From the Division of Pediatric Oncology, The Johns
Hopkins Oncology Center, The Johns Hopkins University, Baltimore,
Maryland 21287 and the § Division of Hematology, Brown
University Department of Medicine and Division of
Hematology/Oncology, The Miriam Hospital, Providence, Rhode
Island 02906
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ABSTRACT |
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The adjacent neutrophil elastase, proteinase 3, and azurocidin genes encode serine proteases expressed specifically in
immature myeloid cells. Subclones of a 17-kilobase (kb) murine
neutrophil elastase genomic clone were assessed for their ability to
stimulate the neutrophil elastase promoter in 32D cl3 myeloid cells.
Region To identify transcriptional events that determine myeloid cell
determination, we have been investigating the regulation of the
neutrophil elastase (NE)1
gene. NE is a microbicidal serine protease present in the primary granules of granulocytes and monocytes (1). In bone marrow, NE mRNA
is present mainly in promyelocytes (2), and transcriptional regulation
plays a key role in its lineage-specific expression (3). We isolated a
17-kb genomic murine NE clone and found that, like the human NE gene,
the murine gene contains five exons and initiates transcription 30 bp
downstream from a TATAA homology (4). We demonstrated that the
5'-flanking region of the NE gene contains a 60-bp proximal enhancer,
located just upstream of the TATAA homology, which is strongly active
in 32D cl3 myeloid cells differentiating in response to
granulocyte-colony-stimulating factor (G-CSF), but only weakly active
in uninduced 32D cl3 cells or in fibroblasts (4). This proximal
enhancer is activated cooperatively by C/EBP, c-Myb, CBF, and Ets
family members such as PU.1 or GABP (4-6). The NE gene is located just
downstream of the proteinase-3 (PR-3) gene, which in turn is located
just downstream of the azurocidin gene (7). These three serine protease genes are expressed specifically and coordinately in immature myeloid
cells (7), and the promoter of each contains conserved binding sites
for C/EBP, c-Myb, and PU.1 (7, 8).
To determine whether the murine NE gene contains additional regulatory
regions, we assessed the activity of NE genomic subclones functionally
after transient transfection into 32D cl3 cells. A 2-kb NE genomic
region extending from Cell Culture and Transfection--
32D cl3 cells (9) were
maintained in Iscove's modified Dulbecco's medium supplemented with
10% HI-FBS, 1 ng/ml IL-3 (R & D Systems), and penicillin/streptomycin.
For induction of granulocytic differentiation, cells were washed twice
with phosphate-buffered saline and placed in Iscove's modified
Dulbecco's medium with 10% HI-FBS supplemented with 1,000 units/ml
G-CSF (Amgen). NIH 3T3 cells were maintained in Dulbecco's modified
Eagle's medium supplemented with 10% calf serum. U937 cells were
cultured in RPMI 1640 with 10% HI-FBS. Schneider cells (ATCC CRL-1963)
were maintained at 22-24 °C in Schneider cell medium (Life
Technologies, Inc.) with 10% FBS.
32D cl3 cells proliferating in IL-3 were transiently transfected by a
DEAE-dextran procedure as described, using 10-15 µg of reporter DNA
(4). After transfection, the 32D cl3 cells were cultured in G-CSF or
split between IL-3- and G-CSF-containing media. NIH 3T3 cells were
transfected by calcium phosphate precipitation using 3 µg of reporter
DNA, followed 18 h later by a 3.5-min, 15% glycerol shock in
phosphate-buffered saline (10). Schneider cells were transfected by
calcium phosphate precipitation with 5 µg of reporter DNAs and 2.5 µg of effector DNAs. pMSV-CAT (0.25-0.5 µg) or pCMV- Nuclear Extracts and EMSA--
32D cl3 and U937 nuclear extracts
were prepared as described (5, 6). GST-PU.1, GST-GABP Plasmids and Oligonucleotides--
A restriction map of the
murine NE genomic locus is presented in Fig. 1A, top
panel. The 1.8-kb BamHI-NcoI segment located just upstream of the first exon was studied previously (4). Six other
segments, designated I-VI, were subcloned upstream of the proximal
enhancer and the luciferase reporter, by insertion between the
HindIII and XhoI sites of pNE(m
NE-C1, NE-C1-mSp5, and NE-C1-mGG1AA2 were also ligated just
upstream of the TATAA homology in the NE promoter by insertion into
HindIII/XhoI digested pNE(m
The sequences of oligonucleotides containing known Sp1- and Ets-binding
sites, and of an irrelevant oligonucleotide, used as competitors in
EMSA are given below.
For trans-activation experiments, Sp1 and the GABP subunits were
expressed using pPac-Sp1 (12) (gift from R. Tjian, University of
California, Berkeley, CA) and pPac-GABP Identification of a Distal NE Enhancer--
Fig.
