroblasts and the RAW264.7 cell line were transfected using Superfect reagent (Qiagen, Chatsworth, CA), according to the manufacturer’s instructions. Briefly, 5 x 104 NIH3T3 cells or 8 x 104 RAW264.7 cells were seeded into six-well plates the day before transfection. A total of 0.6 µg reporter plasmid and 10 µl Superfect reagent was used to transfect the cells. In cotransfections, 0.2 µg of each expression plasmid and/or control plasmid was added to a total of 1 µg DNA. After 2 h, the cells were washed and supplemented with the appropriate medium. The transfected cells were cultivated for 22 h and harvested, and cell lysates were assayed for luciferase activity (Luciferase Reporter Gene Assay; Boehringer Mannheim) on a 1450 MicroBeta TRILUX (Wallac, Gaithersburg, MD). Activity was normalized to protein concentration measured using a modified Bradford assay (Bio-Rad) or in the case of cotransfections on cell number.
Nuclear extracts and electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared with a variation of the method of Osborn et al. (33). rPU.1 was prepared and purified as described elsewhere (34).
Oligonucleotides were end labeled with [
-32P]ATP
using T4 polynucleotide kinase. For the oligonucleotides etsbox1–6 and
PU box, the binding reaction contained 2.5 µg of nuclear extract
protein, 1 µg of poly d(I/C), 20 mM HEPES, pH 7.9, 40 mM KCl, 1 mM
DTT, 1 mM EDTA, pH 8, 5% glycerol, and 40 nmol of probe DNA in a final
volume of 10 µl. Samples were loaded onto 10% polyacrylamide gels
after standing at room temperature for 30 min. Buffers and running
conditions used have been described (34). Gels were fixed in 10%
acetic acid, dried, and autoradiographed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the expression of MiT subfamily members, we initially
performed reverse-transcriptase PCR with degenerate primers covering
the conserved basic helix-loop-helix region. A 269-bp fragment was
generated in murine BMMs and OLCs, cloned, and analyzed by sequencing
and restriction mapping (data not shown). Along with several fragments
corresponding to the known murine TFE3 and Mitf homologues, we
identified fragments for a murine homologue of rat and human TFEC (22, 23). Use of a corresponding control template DNA for TFEB showed that
it was less efficiently amplified with the degenerate primers;
therefore, we also used specific primers to analyze for MiT subfamily
gene expression in macrophage and nonmacrophage cell types. As shown in
Fig. 1, expression of all four members
could be detected in BMMs, TGE-PMs, and OLCs. The fragment for TFEC
could only be amplified in macrophage cell types and to a much lesser
extent in OLCs. The primers used to amplify TFE3 were designed to allow
the separation of the two isoforms, mTFE3-L and mTFE3-S. The latter
lacks the exon encoding the acidic domain that corresponds to a
transcriptional activation domain of mTFE3-L and acts as a
transcriptional repressor (35). In all cases, the larger fragment for
the transactivator TFE3-L was the predominant amplified product.
Sequence analysis of the murine homologue of TFEC
The restricted expression pattern of TFEC had not previously been
described. To allow further analysis of the murine homologue of TFEC, a
full-length cDNA was cloned by a modified RACE-PCR approach that allows
the 5'-mRNA end-dependent, selective amplification of full-length
transcripts. Identical start sites for two independent 5' fragments
indicate a major cap site at the first nucleotide of the cDNA. The
complete sequence is shown in Fig. 2. The
deduced amino acid sequence of the putative mTFEC protein shows 90%
identity with rTFEC and 77% identity with hTFEC-L. Similar to its
homologue in rat, the sequence of mTFEC lacks the exon encoding an
acidic domain that is present in the human homologue of TFEC (22, 23).
This exon corresponds to a transcriptional activation domain of
subfamily members TFE3 and Mitf (36, 37). PCR amplification of a
putative corresponding exon in mTFEC revealed no products for a longer
isoform that would include the activation exon (data not shown).
