Transcription Factors Helix-Loop-Helix Leucine Zipper the Microphthalmia-TFE Subfamily of Basic TFEC Is a Macrophage-Restricted

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TFEC Is a Macrophage-Restricted Member of the Microphthalmia-TFE Subfamily of Basic Helix-Loop-Helix Leucine Zipper Transcription Factors This information is current as of October 3, 2021. Michael Rehli, Agnieszka Lichanska, A. Ian Cassady, Michael C. Ostrowski and David A. Hume J Immunol 1999; 162:1559-1565; ; http://www.jimmunol.org/content/162/3/1559 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. TFEC Is a Macrophage-Restricted Member of the Microphthalmia-TFE Subfamily of Basic Helix-Loop-Helix Leucine Zipper Transcription Factors1

Michael Rehli,* Agnieszka Lichanska,* A. Ian Cassady,* Michael C. Ostrowski,† and David A. Hume2*

The murine homologue of the TFEC was cloned as part of an analysis of the expression of the microphthalmia-TFE (MiT) subfamily of transcription factors in macrophages. TFEC, which most likely acts as a transcriptional repressor in heterodimers with other MiT family members, was identified in cells of the mononuclear phagocyte lineage, coexpressed with all other known MiT subfamily members (Mitf, TFE3, TFEB). Northern blot analysis of several different cell lineages indicated that the expression of murine TFEC (mTFEC) was restricted to macrophages. A 600-bp fragment of the TATA-less putative proximal promoter of Downloaded from TFEC shares features with many known macrophage-specific promoters and preferentially directs luciferase expression in the RAW264.7 macrophage cell line in transient transfection assays. Five of six putative Ets motifs identified in the TFEC promoter bind the macrophage-restricted transcription factor PU.1 under in vitro conditions and in transfected 3T3 fibroblasts; the minimal luciferase activity of the TFEC promoter could be induced by coexpression of PU.1 or the related transcription factor Ets-2. The functional importance of the tissue-restricted expression of TFEC and a possible role in macrophage-specific gene regulation require further investigation, but are likely to be linked to the role of the other MiT family members in this lineage. The Journal http://www.jimmunol.org/ of Immunology, 1999, 162: 1559–1565.

onocytes, macrophages, and their precursors represent entiation signal for macrophages and osteoclasts normally pro- different stages of differentiation in a functionally di- vided from the surrounding tissue (9). The receptor for CSF-1, M verse hemopoietic cell lineage (1). The mature end- c-Fms, is itself macrophage/osteoclast speciﬁc (11), and in the points of this lineage not only include the heterogenous group of absence of CSF-1, macrophage and osteoclast numbers are se- tissue macrophages, but also two other specialized cell types: the verely reduced (12). PU.1 is an Ets family transcription factor multinucleated bone-resorbing osteoclast and the Ag-presenting (13) that binds key elements of macrophage-speciﬁc promoters,