1A diagrams our murine NE
genomic
We chose to focus on I.11, the most active segment of this region, for
further analysis. Using BglII sites inserted by
site-directed mutagenesis, region I.11 was divided into domains X, Y,
and Z. Domain X stimulated the NE promoter 4-fold, domains X and Y
together stimulated activity 2.5-fold, and domains Y and Z stimulated
activity 3-fold. Strikingly, domain Z alone stimulated activity
31-fold. Apparently domain X is minimally active, domain Y is actually repressive, and domain Z is very stimulatory. We designate domain Z as
the core protease enhancer, realizing that nearby regions X and I.12
may also contribute to the activity of the NE enhancer. The sequence of
domain Z is given in Fig. 1B.
To determine whether the myeloid protease enhancer could function in
either orientation, region I.11 was positioned in reverse orientation
upstream of the NE promoter and luciferase. I.11 activated transcription 8-fold in the forward orientation and 18-fold in the
reverse orientation in induced 32D cl3 cells (Fig. 2C).
To determine whether the enhancer could activate a heterologous
promoter, regions I.11 and Z were positioned upstream of the herpes
simplex TK promoter. Region I.11 stimulated the TK promoter 5-fold, and
region Z stimulated the promoter 10-fold in induced 32D cl3 cells (Fig.
2C).
The NE promoter region alone was 5-fold more active when transfected
32D cl3 cells were cultured for 48 h in G-CSF, compared with IL-3
(4). To determine whether the myeloid protease enhancer might also be
more active in G-CSF, the activity of several of these constructs was
also assessed in IL-3 (Fig. 2C). When the observed increased
activities of the NE and TK promoters in cells cultured in G-CSF
compared with IL-3 are taken into account, we conclude that the
protease enhancer is equally active in 32D cl3 cells cultured in G-CSF
or in IL-3.
The activity of the myeloid protease enhancer was also assessed in NIH
3T3 cells. Region I.11 activated the NE promoter 8-fold in induced 32D
cl3 cells and 3-fold in NIH 3T3 cells. Region Z activated the promoter
23-fold in 32D cl3 cells (the average of the data in Fig. 2B
and Fig. 3A) and 7-fold in NIH
3T3 cells. Thus, the protease enhancer was more active in 32D cl3
myeloid cells than in NIH 3T3 fibroblasts. The activity observed in NIH 3T3 cells could indicate binding by factors not normally expressed in
myeloid cells or that do not have access to the enhancer in the
chromatin of non-myeloid cells.
Functional Analysis of the Core Protease Gene Enhancer--
Region
Z was divided into segments A, B, and C by inserting BamHI
sites in place of bp 76-81 or 157-162. Segments ABC (NE-Z), A, AB,
AC, BC, and C were then assessed for their ability to enhance the
activity of the NE promoter region in induced 32D cl3 cells (Fig.
3A). Segment A only activated transcription 2-fold, when assayed alone or when added to segment C. Segment B increased the
activity of segment A 3-fold and of segment C 2-fold. On the other
hand, segment C activated the promoter 10-fold alone and 6-fold with
segment A. Thus the 75-bp segment C was more stimulatory than segments
A or B. To map the active region of segment C, segment C1 was
synthesized and linked to the proximal enhancer. Segment C1 activated
transcription 12-fold (Fig. 3A), indicating that this 39-bp
region was critical to the activity of the core enhancer. Of note,
introduction of a BamHI site at bases 157-162, between segments A and B, disrupted a consensus site for CBF. This mutation did
not decrease the activity of the core
enhancer.3
Inspection of the sequence of C1 revealed an Sp1 consensus site,
GGGCGG, flanked by two GGAA motifs, which are potential binding sites
for Ets family members. Oligonucleotides containing 2-bp mutations in
each of these sites were synthesized and linked to the NE promoter. The
5'-GGAA was mutated to TTAA (GG1), and the 3'-GGAA was mutated to GGTT
(AA2), so that the introduced mutations would be separate from the Sp1
consensus. A double GGAA mutant version of C1 was also prepared
(GG1/AA2). Similarly, the Sp1 consensus was mutated to GTTCGG (Sp2) in
an effort to avoid interfering with the GGAA sites. This
oligonucleotide still weakly bound Sp1 (data not shown), and so a 5-bp
Sp1 site mutation, GTTTTT (Sp5), was also prepared. Finally, an
oligonucleotide containing mutations in both GGAA sites and a 5-bp
mutation in the Sp1 site were also ligated into the basal reporter. The
activities of these constructs relative to that of C1 linked to the NE
promoter were then assessed in induced 32D cl3 cells (Fig.