Macrophage-restricted expression pattern of mTFEC
Northern blot analysis of a wide range of cell lines confirmed our
initial observation that the expression of mTFEC is restricted to cells
of the mononuclear phagocyte lineage. As shown in Fig. 3A, a 1.8-kb RNA transcript
was detectable in BMM, TGE-PM, and the macrophage cell lines RAW264.7
and WR19 M.1, but not in any of the other cell types, including T cell
(WR19L, EL-4), B cell (MPC-11, MOPC31C), melanocyte (B16), and
fibroblast cell lines (NIH3T3, L929). A 3-kb RNA species that had been
shown to hybridize with a probe for rat TFEC could not be detected
(22). In OLCs, the message was barely visible in Northern blot
analysis, but in contrast to nonmyeloid cells, could be detected by PCR
(see Fig. 1). The same blot was probed for expression of the Ets
family transcription factor PU.1 that was expressed in macrophages,
osteoclasts, and B cells (Fig. 3A). To confirm the
association of TFEC with macrophage differentiation in primary cells, a
time course of total RNA was prepared from bone marrow cells cultivated
with CSF-1. Within the limits of detection of Northern blot analysis,
PU.1 was induced rapidly, whereas TFEC was induced later in the
differentiation process corresponding to markers such as
c-fms (Fig. 3B).
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The expression pattern suggested that TFEC might be a
macrophage-specific member of the MiT subfamily of bHLH-ZIP
transcription factors. In many cases, the basal tissue-restricted
expression of genes is controlled by cis-acting elements
located within the 5'-proximal promoter, so we cloned and sequenced a
corresponding region of the murine TFEC gene. Promoter Finder DNA
Walking (Clontech) was used to isolate a 615-bp genomic fragment of the
5'-proximal TFEC promoter that was subsequently sequenced and analyzed
for putative regulatory elements. The sequence, as shown in Fig. 4, shares remarkably similar features
with the promoters of several genes that are specifically expressed in
macrophages (e.g., c-fms (14, 34), PU.1 (38), CD11b (39),
CD18 (40), FcRI (41, 42), FcRIII (43), c-fes (44), the
macrophage scavenger receptor (45), and Nramp-1 (natural
resistance-associated macrophage protein) (46)). As in the promoters of
the above listed genes, the 5'-proximal region of TFEC is characterized
by the absence of TATA boxes, consensus initiator sequences, or GC-rich
regions found in housekeeping genes that normally determine
transcriptional initiation. It also lacks Sp1 or CCAAT-box sequences,
and instead contains six motifs that might be recognized by
transcription factors of the Ets family, including PU.1 (47). Other
identified motifs include putative binding sites for nuclear
factor-
B, C/EBP-ß, c-myc, and AP-1, which might also be
important for myeloid-specific gene expression in some cases (45, 48, 49, 50, 51, 52).
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Growing evidence indicates an important role of PU.1 recognition
motifs for the expression of macrophage-restricted genes (14, 29, 38, 39, 43, 45). Six Ets-like sequences are present in the TFEC promoter
that could possibly bind PU.1. Fig. 6
shows EMSA using double-stranded oligonucleotides of corresponding Ets
sequences in the TFEC promoter. Four of these sequences (etsbox-1, -3,
-4, -6) competed with the binding of rPU.1 to the PU-box
oligonucleotide from the SV40 enhancer (Fig. 6A) that
represents a known, high affinity binding site for PU.1 and was used as
a reference. Subsequently, efficient direct binding of rPU.1 was
demonstrated for etsbox-1, -3, -4, and -6 oligonucleotides (Fig. 6B), whereas etsbox-2 was only weakly bound. No PU.1 binding
of etsbox-5 was detectable. Etsbox-6 represented the highest affinity
binding site. Compared with the SV40 enhancer binding site, all four
binding sites were less effectively bound by PU.1. Similar results were
obtained using nuclear extracts from RAW264.7 cells (data not shown).
The specific binding of each oligonucleotide to PU.1 in nuclear
extracts was further established by competition with cold PU-box
oligonucleotide. The binding activity of this major complex was
reduced, and its mobility was slightly increased by addition of
preimmune serum, but was selectively abolished by specific
anti-PU.1 antiserum. Fig. 6C shows a representative EMSA
using etsbox-6 oligonucleotide and nuclear extracts from RAW264.7
cells. Similar results were obtained for etsbox-1, -3, and -4. No
binding of nuclear proteins could be detected with etsbox-2 or -5
oligonucleotides (data not shown).
