A highly conserved c-fms gene intronic element controls ... - CiteSeerX

A highly conserved c-fms gene intronic element controls macrophage-specific and regulated expression S. Roy Himes,* Hiromi Tagoh,† Nilukshi Goonetilleke,* Tedjo Sasmono,* Delvac Oceandy,* Richard Clark,* Constanze Bonifer,† and David A. Hume* *Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia, 4072 and †University of Leeds, Molecular Medicine Unit, St. James University Hospital, Leeds, United Kingdom

Abstract: The c-fms gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c-fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSs) were identified within mouse intron 2. Sequences of these DHSs were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fms intronic regulatory element (FIRE), which is even more highly conserved than the c-fms proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Sp1, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for highand low-level c-fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fms gene. J. Leukoc. Biol. 70: 812– 820; 2001. Key Words: intron 䡠 enhancer 䡠 transcription 䡠 DNase I hypersensitivity

INTRODUCTION The differentiation of macrophages from bone marrow progenitors requires the coordinate expression of many genes needed for mature cell function. This process is controlled by the lineage-specific growth factor macrophage colonystimulating factor-1 (CSF-1), which acts by binding to cell surface receptors (CSF-1Rs) encoded by the c-fms protooncogene [1]. We have studied the transcriptional regulation of the c-fms gene as a route to understanding cellular differentiation in the macrophage lineage [2–5]. The c-fms 812

Journal of Leukocyte Biology Volume 70, November 2001

gene is of particular interest because its mRNA is detectable in the earliest yolk-sac phagocytes formed during mouse development, prior to many other markers, including the macrophage-restricted transcription factor PU.1, and expression in embryonic and adult mice is largely restricted to macrophage lineage cells [6]. The only other major site of c-fms gene expression is in placental trophoblasts; in humans, c-fms expression is directed by a separate trophoblast-specific promoter that lies at the 3⬘ end of the plateletderived growth factor receptor-B gene, some 20 kb upstream of the first coding exon [7, 8]. The region flanking the first coding exon (exon 2) in both the mouse and human c-fms genes contains the transcription start site used in macrophages. The murine exon 2 c-fms promoter was found to be much more active in a transiently transfected macrophage cell line, RAW264, than in untransformed fibroblasts [5], but it was also determined to be active in a wide range of tumor cell lines that do not express the full-length endogenous mRNA [2, 9]. Tumor cells in which the promoter was active were shown to produce c-fms transcripts that contained exon 2 and extended into the downstream intron 2, but they did not have detectable fulllength c-fms mRNA. Inclusion of intron 2 in reporter gene constructs abolished reporter gene expression in nonmacrophage tumor cells, but significant activity was retained in RAW264 macrophages [2]. In macrophages, agonists that down-modulated c-fms mRNA caused a switch between production of full-length, spliced c-fms transcripts and transcripts containing intron 2 sequences [2]. These findings indicated that production of full-length c-fms mRNA in macrophages is controlled by elements within intron 2 that promote transcription termination. In many examples of regulated transcription elongation, the region of termination has been associated with an open chromatin conformation, detected as deoxyribonuclease I (DNase I)-hypersensitive sites (DHSs) [10,11], and many other genes have enhancer-like elements in the first intron that correspond to DHS [12–23]. In this study, we have characterized DHSs within intron 2 of murine c-fms and have examined their function in c-fms gene expression.

Correspondence: Professor David A. Hume, Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia. E-mail: [email protected] Received April 1, 2001; revised June 11, 2001; accepted June 18, 2001.

http://www.jleukbio.org

Fig. 1. DHS analysis. Nuclei were isolated from the cells shown at top of panel B and treated with various concentrations of DNase I as described in Materials and Methods. Southern blots were probed with a 351-bp PuvIIBamHI fragment, shown below the line in panel A. Panel A also shows the locations of transcription start sites, exons (black bars), and key restriction sites used in analyses. DHS regions are indicated by numbered arrows on both panels.

MATERIALS AND METHODS RAW264 cells RAW264.7 cells were obtained from the American Type Culture Collection (Manassas, VA). The original vial was placed in culture, expanded, and immediately refrozen in ⬃200 aliquots. For the studies described herein, cells were maintained in culture for ⱕ4 weeks from this original stock. RAW264 subclones were produced by limiting-dilution cloning from the parent line. After 3 days in culture, individual wells were inspected visually, and only wells with a single small growth focus were selected. Thirty clones were selected and screened for expression of c-fms (and many other genes) by cDNA microarray analysis. Clone 30 was identified as the clone with the highest level of c-fms expression, while clone 4.5 had the lowest expression level (T. Ravasi, C. Wells, and D. A. Hume, manuscript in preparation).

Plasmids, oligonucleotides, and sequence analysis All luciferase reporter plasmids used were based on the Promega pGL2 series, which includes pGL-2B (promoterless), pGL2-P [simian virus 40 (SV40) proximal promoter], and pGL-2C (SV40 promoter-enhancer). The plasmids pGL6.7fms and pGL3.5fms have been described previously [2]. To eliminate the possibility of translational incompatibility of the c-fms start codon and that of luciferase, which lies downstream in many of the reporter plasmids, an ATG-to-ATA mutation in the c-fms start codon was introduced into the 0.5-kb promoter fragment by splice overlap PCR mutagenesis [2]; the mutated product was subcloned into pGL-2B, and the mutation was confirmed by direct sequencing of the entire amplified region. Subcloning of the mutated 0.5-kb promoter fragment was performed to introduce the mutation into the longer promoter constructs pGL3.5-fms and pGL6.7-fms. The start site mutation had no effect on the activity of either the 3.5-kb or 6.7-kb fms promoter construct assayed in RAW264 cells (data not shown). The fms intronic regulatory element (FIRE) of intron 2—which lies from 2,646 to 3,015 bp downstream of the end of exon 2—was produced by PCR amplification using a high-fidelity enzyme mix (Boehringer Mannheim, Indianapolis, IN) and cloned into the MluI site of pGL.5⌬fms to generate the pGLfmsFIRE(U⫹) and pGLfmsFIRE(U⫺) plasmids and into the SalI site to generate the pGLfmsFIRE(D⫹) and pGLfmsFIRE(D⫺) plasmids. The FIRE fragment was also cloned into the MluI site of the pGL2B plasmid to generate