3B). Mutation of the 5'-GGAA reduced activity 3.6-fold,
mutation of the 3'-GGAA reduced activity 2.6-fold, and mutation of both
Ets sites reduced activity 4.4-fold. The 2- or 5-bp mutation in the Sp1
site had a similar effect, reducing activity 7.6-fold, and mutation of
all three sites reduced activity 6.3-fold. Thus, integrity of the Sp1
site and both Ets sites is important for the activity of segment C1 in
the core myeloid protease enhancer in myeloid cells.
Sp1 and Ets Family Members Bind C1--
A nuclear extract from
induced 32D cl3 cells was prepared and incubated with radiolabeled C1.
The reaction was then resolved on a 5% native acrylamide gel (Fig.
4A). Two gel shift species were observed. Both were competed by 10- or 50-fold excess of unlabeled
C1 or of a consensus Sp1 site. A C1 oligonucleotide carrying a 2-bp
mutation in the Sp1 site competed far less efficiently, whereas the GG1
or AA2 mutations did not prevent efficient competition. When Sp1
antisera was added to the reaction, the more slowly migrating gel shift
species was supershifted efficiently, whereas the lower species was
only mildly affected. The specific peptide prevented the supershift.
Thus, C1 binds Sp1 via the GGGCGG consensus and may bind a less
abundant, Sp1-related protein or degradation product present in 32D cl3
nuclear extracts as well. No additional bands, which might correspond
to Ets family members, were observed, even on long exposures of the
autoradiographs. In particular, we easily detected binding of PU.1 to
the NE proximal enhancer (5), and C1 only weakly competed for this
binding (data not shown).
To further characterize proteins capable of binding C1, we also
employed several purified proteins and U937 nuclear extracts (Fig.
4B). Purified Sp1 bound efficiently (lane 2), and
binding was also detected using bacterially expressed GST-PU.1,
GST-GABP
We next explored the possibility that the endogenous Ets factor, which
bound C1, might do so more efficiently if the Sp1-binding site is
mutated (Fig. 4C). Using radiolabeled C1 and a U937 extract in an EMSA (lane 2) we again identified a minor gel shift
species (dark arrow) between the two Sp1-related species
(open arrows). When the C1-Sp5 oligonucleotide was used as a
probe with the same extract, the Sp1 species were not detected, and the
minor band was more prominent (lanes 4 and 7).
Binding by this species was competed more efficiently by the homologous
probe (lanes 5 and 6) than by C1-mGG1/AA2,
carrying mutations in both Ets consensus sites (lanes 8 and
9). Thus, Sp1 and an Ets factor present in U937 extracts can
simultaneously bind C1. Of note, when purified Sp1 and GST-PU.1 or
GST-GABP C1 Is Activated Cooperatively by Sp1 and GABP--
Sp1 is abundant
in mammalian cells. Therefore, we employed Drosophila
Schneider cells for trans-activation experiments. Initial experiments
suggested that Sp1 could activate the proximal NE enhancer in
pNE(
To verify that cooperation between Sp1 and GABP was dependent upon
binding to the Sp1- and Ets-binding sites in C1, additional transfection experiments were carried out using reporters carrying either C1, C1-mGG1/AA2, or C1-mSp5, linked to NE( We previously studied 1.8 kb of the murine NE 5'-flanking region
and identified a proximal NE enhancer within the first 100 bp, which
activated transcription 300-fold (4). We have now assessed the ability
of an additional 15 kb of the murine NE genomic locus to enhance
transcription from the proximal NE enhancer/promoter in 32D cl3 myeloid
cells as they differentiate in response to G-CSF. Only a 2-kb region
centered at Using a similar functional approach we identified an enhancer at The NE, PR-3, and MPO genes are transcribed specifically in immature
myeloid cells (2-4, 18-20). The MPO distal enhancer is regulated by
PU.1 and C/EBP (21) and perhaps by CBF as well (15). Besides NE and
MPO, the only other gene expressed specifically in myeloid cells known
to contain a distal enhancer is the chicken lysozyme gene. The lysozyme
enhancer, which is most active in mature macrophages, is also activated
by PU.1 (22).
Expression of the linked NE, PR-3, and azurocidin myeloid serine
protease genes is coordinately regulated during myeloid differentiation (6). This coordinate regulation might be accounted for by conserved PU.1-, C/EBP-, and c-Myb-binding sites within their promoters (4, 7, 8,
23), as we found that these three transcription factors cooperatively
activate the NE proximal enhancer (4, 5). Recent genetic mapping
studies indicate that the most 3' PR-3 exon is located 3 kb upstream of
the first NE exon, placing the myeloid protease enhancer within the
large PR-3 second intron, 1 kb downstream of the PR-3
promoter.2 Perhaps coordinate regulation of the myeloid
protease gene cluster also results from activation via this enhancer.