dendritic cell (2, 3). Differentiation of common progenitor cells to including that of c-fms (14), and the targeted disruption of the by guest on October 3, 2021 particular fates is thought to be under the stringent control of the transcription factor PU.1 demonstrated its importance for the surrounding microenvironment and local tissue-specific factors, development of both cell types (6, 15, 16). acting upon specific transcription factors that in turn direct lineage- Three mutations of the transcription factor Mitf selectively af- specific gene expression. In vitro culture systems that allow the fect osteoclast development or function and result in osteopetrosis generation of osteoclast- or dendritic-like cells from monocytes (17, 18). Mitf, which is encoded by the microphthalmia gene (10), reflect the complex structure of this process (3–5). is a member of the large family of basic helix-loop-helix leucine In recent years, the targeted disruption of several genes (e.g., zipper (bHLH-ZIP)3 transcription factors and, like other bHLH- PU.1, c-fos,c-src) and the molecular characterization of natural ZIP proteins, forms homo- or heterodimers that bind a cognate mutations (op/op, mi/mi) have led to new insights into devel- DNA sequence termed the E box (CANNTG) (19). Sequence anal- opmental processes and functions of macrophages and oste- ysis and biochemical studies demonstrated a close relationship be- Ϫ Ϫ oclasts (6–10). In op/op and PU.1 / mice, both cell types are tween Mitf and the three transcription factors, TFE3 (20), TFEB severely affected, probably at the stage of a common precursor. (21), and TFEC (22, 23), which are less well understood in terms The osteopetrotic op/op mouse carries an inactivating mutation of expression pattern and function. Together, these four transcrip- of CSF-1 (macrophage CSF), an important survival and differ- tion 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 *Departments of Microbiology and Biochemistry and Centre for Molecular and Cel- (19). Mitf itself is known to be expressed in melanocytes and to † lular Biology, University of Queensland, Brisbane, Australia; and Department of bind various E boxes with an extended site, termed M boxes, in Molecular Genetics, Ohio State University, Columbus, OH 43210 promoters of several melanocyte-specific genes, e.g., tyrosinase, Received for publication July 27, 1998. Accepted for publication October 26, 1998. tyrosinase-related protein-1, and tyrosinase-related protein-2 (24– The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance 26). Numerous mutant alleles of the murine microphthalmia gene with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Sequence data from this article have been deposited with GenBank/EMBL Data Libraries under Accession Numbers AF077742 and AF077743. This work was sup- ported by the National Health and Medical Research Council of Australia. M.R. is a 3 Abbreviations used in this paper: bHLH-ZIP, basic helix-loop-helix leucine zipper; recipient of a fellowship from the Deutsche Forschungsgemeinschaft. BMM, bone marrow-derived macrophage; EMSA, electrophoretic mobility shift as- 2 Address correspondence and reprint requests to Dr. David A. Hume, Department of say; hTFE, human TFE; mTFE, murine TFE; MiT, microphthalmia-TFE; nt, nucle- Microbiology, University of Queensland, Brisbane, Q4072, Australia. E-mail address: otide; OLC, osteoclast-like cell; RACE, rapid amplification of complementary de- [email protected] oxyribonucleic acid ends; TGE-PM, thioglycolate-elicited peritoneal macrophage.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 1560 MACROPHAGE-RESTRICTED EXPRESSION OF TFEC have been characterized that all affect pigment-producing melano- 72°C for 2 min, repeated for 35 cycles. Amplified cDNA fragments were cytes (17). By contrast, only a subset of Mitf mutations affects cells subcloned, and several independent colonies were used for restriction and of hemopoietic origin (mast cells, osteoclasts, NK cells). These sequencing analysis. The amplification of specific fragments for MiT transcripts was done mutations are subtle variants that produce dominant-negative pro- using the Expand High Fidelity PCR system (Boehringer Mannheim, In- teins that retain their ability to homo- and heterodimerize, but can- dianapolis, IN). Products were sequenced to confirm their identity. Primer not bind to DNA (27). An obvious corollary is that osteoclasts positions and fragment sizes are indicated in Fig. 1. express dimerization partners for Mitf. For the cloning of 5Ј- and 3Ј-cDNA fragments for mTFEC, a modified RACE-PCR approach was used. Full-length cDNA from total BMM RNA Macrophages with mutations in Mitf have not been studied very was generated with the SMART-PCR cDNA Synthesis Kit (Clontech, Palo thoroughly, and dramatic functional or developmental defects in Alto, CA), according to the manufacturer’s instructions. The cDNA con- macrophages have not been reported, with the exception of a re- taining the SMART oligonucleotide, which is added to the 5Ј end