the pGLFIRE(⫹) and pGLFIRE(⫺) plasmids and into the pGL2P plasmid to generate the pGLPFIRE(⫹) and pGLPFIRE(⫺) plasmids. High-fidelity PCR was also used to produce the intron 2 fragment corresponding to DHS 4 to 6 (1,651 to 1,932 bp downstream from the end of exon 2), and that fragment was cloned using the MluI site, as described above, to generate the pGLfmsUE⫹ and pGLfmsUE⫺ plasmids. The plasmid pGL6.7⌬(⫺FIRE) was made by deletion of the FIRE sequence (2,616 to 2,946 bp from the start of intron 2) from pGL6.7⌬fms. The FIRE sequence is contained within a XhoI fragment. Sequences within this XhoI fragment lying on each side of the FIRE sequence were amplified by PCR using primers with overlapping sequences. The sequence overlap was used to splice together the two fragments in a second PCR, and then the mutated XhoI fragment was used to replace the full-length XhoI fragment in pGL6.7⌬fms. Plasmid p6.7⌬fms-EGFP [containing the coding sequence for enhanced green fluorescent protein (EGFP)] and its FIRE-deleted version were constructed by replacing the luciferase gene of the pGL2-based vector with the EGFP gene and SV40 poly(A) fragment of the pEGFP-N1 vector (Clontech). This was achieved by cloning the KpnI-SalI EGFP fragment into the ApaI-SalI fragment of pGL2-6.7fms by utilizing a KpnI-ApaI oligonucleotide linker. Plasmid p3.5fms-EGFP was constructed by removing intron 2 of the p6.7⌬fmsEGFP plasmid by SpeI-NheI digestion.

DHS analysis RAW264 cells were grown in Iscove’s modified minimal essential medium (Gibco) supplemented with 10% fetal calf serum, 2 mM L-glutamine (Glutamax; Gibco BRL, Rockville, MD), and penicillin-streptomycin. Mouse L929 fibroblast cells were grown in Dulbecco’s modified Eagle medium plus 5% fetal calf serum, Glutamax, and penicillin-streptomycin. Thioglycollate-elicited mouse macrophages from the mouse peritoneum were prepared and grown in Iscove’s modified minimal essential medium supplemented with 10% L-cellconditioned medium as described elsewhere [24]. Nuclei from the different cell types were prepared and digested with increasing concentrations of DNase I (Boehringer Mannheim) exactly as described elsewhere [24, 25]. Nuclei from RAW264 cells were digested with 0, 3, 6, 9, or 12 U of DNase I for mapping of the sites at the promoter and with 0, 6, 12, or 24 U for mapping of the intron 2 sites. Macrophage nuclei were digested with 0, 21, 24, or 27 U, and L cells were digested with 0, 6, 12, or 24 U. Genomic DNA was prepared and was digested with either BamHI or PstI. DNA (20 ␮g) was subjected to Southern blot analysis on Hybond N⫹ membranes (Amersham Pharmacia Biotech,

Himes et al. Control of transcription in macrophages

813

Fig. 2. Alignment of DHS regions of the mouse c-fms intron 2 with corresponding regions of the human c-fms gene. (A) A Pustell DNA matrix alignment of the sequences produced with MacVector威 software. (B) Region corresponding to DHS 7 as indicated in Figure 1 and panel A. (C) Region corresponding to DHSs 4 – 6. In both panels B and C the sequences were scanned for consensus transcription factor-binding motifs using Transfac (http://transfac.gbf.de/TRANSFAC/ programs.html). Sites relevant to macrophage gene regulation are annotated.

Uppsala, Sweden), which were hybridized with a 351-bp BamHI-PvuII fragment located downstream of the transcription start sites as indicated on Figure 1.

Cell culture and transfections For transfection studies, RAW264 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Serum Supreme; BioWhittaker, Walkersville, MD) that tested negative for lipopolysaccharide (LPS) contamination by assay of human immunodeficiency virus long terminal repeat promoter activity [26] and 2 mM Glutamax. The NIH 3T3 fibroblast and MOPC B cell lines were maintained in Dulbecco’s modified Eagle medium supplemented as described above. Bone marrow-derived macrophages were produced by isolation of bone marrow cells from the femurs of adult mice and differentiation of cells in the above-described RPMI medium containing 100 U/ml of CSF-1 for 5 days [27]. RAW264 cells (5 ⫻ 106) were transfected with 10 ␮g of each reporter construct by electroporation at 280 V and a capacitance of 960 ␮F, using a Bio-Rad (Hercules, CA) Gene Pulser. Plasmid DNA in transfections was purified by extraction with phenol-chloroform and two rounds of buoyant density ultracentrifugation in cesium chloride-ethidium bromide gradients. To minimize exposure of cells to bacterial plasmid DNA, the DNA was rinsed from cells by resuspension in 5 ml of medium, pelleting by centrifugation, and resuspension in 10 ml of fresh medium. For stable transfections, six