Determining whether the myeloid protease enhancer activates the NE
and/or PR-3 promoters in vivo will be a key question for our
future investigations. As only 10 kb separate these two promoters, it
should prove feasible to develop transgenic murine lines in which this
question can be addressed. On the other hand, transient transfection
assays are not expected to identify insulator or other chromatin
boundary elements, which might prevent this enhancer from activating
the NE or PR-3 promoters.
This core protease enhancer activated the NE proximal region 5-fold in
NIH 3T3 cells, and unlike the proximal NE enhancer, its activity was
not increased when 32D cl3 cells were transferred from IL-3 to G-CSF.
Perhaps in the context of chromatin, greater tissue and developmental
specificity would be observed.
We found that integrity of an Sp1-binding site and of flanking Ets
factor-binding sites were essential for the activity of the core
protease enhancer. Although ubiquitously expressed, Sp1 was found to be
particularly highly expressed in developing murine granulocytic cells
(24). Sp1 was shown to be essential for the myeloid-specific activity
of the CD11b and CD14 promoters (25, 26) and for induction of CD14
expression during monocytic differentiation (27). Sp1 has also been
implicated in the regulation of the c-fes, human MPO, and
CD18 promoters in myeloid cells (28-30). Sp1 DNA binding activity did
not increase during U937 or THP-1 monocytic differentiation (25, 27),
and we similarly did not detect a change in Sp1 DNA binding activity
during 32D cl3 granulocytic differentiation.3 Interaction
with other factors may allow Sp1 to participate in transcriptional
induction during myeloid differentiation.
In addition to Sp1, the CD11b and c-fes promoters are
regulated by PU.1 (28, 31), and the CD18 promoter is regulated by PU.1
and GABP (14, 32, 33). Sp1 and GABP cooperatively activate the CD18
promoter in Schneider cells, the cytochrome c oxidase subunit IV promoter in NIH 3T3 or COS cells, and the folate-binding protein promoter in NIH 3T3 cells (34, 35). In addition, Sp1 and Ets1
cooperatively activate the PTHrP promoter and the human T-cell
lymphotrophic virus, type I long terminal repeat in T cells (36, 37).
Also, cooperative activation of the spleen focus-forming virus enhancer
by CBF and the Ets factor Fli-1 is dependent upon the integrity of an
Sp1-binding site (38). Thus, our finding that Sp1 cooperates with Ets
factors to activate the myeloid protease enhancer is not without precedent.
The active Sp1- and PU.1-binding sites in the CD11b promoter are
separated by 40 bp, and in the c-fes promoter they are
separated by about 50 bp (25, 28, 31). In the PTHrP promoter and in the
human T-cell lymphotrophic virus, type I long terminal repeat, the
central base pairs of the Sp1- and Ets1-binding sites are separated by
15 bp (36, 37). In the myeloid protease enhancer, only 6 bp separates
the centers of the GGAA motifs and the Sp1 consensus. Sp1 binding
partially excluded Ets factor binding from U937 nuclear extracts.
Simultaneous binding by Sp1 and GABP to the protease enhancer was not
observed in vitro, but this assay may not reflect in
vivo conditions. Also, the growth hormone gene promoter contains
overlapping Sp1- and Pit-1-binding sites, both of which are required
for full promoter activity despite mutually exclusive binding in
vitro (39). Perhaps, in vivo additional proteins
mediate cooperative binding by Sp1 and Ets factors to the protease
enhancer. Sp1 and GABP cooperatively activated the myeloid protease
enhancer via their binding sites in Schneider cells, although the Ets
factor which cooperates with Sp1 in vivo to activate the
enhancer remains to be determined. GABP alone did not activate the
enhancer. Similarly, AML1 and C/EBP Most functional Sp1 sites have been identified in promoter regions.
However the immunoglobulin 9.3 to
7.3 kb stimulated transcription 7-fold, whereas other genomic segments were inactive. This enhancer is located in the second
intron of the proteinase-3 gene and so may regulate more than one gene
in the myeloid protease cluster. Deletional analysis of the enhancer
identified several segments which activated the neutrophil elastase and
thymidine kinase promoters 3-6-fold. The most active segment was a
220-base pair region centered at
8.6 kb, which activated
transcription 31-fold. This segment contains an Sp1 consensus site,
which bound Sp1, flanked by two Ets family consensus sequences, which
bound PU.1, GABP, and an Ets factor present in myeloid cell extracts.
