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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3512-3520
Elf-1 and PU.1 Induce Expression of gp91phox Via a
Promoter Element Mutated in a Subset of Chronic Granulomatous
Disease Patients
By
Kui Shin Voo and
David G. Skalnik
From the Herman B Wells Center for Pediatric Research, Section of
Pediatric Hematology/Oncology, Department of Pediatrics, Indiana
University School of Medicine, Indianapolis, IN.
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ABSTRACT |
The cytochrome b heavy chain (gp91phox) is the
redox center of the NADPH-oxidase and is highly expressed in mature
myeloid cells. Point mutations at  57, 55, 53, and 52 bp of
the gp91phox promoter have been detected in
patients with chronic granulomatous disease (CGD; Newburger et al,
J Clin Invest 94:1205, 1994; and Suzuki et al, Proc
Natl Acad Sci USA 95:6085, 1998). We report that Elf-1 and PU.1,
ets family members highly expressed in myeloid cells, bind to
this promoter element. Either factor trans-activates the 102 to
+12 bp gp91phox promoter when overexpressed in
nonhematopoietic HeLa cells or the PLB985 myeloid cell line. However,
no synergy of gp91phox promoter activation occurs
when both Elf-1 and PU.1 are overexpressed. Introduction of the 57
bp or 55 bp CGD mutations into the gp91phox
promoter significantly reduces the binding affinity of Elf-1 and PU.1
and also reduces the ability of these factors to trans-activate the
promoter. These results indicate that Elf-1 and PU.1 contribute to
directing the lineage-restricted expression of the
gp91phox gene in phagocytes and that failure of
these factors to effectively interact with this promoter results in CGD.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE gp91phox GENE IS
HIGHLY expressed in terminally differentiating myeloid cells,
coincident with the transition of a proliferating hematopoietic
progenitor cell to a terminally differentiating cell acquiring mature
properties.1 This gene product is a necessary component of
the superoxide-generating NADPH-oxidase complex that is responsible for
the generation of a respiratory burst and microbicidal activity in
phagocytic cells. The catalytic unit of the oxidase, cytochrome
b558, is a heterodimer composed of
gp91phox and p22phox that is
present in the membranes of phagocytes. Other oxidase components, such
as p47phox and p67phox, are
cytosolic in unstimulated phagocytes but migrate to the membrane upon
phagocyte stimulation and associate with the cytochrome to form a
functional oxidase.2 The gp91phox,
p47phox, and p67phox genes are
transcriptionally inactive until maturation beyond the promyelocyte
stage and are then actively transcribed until cell
death.3-5 Absence of any of these proteins leads to chronic granulomatous disease (CGD), an immunodeficiency syndrome.6
The 450 to +12 bp region of the human
gp91phox promoter directs transcription in a subset
of monocyte/macrophages in transgenic mice7 and also in
response to interferon- (IFN-) in stably transfected PLB985
myeloid cells.8 A number of DNA-binding proteins interact
with this promoter region, including BID (binding increased during
differentiation), IFN regulatory factor-1 (IRF-1), IRF-2, the CCAAT box
binding protein CP1, and the transcriptional repressor CCAAT
displacement protein (CDP; Fig
1).8-12 None of these factors is myeloid cell-specific.
Eklund and Kakar13 reported the cloning of a cDNA that
encodes the BID activity (denoted TF1phox in that
report). However, Yamit-Hezi et al14 have reported that
this cDNA corresponds to a bacterial transcript that contaminates some
commercially available libraries. Therefore, the identity of the BID
DNA-binding factor remains to be determined.
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| Fig 1.
DNA-binding proteins that interact with the 450 to
+12 bp gp91phox promoter. The transcriptional
repressor CDP competes with the binding of transcriptional activators
at multiple elements.10-12 The DNA-binding activity of CDP
is downregulated during terminal phagocyte development, thereby
permitting the interaction of transcriptional activators such as BID,
CP1, IRF-1, and IRF-2 with the promoter. +1 indicates the position of
transcription initiation. Two IFN-stimulated response elements (ISRE)
are denoted by open boxes. The 63 to 33 bp promoter region is
indicated below the diagram.12 The core consensus binding
sequence (GGAA) of the ets family of transcription factors is
underlined, and the positions of point-mutations identified in CGD
patients17,18 are indicated by arrows.