814

Journal of Leukocyte Biology Volume 70, November 2001

electroporations were performed for each construct as describe above, except that 2 ␮g of neomycin resistance plasmid pPNT, a gene-targeting vector containing the phosphoglycerate kinase-neomycin cassette [28], was added. The products of these six transfections were pooled, and cells were pelleted and washed with medium to remove DNA. After resuspension, the cells were split into three independent pools for selection with 200 ␮g/ml of G418 (Geneticin; Gibco BRL) for 7–10 days. NIH 3T3 and MOPC cells were transfected using Superfect威 transfection reagent (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Cells were harvested for luciferase assays by rinsing them with phosphate-buffered saline and then lysing them in 500 mM HEPES buffer containing 1 mM MgCl2, 1 mM dithiothreitol, and 0.2% Triton X-100 detergent. The LucLite Reporter Gene Assay kit (Packard, Groningen, The Netherlands) was used to assay cell lysates. A Packard Trilux plate luminometer was used to measure light emissions for all experiments except assays in the RAW264 subclones, for which a Turner Designs tube luminometer was used. Relative light units correspond to the number of counts per second per microgram of protein, except for experiments with the RAW264 subclones, for which RLU designates light units per microgram of protein. To measure protein concentration in cell extracts, the Bio-Rad protein assay system was used according to the manufacturer’s protocol. EGFP expression was measured as fluorescence intensity using a Becton Dickinson (San Jose, CA) FacsCalibur flow cytometer.

http://www.jleukbio.org

Fig. 3. Diagram of luciferase reporter vectors. The pGL2 series of reporter plasmids was used to analyze c-fms gene regulatory sequences. The diagram at the top shows the lengths and positions of c-fms gene sequences used to drive transcription of the luciferase reporter gene. Conserved sequences that correspond to DHSs are shown as stippled bars, and exon sequences are indicated by hatched bars. The arrows denote the orientation of gene sequences relative to the direction of transcription from the c-fms gene promoter within the endogenous gene. The asterisk indicates a missense mutation within exon 2, for which the ATG start codon of the CSF-1R coding sequence was replaced by ATA.

RESULTS DHS analysis We examined the sensitivity of the c-fms locus to DNase I using nuclei isolated from primary macrophages, the macrophage cell line RAW264, and the fibroblast cell line L929, which lacks c-fms mRNA. Figure 1 shows a map of the DHSs upstream and downstream of exon 2. In both primary macrophages and RAW264 cells, there was a doublet of DHSs (sites 1 and 2) corresponding to the exon 2 proximal-promoter region. The two sites lie between the XbaI site at position ⫺205 and the ATG start codon. The gap between them correlates approximately with a string of 25 adenines around position ⫺140 relative to the ATG, which separates a conserved compound PU.1-C/EBP element from the second PU.1 site and a cluster of transcription start sites [3, 5]. Three DHS regions were evident within intron 2 when primary macrophage or RAW264 nuclei were used. One of these (site 3) lies just downstream of the exon 2-intron 2 boundary. We have previously reported in this region an extended GCrich stem-loop structure that, by analogy with other genes, might control transcriptional elongation [2]. None of the intronic DHSs was detected using L929 nuclei. We also examined the effect of the agonist LPS, which down-modulates c-fms mRNA. In RAW264 cells treated with LPS, no change in the intensity of the two major distal intronic regions (sites 4 – 6 and 7) was observed (Fig. 1).

Sequence of intron 2 Only a small segment of mouse intron 2 had been sequenced previously [29], whereas the human gene has been fully se-

quenced [30]. The sequence of mouse c-fms intron 2 was determined on both strands, as was that of the 3.5-kb sequence upstream of exon 2 (GenBank accession number AF290879). Figure 2A shows a Pustell DNA matrix alignment of the two sequences. The 500-bp flanking exon 2 is conserved between the two species. Further upstream, no obvious homology is detected. However, there is an Alu repeat insertion in the human gene in this region, so the possibility that the alignment continues further can only be addressed with additional mouse and human sequence information. A clear line of relatedness is evident in intron 2, interrupted in the middle of the intron by a 1.5-kb insertion in the human gene relative to that of the mouse. Two regions of extended homology are evident at the distal end of the intron. The more distal of these corresponds precisely to the location of the broad constitutive macrophage-specific DHS 7 in Figure 1. An alignment of the mouse and human sequences in the extended conserved region is shown in Figure 2B. There are numerous, repeated, conserved elements characteristic of macrophage-specific promoters and enhancers. Hence, further experiments addressed the possibility that this conserved region, which we refer to as FIRE, has a function in transcription control. The second major DHS region, which appears as three distinct bands (sites 4 – 6) in Figure 1, lies ⬃1 kb upstream of FIRE. Short segments of conserved sequence in this region can be seen in the Pustell matrix alignment (Fig. 2A). An alignment of the conserved sequences spanning DHSs 4 – 6 is shown in Figure 2C. The most prominent features are two highly conserved purine-rich consensus PU.1/Ets sites. Himes et al. Control of transcription in macrophages