Mutation of the Sp1-binding site reduced enhancer activity 8-fold in
32D cl3 cells, and mutation of either or both Ets-binding sites reduced
activity 3-4-fold. Sp1 activated the distal enhancer 5-fold, GABP
3-fold, and the combination 8-fold in Schneider cells.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
7.3 to
9.3 kb stimulated the NE promoter
7-fold, and stimulated the herpes TK promoter as well, whereas none of
the other subclones, from
7.3 to +7.5 kb, were active. Recent results
indicate that this enhancer maps to the second intron of the adjacent
PR-3 gene,2 and so might
stimulate both the NE and PR-3 promoters in vivo. We
therefore designate it the myeloid protease enhancer. Functional analysis identified a 220-bp "core" enhancer, centered at
8.6 kb,
which activated transcription 15-31-fold. Deletional analysis of this
segment identified an Sp1-binding site and two flanking Ets
family-binding sites, which contributed to its activity in 32D cl3
cells. Also, Sp1 and GABP cooperatively activated transcription via
these sites in Schneider cells. Notably, Sp1 had been shown previously
to cooperate with Ets factors to regulate several myeloid promoters.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
Gal (1 µg) were employed as internal control plasmids where indicated. Total
cell extracts were prepared 42 h after transfection and analyzed
for luciferase and chloramphenicol acetyltransferase or
-galactosidase activities as described (4-6).
, and
GST-GABP
were expressed in Escherichia coli and isolated
with glutathione-Sepharose as described (6). Recombinant human Sp1 was
obtained from Promega. EMSA was carried out as described (5, 6). In
brief, 10-12 µg of nuclear extract, 1 footprinting unit of Sp1, or
approximately 100 ng of the GST fusion proteins were incubated on ice
for 30 min with 1 ng of an oligonucleotide probe, which had been
radiolabeled with [
-32P]dCTP by Klenow fill-in of
5'-overhangs. For detection of Sp1 in 32D cl3 nuclear extracts, the
binding solution was 10 mM Tris (pH 7.5), 50 mM
KCl, 70 mM NaCl, 100 µg/ml poly(dI-dC), 0.1 mg/ml bovine
serum albumin, 0.5 mM EDTA, 1 mM
dithiothreitol, 10% glycerol. For the U937 extracts, recombinant Sp1,
or the GST fusion proteins, the binding solution was 10 mM
Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 50 or 100 µg/ml poly(dI-dC), 1% Ficoll. 10-100-Fold excess of unlabeled
competitor oligonucleotides were added 5 min prior to probe addition,
and 1 µl of Sp1 antisera (Santa Cruz Biotechnology) or Sp1 antisera
and specific peptide were added 30 min prior to probe addition.
Reaction mixtures were then subject to electrophoresis on 5%
acrylamide gels in 0.33-0.5 × Tris borate-EDTA at 180-200 V. The gels were then dried and subjected to autoradiography.
107/
103)LUC
(4). Segments of NE-I were subcloned upstream of the proximal enhancer similarly. These segments are diagrammed in Fig. 1A,
bottom panel. Division of NE-I.11 into NE-X, NE-Y, and NE-Z
was facilitated by insertion of BglII sites into NE-I.11 by
site-directed mutagenesis after annealing mutagenic oligonucleotides
and single-stranded DNA as described (4). pNE-XY(
103)LUC contains
both NE-X and NE-Y upstream of the NE promoter and the luciferase
reporter, and plasmids containing other DNA segment combinations are
designated similarly. The sequence of pNE-Z, the core protease
enhancer, is given in Fig. 1B. NE-Z was further divided into
NE-A, NE-B, and NE-C by insertion of BamHI sites at bp
76-81 or 157-162 using polymerase chain reaction mutagenesis. The
final polymerase chain reaction products were sequenced to confirm that
no additional mutations had been introduced. A 39-bp segment of NE-C,
NE-C1, was ligated upstream of the NE promoter as a synthetic
double-stranded oligonucleotide. Several mutant versions of NE-C1 were
ligated similarly. The sequences of the top strand of these
oligonucleotides are given below. Each has a 4-bp 5'-overhang
compatible with HindIII, and the bottom strand had a similar
overhang compatible with XhoI. The wild-type or mutant Ets
and Sp1 core motifs are underlined,
NE-C1:
5'-AGCTTGGCCTCAAGCAGGAAGGGGCGGGGGAAGGATTGGCGATC-3'
NE-C1mGG1:
5'-AGCTTGGCCTCAAGCATTAAGGGGCGGGGGAAGGATTGGCGATC-3'
NE-C1mAA2:
5'-AGCTTGGCCTCAAGCAGGAAGGGGCGGGGGTTGGATTGGCGATC-3'
NE-C1-mSp2:
5'-AGCTTGGCCTCAAGCAGGAAGGTTCGGGGGAAGGATTGGCGATC-3'
NE-C1-mSp5:
5'-AGCTTGGCCTCAAGCAGGAAGGTTTTTGGGAAGGATTGGCGATC-3'
NE-C1-mGG1AA2:
5'-AGCTTGGCCTCAAGCATTAAGGGGCGGGGGTTGGATTGGCGATC-3'
NE-C1-mGG1AA2Sp5:
5'-AGCTTGGCCTCAAGCATTAAGGTTTTTGGGTTGGATTGGCGATC-3'
47/
42)LUC (4). The
109-bp herpes simplex virus TK promoter was ligated upstream of the
luciferase reporter in p19LUC (11) to create ptk-LUC. NE genomic
segments NE-I.11 and NE-Z were ligated upstream of the TK promoter in
this plasmid to create pNE(I.11)tkLUC and pNE(Z)tkLUC. I.11 was
also subcloned in reverse orientation in front of the 103-bp NE
promoter region, to generate pNE-I.11r(
103)LUC.