of full- duction of superoxide dismutase activity (28). Hence, the most length mRNA transcripts during reverse transcription, was used to amplify Ј Ј obvious explanation for the absence of an overt phenotype in the 5 - and 3 -cDNA fragments for mTFEC. Nested PCR with gene-specific primers (5ЈR1, 5ЈR2Ј)anda5Ј-PCR primer complementary to the SMART macrophage lineage of mi/mi is that the level or pattern of expres- oligonucleotide yielded an 850-bp fragment representing the full-length 5Ј sion of MiT dimerization partners renders them resistant to the end of TFEC, which was subcloned. Two independent clones were se- dominant-negative gene. Of course, the other alternative is that quenced and contained identical 5Ј ends. A similar approach using gene- MiT transcription factors have no function in the macrophage specific primers 3ЈR1 and 3ЈR2 and a 3Ј-oligo(dT) primer allowed the isolation of a 1-kb TFEC 3Ј-end fragment. lineage. To amplify genomic fragments of the proximal promoter region of To address this issue and to investigate a possible role of MiT mTFEC, we used nested gene-specific primers (IN, OUT) and the Promoter subfamily transcription factors in macrophages, we aimed to de- Finder DNA Walking Kit (Clontech), following manufacturer’s instruc- Downloaded from termine which MiT members are expressed during differentiation tions. Positions and sequence of oligonucleotides used for cloning purposes of the monocyte-macrophage lineage and how their expression is are indicated in Fig. 2. controlled. In this study, we show that all four known members of the MiT DNA sequence analysis family are expressed in macrophages, including Mitf, TFE3, The cDNA sequencing was done by Dye Deoxy Terminator Cycle Se- TFEB, and the murine homologue of TFEC, which we cloned quencing (Applied Biosystems, Foster City, CA), according to the manu- during this study. Interestingly, the expression of murine TFEC facturer’s instructions, and sequences were analyzed on the Applied Bio- http://www.jimmunol.org/ appeared restricted to cells of the monocyte-macrophage lineage. systems DNA Sequencing System (model 373A) by the Australian Genome Research Facility. 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-re- Plasmid construction and purification stricted transcription factor PU.1. These findings establish mTFEC PCR fragments were inserted into the plasmid vector pCR2.1 (TA Cloning as a macrophage-specific transcription factor that might itself, in- Kit; Invitrogen, San Diego, CA) for sequencing and subcloning purposes. dividually or in concert with Mitf, TFE3, and TFEB, play a role in To generate the pGL2-TFEC reporter plasmid, a 615-bp fragment of the TFEC proximal promoter was subcloned into pGL2-B (Promega, Madison, macrophage-specific gene regulation. by guest on October 3, 2021 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 Materials and Methods from Dr. Richard Maki, (Burnham Institute, La Jolla, CA). For transient Chemicals transfections, plasmids were isolated by the alkaline-SDS method and purified by two subsequent CsCl density-gradient centrifugations (32). All chemical reagents used were purchased from Ajax Chemicals (Auburn, Australia), unless otherwise noted. Oligonucleotides were synthesized by Pacific Oligo (Lismore, Australia). Transient DNA transfections Cell culture NIH3T3 fibroblasts and the RAW264.7 cell line were transfected using Superfect reagent (Qiagen, Chatsworth, CA), according to the manufac- All cell lines used in this study were obtained from the American Type turer’s instructions. Briefly, 5 ϫ 104 NIH3T3 cells or 8 ϫ 104 RAW264.7 Culture Collection (Manassas, VA). The cell lines J774, WR19 M.1, cells were seeded into six-well plates the day before transfection. A total of WR19L, EL-4, MPC-11, MOPC31C, B16, and NIH3T3 and L929 were 0.6 ␮g reporter plasmid and 10 ␮l Superfect reagent was used to transfect maintained in DMEM (Life Technologies, Gaithersburg, MD) supple- the cells. In cotransfections, 0.2 ␮g of each expression plasmid and/or mented with 10% FCS (Life Technologies). RAW264.7 and M1 cell lines control plasmid was added to a total of 1 ␮g DNA. After 2 h, the cells were were cultivated in RPMI 1640 medium (BioWhittaker, Walkersville, MD) washed and supplemented with the appropriate medium. The transfected plus 10% FCS. Murine bone marrow-derived macrophages (BMMs) and cells were cultivated for 22 h and harvested, and cell lysates were assayed thioglycolate-elicited peritoneal macrophages (TGE-PMs) were obtained for luciferase activity (Luciferase Reporter Gene Assay; Boehringer Mann- and cultured as described previously (29). Osteoclast-like cells (OLCs) heim) on a 1450 MicroBeta TRILUX (Wallac, Gaithersburg, MD). Activ- were generated in a coculture system of primary mouse calvarial osteo- ity was normalized to protein concentration measured using a modified blasts with spleen cells in the presence of 1,25-dihydroxyvitamin D3,as Bradford assay (Bio-Rad) or in the case of cotransfections on cell number. described by Takahashi et al. (5).