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CDP serves as a repressor of the gp91phox gene. It
binds to at least four elements within the promoter and excludes the
binding of transcriptional activators to overlapping binding
sites.10,12 CDP DNA-binding activity is downregulated
during terminal phagocyte differentiation. Importantly, downregulation
of CDP during terminal cell differentiation has also been observed in
tissues that do not express gp91phox, such as
kidney cells15 and myotubes.16 Thus, modulation of CDP DNA-binding activity is insufficient to direct myeloid cell-specific expression of the gp91phox gene but
is a necessary step for the activation of the
gp91phox promoter.12 Presumably
additional transcriptional activating factors, some of which may be
lineage-restricted, direct the transcription of the
gp91phox gene after CDP-mediated repression is
relieved during terminal phagocyte differentiation. The identification
of candidate lineage-restricted activators is the subject of this report.
A consensus binding site for the ets family of transcription
factors (5'-GAGGAAAT-3', lower strand) resides at 57
to 50 bp of the gp91phox
promoter.8 Single base pair mutations at 57,
55, 53, and 52 bp of the
gp91phox promoter have been detected in patients
with CGD (Fig 1).17,18 The gp91phox
protein is absent in the majority of phagocytic cells in these patients, although a subset of phagocytes express
gp91phox normally and generate a respiratory burst
in this variant form of the disease.19,20 We previously
described a DNA-binding protein complex, denoted
hematopoietic-associated factor (HAF-1), which binds to this
site.8 The HAF-1 complex is apparent in electrophoretic
mobility shift assays (EMSA) when nuclear extracts derived from a
variety of hematopoietic lineages are analyzed, including erythroid,
T-cell, B-cell, and phagocytic cells. We now report that Elf-1 and
PU.1, members of the ets family of transcription factors that
are abundantly expressed in myeloid cells, bind to the consensus
ets binding site in the gp91phox promoter
and trans-activate the promoter in cotransfection experiments. Elf-1 is
a component of the HAF-1 complex, whereas PU.1 is present in a
faster-migrating complex. Both the DNA-binding affinity and trans-activation ability of Elf-1 and PU.1 are decreased by
gp91phox promoter mutations identified in CGD patients.
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MATERIALS AND METHODS |
Cell culture and transfections.
The human chronic myelogenous leukemia cell line K562, human
promonocytic cell line U937, human T-cell leukemia cell line Jurkat,
and human cervical carcinoma epithelial cell line HeLa were obtained
from the American Type Culture Collection (Rockville, MD). The
myelomonoblastic cell line PLB98521 was generously provided
by Thomas Rado (Birmingham, AL). Primary human T cells were kindly
provided by Karen Pollok (Indianapolis, IN). HeLa cells were
cultured in Dulbecco's modified Eagle's medium, and PLB985, U937,
Jurkat, and K562 cells were cultured in RPMI 1640 medium. Both media
were supplemented with 10% fetal bovine serum (Sigma Chemical Co, St
Louis, MO) and 50 U/mL penicillin, 50 µg/ mL streptomycin, and 0.2 mmol/L glutamine (GIBCO-BRL, Gaithersburg, MD).
Plasmids were purified using a Maxiprep kit (Promega, Inc, Madison,
WI), followed by ultracentrifugation in a cesium chloride gradient, and
transfected into PLB985 and HeLa cells by electroporation. Briefly,
107 PLB985 cells were suspended in 350 µL of culture
medium and electroporated in the presence of 30 µg of plasmid DNA.
Each transfection contained 10 µg of the gp91phox
promoter/luciferase plasmid and 20 µg of expression vector. When PU.1
or Elf-1 expression constructs were analyzed individually, 10 µg of
the parental expression vector (pcDNA3.1) was added to make a total of
20 µg of expression plasmid. Cells were electroporated at 960 µF
and 220 V in a 4-mm diameter cuvette using a Bio-Rad Gene Pulser
(Bio-Rad, Hercules, CA). For HeLa cell transfections, 0.6 × 107 cells were suspended in 570 µL of culture
medium and electroporated in the presence of 30 µg of plasmid DNA (10 µg of luciferase reporter plasmid and 20 µg of expression
vector[s]) at 960 µF and 250 V.