815

The FIRE sequence has enhancer activity To establish possible functions of FIRE, we examined its activities in ectopic contexts by performing transient transfections of RAW264 macrophage cells. Reporter constructs containing only the 300-bp FIRE sequence cloned in both orientations, either immediately upstream (U) of the c-fms minimal promoter or downstream (D) of the luciferase gene polyadenylation site, were prepared. The sense (⫹) or antisense (⫺) orientation, indicated in the figures, refers to the position of the sequence relative to the c-fms promoter and FIRE in the endogenous gene (Fig. 3). Inclusion of the FIRE sequence upstream of the c-fms promoter increased reporter gene expression 8.5-fold (⫹ orientation) and 15-fold (⫺ orientation) compared with that attained with the promoter alone, i.e., with pGL0.5-fms (Fig. 4A). Downstream, the effect of the FIRE sequence was reduced, and only the ⫺ orientation showed a greater than twofold induction of c-fms promoter activity (Fig. 4B). The enhancer-like activity of FIRE was not promoter specific. When FIRE was cloned upstream of the SV40 minimal promoter in the ⫹ and ⫺ orientations, reporter gene expression was activated by 3.3-fold and 6.8-fold, respectively, compared with the level attained with the promoter alone (Fig. 4C). The enhancer activity of the FIRE sequence showed cell lineage specificity. FIRE enhancer activity, like c-fms promoter activity, is weak in the MOP31C B cell line (Fig. 5A). In the NIH 3T3 fibroblast cell line, inclusion of FIRE had no effect on either the c-fms promoter, which showed no detectable activity, or the SV40 promoter, which is active in fibroblast cells (Fig. 5B). The other major DHSs within intron 2, DHS 4 – 6, were also examined, and constructs incorporating this region upstream of the c-fms promoter showed increased reporter expression compared with that attained with the promoter alone. This enhancer activity was smaller than the observed enhancer activity of FIRE and showed no orientation preference (Fig. 4D).

FIRE can act as a promoter The sequence of FIRE, containing numerous Ets-like elements and GC-rich sequences, is reminiscent of many myeloid-specific promoters [4, 31]. Hence, we examined whether it could act as a promoter. Reporter genes in which FIRE was the only control element and was placed in either orientation [pGLFIRE (⫹) or pGLFIRE (⫺)] were tested for activity in RAW264 cells. Both constructs were active, but pGLFIRE (⫺) was considerably more so, consistent with its orientation dependence in the enhancer assays. In fact, pGLFIRE (⫺) was at least as active as the 0.5fms exon 2 promoter itself in RAW264 cells. (Fig. 6A). The orientation preference suggests that FIRE would act as an antisense promoter with respect to the c-fms gene, which is why we presented the reverse orientation of the FIRE sequence shown in Figure 2. The activity of FIRE as a promoter in transient transfections, like its enhancer activity, was determined to be cell lineage restricted. In contrast to the high-level activity in the macrophage line RAW264, there was little detectable activity in MOPC31C B cells or NIH 3T3 fibroblasts (Fig. 6B and C). 816

Journal of Leukocyte Biology Volume 70, November 2001

Fig. 4. Regulatory function of DHSs within intron 2. (A and B) The FIRE region (DHS 7) was cloned into the pGL.5⌬fms (fms) reporter plasmid in both orientations upstream of the 500-bp conserved region of the c-fms gene promoter [pGLfmsFIREU⫹ and pGLfmsFIREU⫺ (fmsUF⫹ and fmsUF⫺)] (A) or downstream of the luciferase polyadenylation site [pGLfmsFIRED⫹ and pGLfmsFIRED⫺ (fmsDF⫹ and fmsDF⫺)] (B). (C) The FIRE region was also cloned into the pGL2P plasmid (SV40) in both orientations upstream of the SV40 promoter [pGLPFIRE⫹ and pGLPFIRE⫺ (SV40UF⫹ and SV40UF⫺)]. (D) The conserved sequence corresponding to DHSs 4 – 6 was cloned in both orientations upstream of the c-fms promoter within pGL.5⌬fms (fms) [pGLfmsUE⫹ and pGLfmsUE⫺ (fmsUE⫹ and fmsUE⫺)]. In each case, luciferase activity was assayed 24 h after transient transfection of RAW264 cells. ⫹, elements positioned in the sense orientation relative to transcription through the c-fms gene promoter; ⫺, elements positioned in the antisense orientation relative to transcription through the c-fms gene promoter. Values are given in relative light units (RLU), and columns represent the means and error bars indicate the SE of three independent transfections. The level of pGL2B (B) is shown as a background reference. Statistical analysis was applied to determine if small increases in activity were significant. The t-test for small sample size was used, and a P value of ⬍0.05 was considered significant and marked with an asterisk.

Clonal variation in RAW264 cells Although RAW264 cells were originally a clonal cell line, they display considerable phenotypic heterogeneity [32]. In a cDNA microarray study of LPS-inducible gene expression in subclones (T. Ravasi, C. Wells, and D. A. Hume, unpublished data) we noted that there was also reproducible and substantial variation of c-fms mRNA levels. We selected subclones expressing high and low levels, clone 30 and clone 4.5, respectively, whose steady-state levels of c-fms mRNA differed by ⬃10-fold. This clonal variation provided an opportunity to http://www.jleukbio.org

Fig. 5. Macrophage specificity and FIRE enhancer activity. (A and B) Reporter gene activity was assayed in the MOPC31 B cell line (A) or the NIH 3T3 fibroblast cell line (B). The pGL.5⌬fms (fms), pGLfmsFIREU⫺ (fmsUF⫺), pGL2P (SV40), and pGLPFIRE⫺ (SV40UF⫺) plasmids, containing promoters with or without the FIRE region cloned upstream, were transfected into cells, and luciferase activity was measured 24 h post-transfection. A reporter vector containing both the SV40 promoter and enhancer, pGL2C (pGLC), was used as a positive control, and pGL2B, with no promoter (B), was used as a background reference. Values are given in relative light units (RLU), and columns represent the means and error bars indicate the SE of three independent transfections. *, P ⬍ 0.05; **, P ⬎ 0.05 and not considered significant.