RNA preparation and Northern blot analysis Nuclear extracts and electrophoretic mobility shift assay Total RNA was isolated from different cell lines by the guanidine thiocy- (EMSA) anate/acid phenol method (30). Electrophoresis, Northern blotting, and Nuclear extracts were prepared with a variation of the method of Osborn et cDNA hybridization were conducted as described previously (31). The al. (33). rPU.1 was prepared and purified as described elsewhere (34). probes used were: a 1200-bp cDNA of the mTFEC coding region, an Oligonucleotides were end labeled with [␥-32P]ATP using T4 816-bp PU.1 cDNA, and an oligonucleotide complementary to mouse polynucleotide kinase. For the oligonucleotides etsbox1–6 and PU box, 18S rRNA. the binding reaction contained 2.5 ␮g of nuclear extract protein, 1 ␮g PCR amplifications 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 Total RNA from BMMs and in vitro differentiated osteoclasts was reverse of 10 ␮l. Samples were loaded onto 10% polyacrylamide gels after transcribed and, together with degenerate primers (bHLHZIP-S, -AS; Fig. standing at room temperature for 30 min. Buffers and running condi- 2), used as a template to amplify the conserved bHLH-ZIP region of MiT tions used have been described (34). Gels were fixed in 10% acetic acid, subfamily members. Conditions were 94°C for 1 min, 52°C for 1 min, and dried, and autoradiographed. The Journal of Immunology 1561