Sp1:
5'-ATTCGATCGGGGCGGGGCGAGC-3'
Ets:
5'-AACCCACCACTTCCTCCAAGGAGGAGCTGAGAGGAACAGGAAGTGTCAG-3'
irrel:
5'-GCGAAGCTTGCAGTGAGCTGAGATCACGGATCCGCG-3'
+ pPac-GABP
(gift from N. Speck, Dartmouth University, Hanover, NH).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
clone. The five NE exons span 1.7 kb (13). The 1.8-kb
region located just upstream of the first exon was studied previously
and shown to contain a 60-bp proximal NE enhancer located between
90
and
30, just upstream of the TATAA homology (4). Six additional
genomic regions were positioned upstream of
103 in the NE promoter
linked to the luciferase reporter. The activities of these constructs were assessed relative to the
103 promoter segment alone after transient transfection into 32D cl3 myeloid cells and culture in G-CSF
for 2 days (Fig. 2A). Region I
stimulated transcription 7.4-fold, on average, whereas the other
regions were inactive. Several segments of Region I were then also
subcloned upstream of the promoter and luciferase and analyzed
similarly. These segments are diagrammed in the bottom panel
of Fig. 1A, and the results of transient transfection
analysis are shown in Fig. 2B. I.1 stimulated transcription
6.5-fold, whereas I.2 only increased activity 2.2-fold. Also, when I.2
was divided into I.21 and I.22, neither of these DNA segments
stimulated the NE promoter. On the other hand, subdomains of I.1, I.11
and I.12, both retained significant activity. I.11 activated
transcription 14-fold, I.12 activated transcription 6-fold, and both
together stimulated activity 17-fold. Thus, the 1.4-kb genomic region
contained in I.11 and I.12 is the smallest portion of the 16.8-kb
murine NE
clone, which has enhancer activity. As this enhancer maps
in the second intron of the adjacent PR-3 gene,2 we
designated it as the myeloid protease enhancer.
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Fig. 1.
Murine NE genomic locus and subclones and
sequence of core myeloid protease enhancer. A, the
top panel diagrams the 16.8-kb murine NE genomic clone
we isolated (4). The box marks the position of the five
exons. Also indicated are the initial subclones analyzed for enhancer
activity, I (S-Bg, 2.8 kb), II (Bg-S, 2.5 kb), III (S-H, 1.6 kb), IV
(H-B, 0.4 kb), V (N-H, 0.9 kb), and VI (H-S, 6.5 kb). The bottom
panel expands Region I and diagrams additional subclones analyzed,
I.1 (S-Nh, 1.7 kb), I.2 (Nh-Bg, 1.0 kb), I.11 (S-Bs, 0.8 kb), I.12
(Bs-P, 0.6 kb), I.21 (Nh-C, 0.3 kb), I.22 (C-Bg, 0.7 kb), X (S-Bg, 0.32 kb), Y (Bg-Bg, 0.25 kb), Z (Bg-Bs, 0.23 kb), A (B-Bs, 64 bp), B (B-B,
82 bp), C (Bg-B, 81 bp), and C1 (39 bp). S, SalI;
Bg, BglII; H, HindIII; B,
BamHI; N, NcoI; Nh,
NheI; Bs, BstX1; P,
PstI; C, ClaI. Underlined
B and Bg sites were introduced by mutagenesis.
B, sequence of Region Z, which we designate as the core of
the enhancer. The start of subregions C, B, and A are indicated to the
left, and the top line is the sequence of C1, as
indicated to the right. Potential binding sites for Sp1,
c-Myb, CBF, and Ets family members are underlined.
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Fig. 2.