assess whether the activity of the promoter constructs correlates with expression of the c-fms gene. Figure 7 compares the activities in transient transfections of all constructs described above in the two clones. In the high-level-expressing clone 30, the proximal promoter (0.5fms or 3.5fms) was more active, the inclusion of intron 2 was less inhibitory (6.7⌬fms compared with 3.5⌬fms), and FIRE had greater enhancer activity (0.5fmsUF⫺ versus 0.5fms) than in clone 4.5 cells. By contrast, the promoter activity of FIRE was much lower in the high-level-expressing clone. It is interesting that removal of FIRE from the intron-containing 6.7fms plasmid resulted in the loss of most of the reporter activity in the high-level-expressing clone 30 but had no significant effect on the already low level of expression in clone 4.5 (Fig. 7A). The difficulty with transient-transfection analysis of c-fms is that plasmid DNA added during transfection is a confounding variable because it activates macrophages [33]. To avoid this variable, and to examine the role of FIRE in a chromatin context, we produced stable transfectants of both the parent RAW264 line and the high-level-expressing clone 30. For this purpose, we transferred the promoters to an EGFP reporter and examined the transfected cells by flow cytometry to obtain single-cell information. Figure 8 shows the results of this experiment. 6.7fms-EGFP had detectable expression in almost all transfected RAW264 cells. There was a clear separation into three populations: those that were only marginally more fluorescent than the untransfected control, those that exhibited a moderate level of expression, and a small population that expressed EGFP at a very high level. In RAW264 cells transfected with 3.5fms-EGFP, far fewer cells expressed high levels

of EGFP, and the level of expression per cell was also reduced in both high- and low-level-expressing pools. In clone 30, both 3.5fms-EGFP and 6.7fms-EGFP were expressed in a greater proportion of cells, and 6.7fms-EGFP also directed a significantly higher median level of EGFP expression. The most striking finding was that omission of FIRE completely abolished expression of the reporter gene in either the parent RAW264 line or clone 30 cells (Fig. 8).

DISCUSSION In an attempt to identify elements that might regulate transcription elongation, we identified macrophage-specific DHSs within intron 2 of the murine c-fms gene (Fig. 1). Sequences around the DHSs were conserved between the mouse and human c-fms genes (Fig. 2), particularly the sequence we have named FIRE (Fig. 2B), and represent the only conserved sequences in the entire 3.5-kb intron. DHSs over the proximalpromoter region of exon 2 (DHSs 1 and 2) were also detected in RAW264 cells and primary macrophages, but not in L929 cells. This is somewhat surprising given the previously obtained evidence that the promoter is active in a wide range of nonmacrophage cell lines [9]. However, as noted in the introduction, promoter activity in nonmacrophage lines is growth factor responsive. We have not attempted to optimize detection of DHSs 1 and 2 in nonmacrophage cells by manipulating growth conditions because this is not central to the present study.

Fig. 6. Analysis of FIRE promoter activity. The FIRE region of intron 2 was cloned in both orientations immediately upstream of the luciferase coding region, yielding pGLFIRE⫹ and pGLFIRE⫺ (with FIRE oriented in the sense and antisense orientations, respectively, relative to transcription through the c-fms gene promoter). (A) Luciferase expression from 500 bp of the proximal c-fms gene promoter (fms) or FIRE plasmid (FIRE⫹, FIRE⫺) constructs was assayed 24 h after transient transfection into RAW264 cells. (B and C) The fms promoter or FIRE antisense promoter activity was also assayed in nonmacrophage cell lines by transient transfection of MOPC B cells and NIH 3T3 fibroblasts. The pGL2P plasmid (SV40) was used as a positive control and the pGL2B plasmid was employed as a background reference in each assay. Values are given in relative light units (RLU), and columns represent the means and error bars indicate the SE of three independent transfections. *, P ⬍ 0.05.

Himes et al. Control of transcription in macrophages

817

Fig. 7. Variation in reporter gene activity in RAW264 cell clones. (A) Reporter plasmids contained the c-fms promoter alone [pGL3.5⌬fms (3.5⌬)], the promoter with intron 2 [pGL6.7⌬fms (6.7⌬)], or intron 2 with a specific deletion of the FIRE sequence [pGL6.7⌬fms-FIRE (6.7⌬-FIRE)]. Luciferase activity was assayed 24 h after transient transfection of RAW264 subclones, clone 4.5 (which expresses c-fms mRNA at a low level) and clone 30 (which expresses high levels of c-fms). (B) Plasmids containing the 500-bp c-fms promoter [pGL.5⌬fms (fms)], the FIRE antisense promoter [pGLFIRE- (FIRE⫺)], or the cfms promoter with FIRE cloned upstream [pGLfmsFIREU⫹ (fmsUF⫹)] were also assayed in the RAW264 subclones. Values are given in relative light units (RLU), and columns represent the means and error bars indicate the SE of three independent transfections. The level of pGL2B (B) is shown as a background reference.