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, FIGURE 1. Gene expression of MiT family members. Reverse-tran- but in contrast to nonmyeloid cells, could be detected by PCR (see scriptase PCR was performed on RNA from indicated cell types using Fig. 1). The same blot was probed for expression of the Ets family gene-specific primers for Mitf (nt 464..487, nt 847..873; Accession Num- transcription factor PU.1 that was expressed in macrophages, os- ber Z23066), TFE3 (nt 71..97, nt 729..755; Accession Number S76673), teoclasts, and B cells (Fig. 3A). To confirm the association of TFEB (nt 729..754, nt 1113..1134; Accession Number AF079095), TFEC TFEC with macrophage differentiation in primary cells, a time (nt 557..581, nt 876..900; Accession Number AF077742), and ␤-actin (nt Downloaded from 414..435, nt 781..808; Accession Number X03672). PCR reactions for in- course of total RNA was prepared from bone marrow cells culti- dividual genes were performed in parallel, with increasing cycle numbers vated with CSF-1. Within the limits of detection of Northern blot to allow for an analysis in the exponential range. analysis, PU.1 was induced rapidly, whereas TFEC was induced later in the differentiation process corresponding to markers such as c-fms (Fig. 3B).

Results Isolation and macrophage-speciﬁc activity of the proximal http://www.jimmunol.org/ MiT subfamily gene expression in macrophages and osteoclasts TFEC promoter To assess the expression of MiT subfamily members, we initially The expression pattern suggested that TFEC might be a mac- performed reverse-transcriptase PCR with degenerate primers cov- rophage-speciﬁc member of the MiT subfamily of bHLH-ZIP ering 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 by guest on October 3, 2021 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 5Ј-mRNA end-dependent, selective amplification of full-length transcripts. Identical start sites for two independent 5Ј fragments indicate a major cap site at the first nu- cleotide of the cDNA. The complete sequence is shown in Fig. 2. Ј Ј The deduced amino acid sequence of the putative mTFEC protein FIGURE 2. cDNA sequence of mTFEC. 5 and 3 fragments of the mTFEC transcript were amplified by a modified RACE-PCR technique, shows 90% identity with rTFEC and 77% identity with hTFEC-L. cloned, and sequenced. The complete cDNA is shown and contains an open Similar to its homologue in rat, the sequence of mTFEC lacks the reading frame encoding a protein of 317 amino acids in length. The basic exon encoding an acidic domain that is present in the human ho- helix-loop-helix region is marked as a grey box, the leucine residues of the mologue of TFEC (22, 23). This exon corresponds to a transcrip- zipper as grey spheres. The position of a consensus polyadenylation site is tional activation domain of subfamily members TFE3 and Mitf underlined, and sequences and positions of primers used for cloning pur- (36, 37). PCR amplification of a putative corresponding exon in poses are indicated. 1562 MACROPHAGE-RESTRICTED EXPRESSION OF TFEC

FIGURE 4. Sequence of the proximal promoter region of TFEC. A genomic fragment of the 5Ј-proximal TFEC promoter was cloned via Pro- moter Finder DNA Walking and sequenced. Putative binding sites for several transcription factors that were identiﬁed using the TRANSFAC data- base (57) are indicated. Dashed lines mark the positions and sequences of etsbox-1–6 oligonucleotides, which were used for EMSA.

the macrophage cell line RAW264.7, approximately 60 times over

the activity of pGL2-B (Fig. 5). Downloaded from

PU.1 binding and transactivation of the TFEC promoter 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 http://www.jimmunol.org/ double-stranded oligonucleotides of corresponding Ets sequences FIGURE 3. Northern blot analysis of TFEC expression. A, Total RNA in the TFEC promoter. Four of these sequences (etsbox-1, -3, -4, from different macrophage and nonmacrophage cell types, or B,anRNA -6) competed with the binding of rPU.1 to the PU-box oligonu- time course of CSF-1-induced macrophage differentiation from bone mar- cleotide from the SV40 enhancer (Fig. 6A) that represents a row cells was probed for TFEC, PU.1, and 18S rRNA to normalize loading. known, high afﬁnity binding site for PU.1 and was used as a ref- erence. 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

transcription factors. In many cases, the basal tissue-restricted by guest on October 3, 2021 etsbox-5 was detectable. Etsbox-6 represented the highest affinity expression of genes is controlled by cis-acting elements located binding site. Compared with the SV40 enhancer binding site, all within the 5Ј-proximal promoter, so we cloned and sequenced a four binding sites were less effectively bound by PU.1. Similar corresponding region of the murine TFEC gene. Promoter results were obtained using nuclear extracts from RAW264.7 cells Finder DNA Walking (Clontech) was used to isolate a 615-bp (data not shown). The specific binding of each oligonucleotide to genomic fragment of the 5Ј-proximal TFEC promoter that was PU.1 in nuclear extracts was further established by competition subsequently sequenced and analyzed for putative regulatory with cold PU-box oligonucleotide. The binding activity of this elements. The sequence, as shown in Fig. 4, shares remarkably major complex was reduced, and its mobility was slightly in- similar features with the promoters of several genes that are creased by addition of preimmune serum, but was selectively abol- specifically expressed in macrophages (e.g., c-fms (14, 34), ished by specific anti-PU.1 antiserum. Fig. 6C shows a represen- PU.1 (38), CD11b (39), CD18 (40), Fc␥RI (41, 42), Fc␥RIII tative EMSA using etsbox-6 oligonucleotide and nuclear extracts (43), c-fes (44), the macrophage scavenger receptor (45), and from RAW264.7 cells. Similar results were obtained for etsbox-1, Nramp-1 (natural resistance-associated macrophage protein) -3, and -4. No binding of nuclear proteins could be detected with (46)). As in the promoters of the above listed genes, the 5Ј- etsbox-2 or -5 oligonucleotides (data not shown). proximal region of TFEC is characterized by the absence of Our group has shown recently that in NIH3T3 fibroblasts, which TATA boxes, consensus initiator sequences, or GC-rich regions lack nuclear PU.1, the coexpression of PU.1 and/or Ets-2 activated 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-␤,c-myc, and AP-1, which might also be important for myeloid-specific gene expression in some cases (45, 48–52). The sequence structure of the proximal promoter suggested that this region might be implicated in the tissue-restricted expression FIGURE 5. The TFEC promoter is activated specifically in macro- of the TFEC gene. To test this possibility, we inserted the TFEC- phages. The fibroblast cell line NIH3T3 and the macrophage cell line promoter fragment into the promoterless vector pGL2-B and as- RAW264.7 were transfected with the TFEC-promoter construct, as de- sayed its reporter activity in transient transfections. Whereas in scribed in Materials and Methods. Values represent the luciferase activity NIH3T3 fibroblasts the construct was only slightly more active relative to the empty control vector pGL2-B, and are the mean ϩ SD than the promoterless control vector, it was effectively activated in obtained from three independent experiments. The Journal of Immunology 1563