Functional identification of a myeloid
protease enhancer. A, the activities of genomic regions
I-VI linked to the NE promoter at bp 103 and to the luciferase
reporter were assessed relative to the promoter alone, the activity of
which was set as 1 in each experiment. All transfections included
pMSVCAT as an internal control. B, the activities of
segments of genomic region I were assessed similarly. C,
plasmids containing segment I.11 linked in the forward or reverse
(I.11r) orientation to the NE promoter (NE) in pNE(
103)LUC, or
segments I.11 or Z linked to the tk promoter (TK) in ptkLUC, were
transiently transfected into 32D cl3 cells proliferating in IL-3.
Transfected cells were then split into IL-3 or G-CSF and cultured for
an additional 2 days. Luciferase and chloramphenicol acetyltransferase
activities were then determined. The activity of each construct is
shown relative to that of the NE or TK promoter alone in G-CSF. The
activity of region I.11 was assessed also in NIH 3T3 cells, and the
activity of region Z linked to the NE promoter was assessed in 32D cl3
cells cultured in G-CSF and in NIH 3T3 cells. The mean and S.E. from
three experiments are shown.
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Fig. 3.
Identification of functional sites within the
core enhancer. A, the activities of enhancer segments
ABC (NE-Z), A, AB, AC, BC, C, and C1 linked to the NE promoter at bp
103 and to the luciferase reporter were assessed relative to the NE
promoter alone, the activity of which was set as 1 in each experiment.
All transfections included pMSVCAT or pCMV-
Gal as an internal
control. B, the activities of segment C1 carrying mutations
in the 5'-GGAA (GG1), the 3'-GGAA (AA2), or both (GG1/AA2), carrying 2- or 5-bp mutations in the Sp1 site (Sp2 or Sp5), or carrying mutations
in all three sites (Sp5+GG1/AA2) were assessed in induced 32D cl3
cells. The activity of C1 was set at 100% in each experiment. The mean
and S.E. from three determinations are shown.
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Fig. 4.
Enhancer segment C1 binds Sp1 and Ets
factors. A, 12 µg of induced 32D cl3 nuclear extract
was incubated with radiolabeled C1 in the presence of no competitor
(NC) or 10- or 50-fold excess of C1, of an Sp1 consensus
oligonucleotide (Sp1), of C1 carrying a 2-bp mutation in the Sp1
consensus (C1-mSp1), or of C1 carrying 2-bp mutations in the 5'- or 3'-
GGAA sites (C1-mGG1 and C1-mAA2). After 30 min on ice the reaction
mixtures were resolved on a 5% acrylamide gel and visualized by
autoradiography. For supershift assay, 1 µl of Sp1 antisera or 1 µl
of antisera and 4 µg of Sp1 peptide were incubated with the nuclear
extracts for 30 min prior to probe addition. The positions of Sp1 and
of a supershift species (arrow) are indicated. B,
radiolabeled C1 was incubated alone (lane 1), with 1 footprinting unit affinity-purified Sp1 (lane 2), 100 ng of
GST-PU.1 (lane 3), 100 ng of GST-GABP (lane
4), 100 ng each of GST-GABP
and GST-GABP
(lane
5), 10 µg of U937 nuclear extract (lane 6), or U937
extract and 100-fold excess of C1 (lane 7), a consensus Sp1 oligonucleotide (lane 8), a
known Ets factor-binding site (lane 9), or an irrelevant
oligonucleotide (lane 10). C, C1 probe was again
incubated alone (lane 1) or with U937 extract (lane
2). C1-mSp5 (mut Sp1 probe) was incubated alone (lane
3), with U937 extract (lanes 4 and 7), or
with 10- or 100-fold excess of C1-mSp5 (lanes 5 and
6) or C1-mSp5+mGG1/AA2 containing mutations in the Sp1 and
in the two Ets sites (lanes 8 and 9).
, or GST-GABP
with GST-GABP
(lanes 3-5).
GABP is an Ets family member, which also binds and activates the NE
promoter (6). Several bands were detected in EMSA using a U937 extract,
including a prominent doublet (open arrows) reminiscent of
the doublet detected using 32D cl3 extracts (lane 6).
Interestingly, competition with a consensus Sp1 oligonucleotide
inhibited binding by all of the bands except for a minor band present
between the doublet (lane 8, filled arrow). And
competition with an oligonucleotide derived from the CD18 promoter,
which is known to bind PU.1 and GABP (14), prevented binding by this
minor species but not by the major doublet (lane 9). An
irrelevant oligonucleotide competitor had no effect. These results
indicate that the Ets factors PU.1 and GABP can bind C1. They also
identify an endogenous Ets factor that can bind C1. This endogenous
factor was not PU.1 or GABP, as it did not supershift with the
corresponding antisera (data not shown).