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, both PU.1 and Ets-2 were also able to
activate the TFEC promoter construct, and coexpression of PU.1 with
Ets-2 had an additive effect in the fibroblast cell line.
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Fig. 8 highlights major
characteristics found in the region of transcriptional initiation of a
range of well-characterized myeloid-specific promoters. The aligned
sequences generally contain multiple Ets-like recognition motifs. In
many cases, these purine-rich sequences have been shown to be strictly
necessary for basal promoter activity. The comparison also indicates
another common, yet uncharacterized, motif (CCAGTG) that can be found
in many myeloid promoters. This region will be subject to further
investigations. Initial experiments do not indicate the binding of
macrophage-restricted proteins from nuclear extracts to this site. The
macrophage-specific promoters of c-fms and TFEC share the
strongest similarity, including number and position of Ets-like motifs.
A weak Ets-like binding motif (CAGGAA) that is essential for the
activity of the c-fms promoter (29) and the promoter of PU.1
(38) itself is also present in the TFEC promoter at a similar position.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A proposed role of Mitf and related transcription factors in osteoclast development (18, 27) tempted us to investigate a possible role of the MiT transcription factor family in macrophages that share a common precursor with osteoclasts (2). The PCR-based approach, which was designed to identify new and already known members of the MiT subfamily in macrophages, resulted in several interesting observations. In general, transcripts for all known MiT members could be identified in macrophages and OLCs. The presence of TFE3 in osteoclasts confirms recent findings of Weilbaecher et al. (18), who identified TFE3 as a dimerization partner of Mitf in these cells. Although the PCR-based approach was able to identify the mRNA of MiT members in macrophages and OLCs, it does not allow quantitative statements about transcript levels or extent of protein expression. In the case of cocultured OLCs, the detected fragments could conceivably originate from 5–10% residual primary osteoblasts that may contaminate the OLC-RNA preparation. Although no TFEC expression and only low levels of TFEB expression were detectable in RNA from primary osteoblasts (data not shown), we cannot rule out that their expression is induced in coculture with osteoclast precursors. However, these results suggest that TFEB or TFEC might also be possible dimerization partners for Mitf and TFE3 in osteoclasts and certainly in macrophages. Interestingly, transcripts for TFE3 and TFEB could also be detected in the melanoma cell line B16, which was used as a positive control for the expression of Mitf. This observation suggests that mature melanocytes may be able to express other MiT factors together with Mitf.
The previously undescribed murine homologue of TFEC appears restricted to macrophage-type cells, and this observation became the major focus of this study. Cloning and sequence analysis of mTFEC revealed a very similar structure and close homology with rat TFEC (22). The sequence structure of the human homologue TFEC-L is less homologous and includes an exon encoding an acidic activation domain that corresponds to a transcriptional activation domain of subfamily members TFE3 and Mitf (23). Transient transfection studies revealed that hTFEC-L could slightly activate, although much less than Mitf or TFE3, or inhibit transactivation of different reporters, depending on the cell lines used (23). Based on sequence comparison, the properties of mTFEC probably resemble those of the rat homologue, which also lacks the exon encoding the acidic domain, and acts as a transcriptional repressor of TFE3 in transient cotransfections (22).
Northern blot analysis of different cell lines confirmed macrophage specificity of TFEC. The limited data on rat and human TFEC are not inconsistent with a macrophage-restricted expression pattern. The rat homologue of mTFEC was cloned from a chondrosarcoma, but with the exception of the primary tumor, no TFEC expression could be detected in any specific cell type, including chondrosarcoma, fibroblast, myoblast, and myeloma cell lines (22). A major portion of tumor-infiltrating leukocytes are macrophages, which could explain the expression of TFEC in the primary tumor. The pattern of tissue expression described for 8-wk-old rats, with the highest mRNA levels found in kidney, spleen, and lung; lower levels in liver, testis, and muscle; and even less in heart or brain (22), could be attributed to the appearance of macrophages in those tissues. Finally, the human homologue TFEC-L was recently cloned from the promonocytic leukemia cell line THP-1 (23).