FIGURE 6. PU.1 binding to Ets motifs of the TFEC promoter in EMSA. A, rPU.1 protein and the labeled SV40 PU-box oligonucleotide were used in competition tests with unlabeled etsbox oligonucleotides 1–6. B, EMSA of rPU.1 with individual ets boxes compared with SV40 PU box. C, EMSA of RAW264.7 nuclear proteins with labeled etsbox-6 oligonucleotide. PU.1 was competed by cold etsbox-6 itself and by the SV40 PU box. The anti-PU.1 serum significantly interrupted complex formation. The top of the gel and positions of free probe and shifted PU.1 are indicated in each case. reporter constructs containing the proximal promoters of human does not allow quantitative statements about transcript levels or and mouse c-fms or an artificial minimal promoter consisting of extent of protein expression. In the case of cocultured OLCs, the Downloaded from two copies of the SV40 PU-box element (29). As shown in Fig. 7, detected fragments could conceivably originate from 5–10% re- both PU.1 and Ets-2 were also able to activate the TFEC promoter sidual primary osteoblasts that may contaminate the OLC-RNA construct, and coexpression of PU.1 with Ets-2 had an additive preparation. Although no TFEC expression and only low levels of effect in the fibroblast cell line. TFEB expression were detectable in RNA from primary osteoblasts (data not shown), we cannot rule out that their expression is Comparison with other macrophage-type promoters induced in coculture with osteoclast precursors. However, these http://www.jimmunol.org/ Fig. 8 highlights major characteristics found in the region of tran- results suggest that TFEB or TFEC might also be possible dimer- scriptional initiation of a range of well-characterized myeloid-spe- ization partners for Mitf and TFE3 in osteoclasts and certainly in cific promoters. The aligned sequences generally contain multiple macrophages. Interestingly, transcripts for TFE3 and TFEB could Ets-like recognition motifs. In many cases, these purine-rich se- also be detected in the melanoma cell line B16, which was used as quences have been shown to be strictly necessary for basal pro- a positive control for the expression of Mitf. This observation sug- moter activity. The comparison also indicates another common, gests that mature melanocytes may be able to express other MiT yet uncharacterized, motif (CCAGTG) that can be found in many factors together with Mitf. myeloid promoters. This region will be subject to further investi- The previously undescribed murine homologue of TFEC ap- gations. Initial experiments do not indicate the binding of mac- pears restricted to macrophage-type cells, and this observation be- by guest on October 3, 2021 rophage-restricted proteins from nuclear extracts to this site. The came the major focus of this study. Cloning and sequence analysis macrophage-specific promoters of c-fms and TFEC share the stron- of mTFEC revealed a very similar structure and close homology gest similarity, including number and position of Ets-like motifs. A with rat TFEC (22). The sequence structure of the human homo- weak Ets-like binding motif (CAGGAA) that is essential for the logue TFEC-L is less homologous and includes an exon encoding activity of the c-fms promoter (29) and the promoter of PU.1 (38) an acidic activation domain that corresponds to a transcriptional itself is also present in the TFEC promoter at a similar position. activation domain of subfamily members TFE3 and Mitf (23). Transient transfection studies revealed that hTFEC-L could Discussion slightly activate, although much less than Mitf or TFE3, or inhibit transactivation of different reporters, depending on the cell lines In this study, we present evidence for a novel macrophage-re- used (23). Based on sequence comparison, the properties of stricted transcription factor. We cloned the murine homologue of mTFEC probably resemble those of the rat homologue, which also TFEC during an attempt to characterize the MiT subfamily of bHLH-ZIP transcription factors in macrophages, and show in this work that its expression is highly restricted to cells of monocyte/ macrophage origin. Characterization of the putative proximal promoter of mTFEC reveals typical macrophage-specific features and supports placement of TFEC within the group of macrophage-specific genes. 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 FIGURE 7. PU.1 and Ets-2 transactivate the TFEC promoter in fibro- members could be identified in macrophages and OLCs. The pres- blasts. The fibroblast cell line NIH3T3 was cotransfected with the TFEC- ence of TFE3 in osteoclasts confirms recent findings of Weilbae- promoter construct and expression plasmids of PU.1 and Ets-2, as de- cher et al. (18), who identified TFE3 as a dimerization partner of scribed in Materials and Methods. Values represent the luciferase activity Mitf in these cells. Although the PCR-based approach was able to relative to the empty control vector pECE and are the mean ϩ SD obtained identify the mRNA of MiT members in macrophages and OLCs, it from three independent experiments. 1564 MACROPHAGE-RESTRICTED EXPRESSION OF TFEC