/
were used in an EMSA with C1 as probe, no additional,
more slowly migrating species was detected, indicating that Sp1 and
PU.1 or GABP did not bind C1 simultaneously under these
conditions.3
103)LUC. Therefore, for these experiments we positioned C1
just upstream of the TATAA homology in the NE promoter, followed by the
luciferase reporter. The empty pPac Drosophila expression
vector did not affect the activity of this
reporter.4 Sp1 did not
activate the NE promoter in pNE(
42)LUC alone, but activated the C1
segment of pC1-NE(
42)LUC 3-fold, on average (Fig.
5A). Neither PU.1 nor GABP
alone activated segment C1 in Schneider cells, and PU.1 did not
increase stimulation by Sp1. On the other hand, the combination of GABP
and Sp1 stimulated pC1-NE(
42)LUC 8-fold, but did not stimulate
pNE(
42)LUC. Thus, Sp1 and GABP cooperated to stimulate
transcription via the enhancer segment C1 in Schneider cells.
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Fig. 5.
Sp1 and GABP cooperatively activate the core
myeloid protease enhancer in Schneider cells. A,
Schneider cells were transfected with pNE( 42)LUC or
pC1-NE(
42)LUC and pPac, pPac-Sp1, pPac-PU.1, pPac-GABP
+ pPac-GABP
(GABP), pPac-Sp1 + pPac-PU.1, or pPac-Sp1 + pPac-GABP
+ pPac-GABP
. 2.5 µg of each expression vector was used, and
additional pPac was employed where needed so that each transfection had
a total of 7.5 µg of expression vectors. Two days after transfection
cell extracts were analyzed for luciferase activities. Fold activation
relative to the activity of each reporter cotransfected with pPac alone
is shown. B, Schneider cells were transfected with
pC1-NE(
42)LUC, pC1-mGG1AA2-NE (
42)LUC, or
pC1-mSp5-NE(
42)LUC and pPac, pPac-Sp1, pPac-GABP
+ pPac-GABP
, or pPac-Sp1 + pPac-GABP
+ pPac-GABP
. Fold
activation relative to the activity of each reporter cotransfected with
pPac alone is shown (mean and S.E. from four determinations).
42)LUC (Fig. 5B). In this set of experiments, Sp1 activated C1 5-fold,
and Sp1 + GABP activated C1 12-fold. Mutation of the two Ets-binding sites did not prevent activation by Sp1, but prevented additional activation by GABP. Mutation of the Sp1-binding site prevented activation by Sp1, alone or with GABP.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
8.3 kb was active, and this region includes a highly
active 220-bp core enhancer, which activated the proximal
enhancer/promoter 30-fold in 32D cl3 cells.
3 kb
in the murine myeloperoxidase (MPO) gene, which was also localized
using a DNase I hypersensitivity assay (15, 16). A distal enhancer in
the MyoD gene was also identified by functional assay of multiple
genomic subclones (17). We made numerous attempts to identify DNase I
hypersensitive sites within the murine NE locus. Although most were
unsuccessful, two experiments indicated a hypersensitive site in the
region that we have now identified as functionally
important.3
synergistically activate the
M-CSF receptor gene, but C/EBP
alone is inactive (40).
, collagen I, collagen II,
-globin,
CD13, and gb110 genes contain more distal, functional Sp1 sites
(41-46). We anticipate that further characterization of the myeloid
protease enhancer and its interaction with the NE and PR-3 promoters
in vivo will contribute to our understanding of early
myeloid differentiation and of transcriptional mechanisms which
enable coordinate gene activation.
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ACKNOWLEDGEMENTS |
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We thank Martin Britos-Bray for technical
assistance, R. Brown for GABP antisera; N. Speck for PU.1, GABP, and
GABP
expression vectors; and R. Tjian for the Sp1 expression vector.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL51388 (to A. D. F.) and R29DK44728 (to A. G. R.), a National Research Service Award (to I. N.), an American Heart Association Rhode Island Affiliate Fellowship (to C. P. S.), and American Cancer Society Grant DHP-84242 (to A. G. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Leukemia Society Scholar. To whom correspondence should be addressed: Johns Hopkins Oncology Center, Rm. 3-109, 600 North Wolfe St., Baltimore, MD 21287. Tel.: 410-955-2095; Fax: 410-955-8897; E-mail: adfrdman{at}jhmi.edu.
The abbreviations used are: NE, neutrophil elastase; PR-3, proteinase-3; IL-3, interleukin-3; G-CSF, granulocyte-colony-stimulating factor; HI-FBS, heat-inactivated fetal bovine serum; TK, thymidine kinase; LUC, luciferase; MPO, myeloperoxidase; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; kb, kilobase(s); bp, base pair(s).
2 S. Shapiro, personal communication.
3 I. Nuchprayoon and A. D. Friedman, unpublished data.
4 C. P. Simkevich, M. Luo, and A. G. Rosmarin, unpublished data.
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REFERENCES |
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