Electroporated cells were transferred to 150-mm tissue culture dishes
containing 12 mL of prewarmed media and incubated at 37°C and 5%
CO2. After an incubation of 12 hours (PLB985 cells) or 15 hours (HeLa cells), cells were harvested and washed with phosphate-buffered saline, and cell pellets were collected and resuspended in 100 µL of lysis buffer (Promega). After incubation at
room temperature for 15 minutes, cell extracts were centrifuged for 4 minutes in a microcentrifuge and 30 to 40 µL of the supernatant was
used to determine luciferase activity using a Promega luciferase assay
kit and a Lumat 9210 luminometer, Wallac Inc, Gaithersburg, MD.
Luciferase activities in all transfection experiments are expressed in
relative light units, corrected for protein concentration, where the
level of reporter expression produced by cotransfection of the
102 to +12 bp gp91phox/luciferase reporter
plasmid and empty expression vector is defined as 100%. Multiple
independent plasmid preparations of promoter/reporter constructs and
expression vectors were analyzed.
EMSA.
Nuclear extracts were isolated as described by Dignam et
al,22 except that PLB985, U937, and Jurkat cells were
treated with 2 mmol/L diisopropylfluorophosphate (DFP; Sigma Chemical
Co) before homogenization. Total cellular protein extracts of K562
cells were prepared by lysing cells in Promega lysis buffer as
described above for luciferase assays. Oligonucleotide probes were
radiolabeled by T4 polynucleotide kinase using
[32P]ATP, followed by annealing with the complementary
strand oligonucleotides. Radiolabeled probes were resolved by
polyacrylamide gel electrophoresis and eluted by the crush and soak
method.23 EMSA was performed as described
previously12 with slight modifications. Briefly, 3 µg of
nuclear extract was mixed with 0.25 µg of herring sperm DNA and
competitor double-stranded oligonucleotides where indicated in a 40 µL reaction volume. The mixture was incubated on ice for 15 minutes
before the addition of 10,000 cpm of probe. After another 30 minutes
incubation on ice, samples were loaded onto a 0.5× Tris-Borate/EDTA, 5% nondenaturing polyacrylamide gel and
electrophoresis was performed at 200 V (constant voltage) at 4°C
for 2.5 hours. Antisera raised against Elf-1 and PU.1 and corresponding
blocking peptides were obtained from Santa Cruz Biotech (Santa Cruz,
CA), and 0.15 µg of the antibodies was incubated with nuclear
extracts for 1 hour before loading onto the gel in supershift assays.
Additional antisera directed against full-length PU.1 were provided by
David Kabat (Portland, OR) and Richard Maki (La Jolla, CA). Furguson plots were performed as described24,25 to estimate the mass of the HAF-1 complex. Briefly, protein standards (1 µg each) or EMSA
reactions were subjected to electrophoresis on a series of native
polyacrylamide gels containing 0.25× TBE and acrylamide concentrations in the range of 4% to 10%. The portion of the gel containing radioactive EMSA samples was dried and autoradiography was
performed. The portion of the gel containing protein standards was
stained with Coomassie Blue. The distance migrated by the protein-DNA
complex and by each of the protein standards was divided by the
distance of bromophenol blue dye migration to calculate the relative
mobility (Rf). The logarithm of Rf was plotted versus acrylamide
concentration to determine the mobility response slope for each
species. The determined slope for each standard protein was then
plotted against the known mass of each standard. The resulting linear
Furguson plot was used to determine the mass of the HAF-1 complex as
deduced by its mobility response slope, calculated as described above.
The predicted mass contributed by the DNA probe was subtracted to yield
a value for the mass of the protein components of the HAF-1 complex.
Oligonucleotides used in EMSA.