FIGURE 8. Comparison of proximal promoter regions of several macrophage-restricted genes. The sequence of promoter regions of indicated genes was aligned using the CCAGNG motif that was found in proximal promoters of many myeloid-restricted genes. Published transcription initiation sites are indicated by arrows, and Ets-like GGAA motifs on either strand are marked as grey spheres. Black spheres indicate the position of motifs that greatly reduce myeloid-specific activity when mutated. lacks the exon encoding the acidic domain, and acts as a transcrip- The typical absence of TATA boxes or other conventional initiator Downloaded from tional repressor of TFE3 in transient cotransfections (22). sequences and the ability of PU.1 to bind directly to TFIID or the Northern blot analysis of different cell lines confirmed macro- retinoblastoma gene product (56) suggest an alternative, macrophage specificity of TFEC. The limited data on rat and human phage-specific mechanism for transcriptional initiation that might TFEC are not inconsistent with a macrophage-restricted expres- involve interactions of different Ets family transcription factors sion pattern. The rat homologue of mTFEC was cloned from a and a myeloid-specific initiator element such as the CAGGAA chondrosarcoma, but with the exception of the primary tumor, no sequence in c-fms,c-fes, PU.1, and TFEC. TFEC expression could be detected in any specific cell type, in- Further studies will be needed to determine a functional role http://www.jimmunol.org/ cluding chondrosarcoma, fibroblast, myoblast, and myeloma cell for mTFEC in macrophages. The published observations on rT- lines (22). A major portion of tumor-infiltrating leukocytes are FEC and hTFEC-L suggest that mTFEC would most likely act macrophages, which could explain the expression of TFEC in the as a transcriptional repressor of other coexpressed MiT family primary tumor. The pattern of tissue expression described for members (22, 23). In all macrophage cell lines studied to date, 8-wk-old rats, with the highest mRNA levels found in kidney, TFEC was coexpressed with other subfamily members, and in spleen, and lung; lower levels in liver, testis, and muscle; and even certain macrophage cell types (e.g., BMM, TGE-PM), all four less in heart or brain (22), could be attributed to the appearance of members, TFE3, TFEB, Mitf, and TFEC, can be detected. In

