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* Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia, 4072 and
University of Leeds, Molecular Medicine Unit, St. James University Hospital, Leeds, United Kingdom
Correspondence: Professor David A. Hume, Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia. E-mail: D.Hume{at}cmcb.uq.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: intron • enhancer • transcription • DNase I hypersensitivity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 full-length 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 13 14 15 16 17 18 19 20 21 22 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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200 aliquots. For the studies
described herein, cells were maintained in culture for 
4 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
pGL6.7fms. 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 pGL6.7fms.
Plasmid p6.7fms-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
p6.7fms-EGFP 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-cell-conditioned
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 µg) was subjected to Southern blot analysis
on Hybond N+ membranes (Amersham Pharmacia Biotech, Uppsala,
Sweden), which were hybridized with a 351-bp
BamHI-PvuII fragment located downstream of
the transcription start sites as indicated on Figure 1
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 GC-rich 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
sequenced [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.
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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.
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) .
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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
.
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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 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
(6.7fms compared with 3.5fms), 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) .
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 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 36 37 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.
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ACKNOWLEDGEMENTS |
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Received April 1, 2001; revised June 11, 2001; accepted June 18, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene in the macrophage cell line ANA-1 is regulated at the level of transcription processivity J. Immunol. 155,674-683[Abstract]
gene by promoter-proximal pausing of RNA polymerase II J. Immunol. 163,4375-4382This article has been cited by other articles:
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 L. Zhang, G. M. Vincent, M. Baralle, F. E. Baralle, B. D. Anson, D. W. Benson, B. Whiting, K. W. Timothy, J. Carlquist, C. T. January, et al. An intronic mutation causes long QT syndrome J. Am. Coll. Cardiol., September 15, 2004; 44(6): 1283 - 1291. [Abstract] [Full Text] [PDF]
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B. Burke, S. Sumner, N. Maitland, and C. E. Lewis Macrophages in gene therapy: cellular delivery vehicles and in vivo targets J. Leukoc. Biol., September 1, 2002; 72(3): 417 - 428. [Abstract] [Full Text] [PDF]
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