Within the proximal intronic DHS region, encompassing sites 4 – 6 in Figure 1, the most prominent conserved features are two strong consensus PU.1 sites (Fig. 2C). We have shown that DHS 4 – 6 in combination possess macrophage-specific enhancer activity in transient-transfection assays of RAW264 cells, which is entirely consistent with the known biology of PU.1. We focused in more detail on the DHS7 region (FIRE) because of the remarkable extended sequence conservation, including numerous binding sites for known macrophage-expressed transcription factors (Fig. 2B). The FIRE sequence was found to possess macrophage-specific enhancer activity on either the c-fms promoter or an unrelated TATA-containing promoter. In the stable transfections performed with the EGFP reporter, the intron was clearly a powerful stimulatory element. A major effect of the intron appeared to be to increase the probability and/or frequency of expression of the EGFP reporter gene, consistent with an emerging view of the function of transcriptional enhancers (reviewed in ref. 34). The activity of

6.7fms-EGFP correlated well with c-fms expression in RAW264 cells, displaying heterogeneity in the parent line and a much more uniform, high-level expression in clone 30. The consistency of EGFP expression in a pool of transfectants suggests that promoter activity is in large measure insulated from the effect of position of integration into chromosomal DNA. In previous descriptions of the function of intron 2, its inclusion caused a relatively small inhibitory effect in RAW264 cells [2, 9]. The current stock of RAW264 cells was obtained directly from the American Type Culture Collection and used after a single additional passage, whereas early studies utilized cells that had been maintained in culture for longer periods. Variable gene expression profiles in the RAW264 line may lead to phenotypic selection, depending on culture conditions. For example, we maintain cells nonadherent on bacteriological plastic. Not all cells adhere to tissue culture plastic, and this may lead to selection for adherence

Fig. 8. Role of FIRE in stable transfection. RAW264 cells, or the clone 30 cells, were stably transfected with enhanced green fluorescent protein (EGFP) expression plasmids containing the c-fms promoter alone (3.5fmsEGFP), the promoter with intron 2␮ (6.7fmsEGFP), or the promoter with intron 2 with a specific deletion of the FIRE sequence (6.7fms⌬FIRE-EGFP). Fluorescence-activated cell sorter profiles of the transfected cells are shown; the dotted lines in each panel represent the profile obtained with untransfected cells. The vertical axis shows the number of events, and the horizontal axis indicates EGFP fluorescence. Each profile is representative of two independent pools in this experiment. The same patterns of relative activity were obtained in an independent set of transfectants.

818

Journal of Leukocyte Biology Volume 70, November 2001

http://www.jleukbio.org

after a small number of passages. The fact that c-fms promoter activity correlates with c-fms mRNA expression implies that the basis of the variation lies in transcription factor expression. Indeed, the cDNA microarray analysis reveals that key transcription factors also differ between clones (T. Ravasi, C. Wells, and D. A. Hume, unpublished data), and such an analysis will provide additional insight into the regulatory hierarchies that control expression of c-fms and other macrophage-specific genes. Removal of FIRE from the 6.7fms-EGFP plasmid abolished reporter gene expression and revealed the suppressive effect of the remainder of the intron (Fig. 8). Hence, the DHS sequence conservation and functional analysis support the view that FIRE is a key regulatory element in the c-fms gene. On the basis of our previous evidence of transcription termination within intron 2, we suggest that the ultimate function of FIRE is to relieve a block to elongation. At least two possible mechanisms can be envisaged. First, the open chromatin structure in the vicinity of FIRE that is evident from the DHS may act directly to permit RNA polymerase to read through the distal end of intron 2. Second, events occurring at the promoter, influenced by the FIRE enhancer activity, could control transcript elongation because the processivity of RNA polymerase II can be regulated and is an important component of transcriptional regulation [35–38]. How does the role of intron 2 implied by this study relate to regulation of the gene in vivo? Ultimately, the proof will have to come from a targeted removal of the FIRE sequence from the mouse germ line. There has been a study using the human c-fms promoter without intron 2 to drive transgenes in mice, but the level of expression attained was very low and tissue specificity was not conclusively demonstrated [39]. Our own studies have demonstrated that the intron-containing 6.7-kb mouse fms promoter can direct high-level expression of the cystic fibrosis transmembrane conductance regulator to peritoneal macrophages in transgenic mice (D. Oceandy, B. Wainwright, and D. A. Hume, unpublished data). We have also produced mice using the 6.7fms-EGFP construct that was active in RAW264 cells (Fig. 8) and have demonstrated constitutive expression in bone marrow-derived macrophages and peritoneal macrophages (T. Sasmono, S. R. Himes, and D. A. Hume, unpublished data). By contrast, the 3.5-kb promoter alone generated no transgenic animals with detectable EGFP expression. Apart from the enhancer/antirepressor function, FIRE was also shown to possess directional promoter activity when assayed by transient transfection in RAW264 cells (Fig. 6). We have looked for evidence of antisense transcripts and found none detectable in RAW264 cells by RNase protection assay. The promoter activity of FIRE correlated inversely with c-fms promoter activity when high- and low-level-expressing RAW264 cell clones were compared. It is interesting that FIRE contains numerous consensus binding sites, for transcription factors such as Sp1, AP1, C/EBP, and Ets2 (Fig. 2), that are induced by repressors of c-fms transcription elongation such LPS, CSF-1, and phorbol esters [27, 40, 41]. These observations led us to speculate that such inducible transcription factors interfere in some way with the ability of FIRE to relieve intronic repression.

Each of the elements we have described in this paper, the proximal promoter, the intronic DHSs, and the intronic repressor elements (which could include DHS 3), contains multiple conserved sites that are likely occupied by sequence-specific DNA-binding proteins. We have established systems in which each of these elements can be studied in isolation, emphasizing the importance of stable transfection and identification of subclones of RAW264 cells that express high or low levels of c-fms. A systematic dissection of the elements required for repression by intron 2 and its relief by FIRE is currently under way.