macrophages in those tissues. Finally, the human homologue theory, this would allow the formation of many different het- by guest on October 3, 2021 TFEC-L was recently cloned from the promonocytic leukemia cell erodimer combinations that could, on one hand, have individual line THP-1 (23). properties, but, on the other hand, may also have overlapping With the analysis of a growing number of macrophage-specific functions. In this context, TFEC could be important in balanc- genes, it has become clear that their 5Ј-proximal promoter regions ing the actions of coexpressed transactivators Mitf, TFE3, and share several features that distinguish them from other genes. A TFEB at certain stages of development or in special macro- typical macrophage-type promoter appears to lack TATA boxes, phage subpopulations. It will be of interest to determine consensus initiator sequences, or GC-rich regions found in house- whether the relative levels of these four factors are regulated by keeping genes, and instead contains multiple purine-rich sequence the wide range of lymphokines, bacterial products, and other motifs that are recognized by transcription factors of the Ets family agents that modulate macrophage differentiation. (14, 29, 38, 39, 42–44, 51, 53–55). We cloned and sequenced a Clearly, further studies are required to fully understand the func- 600-bp region of the proximal TFEC promoter and found exactly tion of MiT transcription factors in osteoclasts or macrophages. these characteristics of macrophage-restricted promoters. In addi- These studies may include knockout mutations of all members and tion, the 600-bp promoter was efficiently active in a macrophage their combinatorial analysis. Other important issues include the cell line, but not in fibroblasts, indicating that it contains the reg- regulation of MiT-protein expression in macrophages and oste- ulatory elements necessary for basal macrophage-specific expres- oclasts and, equally important, the identification of transcriptional sion. These regulatory elements most likely include at least one of targets in osteoclasts or macrophages that are currently unknown. the five binding sites for the Ets family transcription factor PU.1 that were identified by EMSA. In fibroblasts, PU.1 was able to Acknowledgments transactivate the TFEC promoter alone and cooperatively with a We thank M. Cahill for technical assistance; L. Fowles for reading the second Ets factor (Ets-2), a characteristic described previously for manuscript; and all of the members of the Hume Laboratory who contrib- the c-fms promoter (29). The temporal sequence of appearance of uted to this work by helpful discussion and suggestions. PU.1 and TFEC in CSF-1-stimulated bone marrow cultures also supports the view that PU.1 is a regulator of TFEC expression. References A direct comparison of proximal promoter regions of myeloid- 1. Van Furth, R. 1982. Current view on the mononuclear phagocyte system. Immu- restricted genes with the promoter of TFEC revealed their common nobiology 161:178. structure, with marked structural similarities especially between 2. Udagawa, N., N. Takahashi, T. Akatsu, H. Tanaka, T. Sasaki, T. Nishihara, T. Koga, T. J. Martin, and T. Suda. 1990. Origin of osteoclasts: mature mono- c-fms and TFEC. As in the TFEC promoter, transcription initiation cytes and macrophages are capable of differentiating into osteoclasts under a in the promoters of c-fes (44), c-fms (14), and PU.1 (38) occurs suitable microenvironment prepared by bone marrow-derived stromal cells. Proc. Natl. Acad. Sci. USA 87:7260. around a CAGGAA sequence, and mutation of this element greatly 3. Akagawa, K. S., N. Takasuka, Y. Nozaki, I. Komuro, M. Azuma, M. Ueda, reduced the activity of the latter three promoters in myeloid cells. M. Naito, and K. Takahashi. 1996. Generation of CD1ϩ RelBϩ dendritic cells The Journal of Immunology 1565

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