With the analysis of a growing number of macrophage-specific genes, it has become clear that their 5'-proximal promoter regions share several features that distinguish them from other genes. A typical macrophage-type promoter appears to lack TATA boxes, consensus initiator sequences, or GC-rich regions found in housekeeping genes, and instead contains multiple purine-rich sequence motifs that are recognized by transcription factors of the Ets family (14, 29, 38, 39, 42, 43, 44, 51, 53, 54, 55). We cloned and sequenced a 600-bp region of the proximal TFEC promoter and found exactly these characteristics of macrophage-restricted promoters. In addition, the 600-bp promoter was efficiently active in a macrophage cell line, but not in fibroblasts, indicating that it contains the regulatory elements necessary for basal macrophage-specific expression. These regulatory elements most likely include at least one of the five binding sites for the Ets family transcription factor PU.1 that were identified by EMSA. In fibroblasts, PU.1 was able to transactivate the TFEC promoter alone and cooperatively with a second Ets factor (Ets-2), a characteristic described previously for the c-fms promoter (29). The temporal sequence of appearance of PU.1 and TFEC in CSF-1-stimulated bone marrow cultures also supports the view that PU.1 is a regulator of TFEC expression.
A direct comparison of proximal promoter regions of myeloid-restricted genes with the promoter of TFEC revealed their common structure, with marked structural similarities especially between c-fms and TFEC. As in the TFEC promoter, transcription initiation in the promoters of c-fes (44), c-fms (14), and PU.1 (38) occurs around a CAGGAA sequence, and mutation of this element greatly reduced the activity of the latter three promoters in myeloid cells. The typical absence of TATA boxes or other conventional initiator sequences and the ability of PU.1 to bind directly to TFIID or the retinoblastoma gene product (56) suggest an alternative, macrophage-specific mechanism for transcriptional initiation that might involve interactions of different Ets family transcription factors and a myeloid-specific initiator element such as the CAGGAA sequence in c-fms, c-fes, PU.1, and TFEC.
Further studies will be needed to determine a functional role for mTFEC in macrophages. The published observations on rTFEC and hTFEC-L suggest that mTFEC would most likely act as a transcriptional repressor of other coexpressed MiT family members (22, 23). In all macrophage cell lines studied to date, TFEC was coexpressed with other subfamily members, and in certain macrophage cell types (e.g., BMM, TGE-PM), all four members, TFE3, TFEB, Mitf, and TFEC, can be detected. In theory, this would allow the formation of many different heterodimer combinations that could, on one hand, have individual properties, but, on the other hand, may also have overlapping functions. In this context, TFEC could be important in balancing the actions of coexpressed transactivators Mitf, TFE3, and TFEB at certain stages of development or in special macrophage subpopulations. It will be of interest to determine whether the relative levels of these four factors are regulated by the wide range of lymphokines, bacterial products, and other agents that modulate macrophage differentiation.
Clearly, further studies are required to fully understand the function of MiT transcription factors in osteoclasts or macrophages. These studies may include knockout mutations of all members and their combinatorial analysis. Other important issues include the regulation of MiT-protein expression in macrophages and osteoclasts and, equally important, the identification of transcriptional targets in osteoclasts or macrophages that are currently unknown.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David A. Hume, Department of Microbiology, University of Queensland, Brisbane, Q4072, Australia. E-mail address:
3 Abbreviations used in this paper: bHLH-ZIP, basic helix-loop-helix leucine zipper; BMM, bone marrow-derived macrophage; EMSA, electrophoretic mobility shift assay; hTFE, human TFE; mTFE, murine TFE; MiT, microphthalmia-TFE; nt, nucleotide; OLC, osteoclast-like cell; RACE, rapid amplification of complementary deoxyribonucleic acid ends; TGE-PM, thioglycolate-elicited peritoneal macrophage.
Received for publication July 27, 1998. Accepted for publication October 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor B. Proc. Natl. Acad. Sci. USA 86:2336. receptor. Mol. Cell. Biol. 14:5023. responsiveness and basal expression of the myeloid human FcR1b gene is mediated by a functional PU.1 site and a transcription initiator consensus. J. Exp. Med. 179:1985.RIIIA promoter. EMBO J. 13:3852.[Medline]
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