ACKNOWLEDGMENTS This project was funded in part by grants from the National Health and Medical Research Council of Australia and the Queensland Cancer Fund to D.A.H. Experiments performed in C. Bonifer’s laboratory were funded by the Wellcome Trust and by the BBSRC. The Centre for Molecular and Cellular Biology is a Special Research Centre of the Australian Research Council.

REFERENCES 1. Roth, P., Stanley, E. (1992) The biology of CSF-1 and its receptor. Curr. Top. Microbiol. Immunol. 181, 141–167. 2. Xie, Y., Favot, P., Dunn, T. L., Cassady, A. I., Hume, D. A. (1993) Expression of mRNA encoding the macrophage colony-stimulating factor receptor (c-fms) is controlled by a constitutive promoter and tissuespecific transcription elongation. Mol. Cell. Biol. 13, 3191–3201. 3. Ross, I. L., Yue, X., Ostrowski, M. C., Hume, D. A. (1998) Interaction between PU.1 and another Ets family transcription factor promotes macrophage-specific basal transcription initiation. J. Biol. Chem. 273, 6662– 6669. 4. Rehli, M., Lichanska, A., Cassady, A. I., Ostrowski, M. C., Hume, D. A. (1999) TFEC is a macrophage-restricted member of the microphthalmiaTFE subfamily of basic helix-loop-helix leucine zipper transcription factors. J. Immunol. 162, 1559 –1565. 5. Reddy, M. A., Yang, B. S., Yue, X., Barnett, C. J., Ross, I. L., Sweet, M. J., Hume, D. A., Ostrowski, M. C. (1994) Opposing actions of c-ets/PU.1 and c-myb protooncogene products in regulating the macrophage-specific promoters of the human and mouse colony-stimulating factor-1 receptor (c-fms) genes. J. Exp. Med. 180, 2309 –2319. 6. Lichanska, A. M., Browne, C. M., Henkel, G. W., Murphy, K. M., Ostrowski, M. C., McKercher, S. R., Maki, R. A., Hume, D. A. (1999) Differentiation of the mononuclear phagocyte system during mouse embryogenesis: the role of transcription factor PU.1. Blood 94, 127–138. 7. Roberts, W., Shapiro, L., Ashmun, R., Look, A. (1992) Transcription of the human colony-stimulating factor-1 receptor gene is regulated by separate tissue-specific promoters. Blood 79, 586 –593. 8. Visvader, J., Verma, I. M. (1989) Differential transcription of exon 1 of the human c-fms gene in placental trophoblasts and monocytes. Mol. Cell. Biol. 9, 1336 –1341. 9. Favot, P., Yue, X., Hume, D. A. (1995) Regulation of the c-fms promoter in murine tumour cell lines. Oncogene 11, 1371–1381. 10. Haley, J. D., Waterfield, M. D. (1991) Contributory effects of de novo transcription and premature transcript termination in the regulation of human epidermal growth factor receptor proto-oncogene RNA synthesis. J. Biol. Chem. 266, 1746 –1753. 11. Porcher, C., Picat, C., Daegelen, D., Beaumont, C., Grandchamp, B. (1995) Functional analysis of DNase-I hypersensitive sites at the mouse porphobilinogen deaminase gene locus. Different requirements for position-independent expression from its two promoters. J. Biol. Chem. 270, 17368 –17374. 12. Bertani, A., Polentarutti, N., Sica, A., Rambaldi, A., Mantovani, A., Colotta, F. (1989) Expression of c-jun protooncogene in human myelomonocytic cells. Blood 74, 1811–1816.

Himes et al. Control of transcription in macrophages

819

13. Brooks, A. R., Nagy, B. P., Taylor, S., Simonet, W. S., Taylor, J. M., Levy-Wilson, B. (1994) Sequences containing the second-intron enhancer are essential for transcription of the human apolipoprotein B gene in the livers of transgenic mice. Mol. Cell. Biol. 14, 2243–2256. 14. Gaasenbeek, M., Gellersen, B., DiMattia, G. E. (1999) DNase I hypersensitivity analysis of non-pituitary human prolactin gene expression. Mol. Cell. Endocrinol. 152, 147–159. 15. Gaunitz, F., Gaunitz, C., Papke, M., Gebhardt, R. (1997) cis-regulatory sequences from the first intron of the rat glutamine synthetase gene are involved in hepatocyte specific expression of the enzyme. Biol. Chem. 378, 11–18. 16. Lehmann, U., Brocke, P., Dittmer, J., Nordheim, A. (1999) Characterization of the human elk-1 promoter. Potential role of a downstream intronic sequence for elk-1 gene expression in monocytes. J. Biol. Chem. 274, 1736 –1744. 17. Makar, K. W., Pham, C. T., Dehoff, M. H., O’Connor, S. M., Jacobi, S. M., Holers, V. M. (1998) An intronic silencer regulates B lymphocyte cell- and stage-specific expression of the human complement receptor type 2 (CR2, CD21) gene. J. Immunol. 160, 1268 –1278. 18. McCready, P. M., Hansen, R. K., Burke, S. L., Sands, J. F. (1997) Multiple negative and positive cis-acting elements control the expression of the murine CD4 gene. Biochim. Biophys. Acta 1351, 181–191. 19. Moulin, P. S., Manson, A. L., Nuthall, H. N., Smith, D. J., Huxley, C., Harris, A. (1999) In vivo analysis of DNase I hypersensitive sites in the human CFTR gene. Mol. Med. 5(4), 211–223. 20. Sakamoto, S., Morisawa, K., Ota, K., Nie, J., Taniguchi, T. (1999) A binding protein to the DNase I hypersensitive site II in HLA-DR alpha gene was identified as NF90. Biochemistry 38, 3355–3361. 21. van Dijk, T. B., Baltus, B., Raaijmakers, J. A., Lammers, J. W., Koenderman, L., de Groot, R. P. (1999) A composite C/EBP binding site is essential for the activity of the promoter of the IL-3/IL-5/granulocytemacrophage colony-stimulating factor receptor beta c gene. J. Immunol. 163, 2674 –2680. 22. Winston, J. H., Hong, L. , Datta, S. K., Kellems, R. E. (1996) An intron 1 regulatory region from the murine adenosine deaminase gene can activate heterologous promoters for ubiquitous expression in transgenic mice. Somat. Cell Mol. Genet. 22, 261–278. 23. Amaravadi, L., Klemsz, M. J. (1999) DNA methylation and chromatin structure regulate PU.1 expression. DNA Cell Biol. 18, 875– 884. 24. Huber, M. C., Bosch, F., Sippel, A. E., Bonifer, C. (1994) Chromosomal position effects in chicken lysozyme gene transgenic mice are correlated with suppression of DNase I hypersensitive site formation. Nucleic Acids Res. 22, 4195– 4201. 25. Sippel, A. E., Saueressig, H., Huber, M. C., Hoefer, H. C., Stief, A., Borgmeyer, U., Bonifer, C. (1996) Identification of cis-acting elements as DNase I hypersensitive sites in lysozyme chromatin. Methods Enzymol. 274, 233–246. 26. Sweet, M. J., Hume, D. A. (1995) RAW264 macrophages stably transfected with an HIV-1 LTR reporter gene provide a sensitive bioassay for analysis of signalling pathways in macrophages stimulated with lipopolysaccharide, TNF-alpha or taxol. J. Inflamm. 45, 126 –135. 27. Stacey, K. J., Fowles, L. F., Colman, M. S., Ostrowski, M. C., Hume, D. A. (1995) Regulation of urokinase-type plasminogen activator gene transcrip-

820

Journal of Leukocyte Biology Volume 70, November 2001

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

tion by macrophage colony-stimulating factor. Mol. Cell. Biol. 15, 3430 – 3441. Passey, R. J., Williams, E., Lichanska, A. M., Wells, C., Hu, S., Geczy, C. L., Little, M. H., Hume, D. A. (1999) A null mutation in the inflammation-associated S100 protein S100A8 causes early resorption of the mouse embryo. J. Immunol. 163, 2209 –2216. Gisselbrecht, S., Fichelson, S., Sola, B., Bordereaux, D., Hampe, A., Andre, C., Galibert, F., Tambourin, P. (1987) Frequent c-fms activation by proviral insertion in mouse myeloblastic leukaemias. Nature 329, 259 – 261. Andre, C., Hampe, A., Lachaume, P., Martin, E., Wang, X. P., Manus, V., Hu, W. X., Galibert, F. (1997) Sequence analysis of two genomic regions containing the KIT and the FMS receptor tyrosine kinase genes. Genomics 39, 216 –226. Zhang, D. E., Hohaus, S., Voso, M. T., Chen, H. M., Smith, L. T., Hetherington, C. J., Tenen, D. E. (1996) Function of PU.1 (Spi-1), C/EBP, and AML1 in early myelopoiesis: regulation of multiple myeloid CSF receptor promoters. Curr. Top. Microbiol. Immunol. 211, 137–147. Costelloe, E. O., Stacey, K. J., Antalis, T. M., Hume, D. A. (1999) Regulation of the plasminogen activator inhibitor-2 (PAI-2) gene in murine macrophages. Demonstration of a novel pattern of responsiveness to bacterial endotoxin. J. Leukoc. Biol. 66, 172–182. Stacey, K. J., Sester, D. P., Sweet, M. J., Hume, D. A. (1999) Macrophage activation by immunostimulatory DNA. Curr. Top. Microbiol. Immunol. 247, 41–58. Hume, D. A. (2000) Probability in transcriptional regulation and its implications for leukocyte differentiation and inducible gene expression. Blood 96, 2323–2328. Biragyn, A., Nedospasov, S. A. (1995) Lipopolysaccharide-induced expression of TNF-␣ gene in the macrophage cell line ANA-1 is regulated at the level of transcription processivity. J. Immunol. 155, 674 – 683. Parada, C. A., Roeder, R. G. (1996) Enhanced processivity of RNA polymerase II triggered by Tat-induced phosphorylation of its carboxyterminal domain. Nature 384, 375–378. Raschke, E. E., Albert, T., Eick, D. (1999) Transcriptional regulation of the Ig␬ gene by promoter-proximal pausing of RNA polymerase II. J. Immunol. 163, 4375– 4382. Yankulov, K., Blau, J., Purton, T., Roberts, S., Bentley, D. L. (1994) Transcriptional elongation by RNA polymerase II is stimulated by transactivators. Cell 77, 749 –759. Langer, S. J., Bortner, D. M., Roussel, M. F., Sherr, C. J., Ostrowski, M. C. (1992) Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression. Mol. Cell. Biol. 12, 5355–5362. Fowles, L. F., Martin, M. L., Nelsen, L., Stacey, K. J., Redd, D., Clark, Y. M., Nagamine, Y., McMahon, M., Hume, D. A., Ostrowski, M. C. (1998) Persistent activation of mitogen-activated protein kinases p42 and p44 and ets-2 phosphorylation in response to colony-stimulating factor 1/c-fms signaling. Mol. Cell. Biol. 18, 5148 –5156. Sweet, M. J., Hume, D. A. (1996) Endotoxin signal transduction in macrophages. J. Leukoc. Biol. 60, 8 –26.

http://www.jleukbio.org