Complementary oligonucleotides were synthesized on an Applied
Biosystems model 394 synthesizer (Applied Biosystems, Foster City,
CA). 68 to 30 bp of the
gp91phox promoter,8
5'-CTATGCTTCTTCTTCCAATGAGGAAATGAAAACAGCAG-3';
57 CGD,17
5'-CTATGCTTCTTCTTCCAATGAGGAGATGAAAACAGCAG-3'; 55
CGD,17 5'-CTATGCTTCTTCTTCCAATGAGGAAAGGAAAACAGCAG-3';
Elf-1,26 5'-AAACAGGAAGTCCTGCCCCC-3'; PU.1,27
5'-GATCCTGAAATAACCTCTGAAAGAGGAACTTGGTTAGGTAG-3'; and E36,28 5'-CGGATCCGAATTCATCGATAATCGATTAT-3'.
Plasmid construction.
The wild-type 102 to +12 bp gp91phox
promoter/luciferase reporter construct was previously
described.11 CGD mutations (57 bp and 55 bp)
were introduced into the 102 to +12 bp
gp91phox promoter by polymerase chain reaction
(PCR)-mediated mutagenesis, using the 450 to +12 bp region of
the gp91phox promoter as a template.8
PCR products were digested with Sal I and BamHI and
subcloned into Sal I/Bgl II-digested luciferase reporter gene vector (pXP2).29 The nucleotide sequence of
each gp91phox promoter/reporter construct was
confirmed by the dideoxy chain termination DNA sequencing method using
a Perkin Elmer cycle sequencing kit (Perkin Elmer, Norwalk, CT).
Expression constructs were generated by transferring the human Elf-1 or
PU.1 cDNAs into HindIII/Kpn I- and
EcoRV/Xho I-digested pcDNA3.1(+) plasmid (Invitrogen,
Carlsbad, CA), respectively. The PU.1 and Elf-1 cDNA vectors were
generously provided by Michael Klemsz (Indianapolis, IN) and Jeffrey
Leiden (Chicago, IL).
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RESULTS |
Elf-1 and PU.1 interact with the proximal gp91phox
promoter.
We have previously demonstrated the binding of HAF-1, a hematopoietic
cell-restricted DNA-binding complex, to the 68 to 30 bp
region of the gp91phox promoter (Fig
1).8 Four distinct single base pair mutations of this
element were identified in variant CGD kindreds, each of which inhibits
formation of the HAF-1 complex.17,18 Although the HAF-1
binding site contains the GGAA core consensus binding site for the
ets family of transcription factors, antiserum directed against
the highly conserved ETS DNA-binding domain failed to disrupt the HAF-1
complex.8 However, in control experiments, the anti-ETS
domain antibody disrupted EMSA complexes containing Ets-1, PU.1, or
Fli-1.8
HAF-1 is the dominant EMSA complex formed after incubation of the
68 to 30 bp gp91phox promoter probe
with nuclear extract isolated from PLB985 cells (Fig 2). A shorter autoradiogram exposure
shows a doublet of complexes (bottom of Fig 2). The upper complex was
previously identified as containing the CCAAT-box binding factor
CP18 and is disrupted by both the wild-type 68 to
30 bp gp91phox promoter oligonucleotide as
well as by oligonucleotides containing either the 57 bp or
55 bp CGD mutations. Hence, the formation of the CP1 complex is
unaffected by promoter mutations identified in CGD patients. In
contrast, the HAF-1 complex is efficiently disrupted by homologous
competition but is competed less efficiently by an oligonucleotide
containing the 57 bp CGD mutation and poorly by an
oligonucleotide that contains the 55 bp CGD mutation (which disrupts the core ets consensus binding site). It is also
poorly competed by an oligonucleotide that contains a binding site for the ets factor PU.1. An oligonucleotide containing an Elf-1
binding site26 exhibits moderate competition of the HAF-1
complex (comparable to that of the 57 bp CGD mutation). An
indistinguishable HAF-1 complex was also detected using nuclear
extracts derived from the promonocytic cell line U937 and the T-cell
leukemia cell line Jurkat (Fig 2, middle and right-hand panels).
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| Fig 2.
DNA-binding proteins that interact with the 68 to
30 bp gp91phox promoter. EMSA was performed as
described in Materials and Methods using the 68 to 30 bp region
of the gp91phox promoter as a probe. Probe was
incubated with 3 µg of nuclear extract isolated from PLB985, U937,
and Jurkat cells after preincubation with a 100-fold molar excess of
unlabeled double-stranded competitor oligonucleotide where indicated.
The left-hand autoradiogram is overexposed to visualize the faint
faster-migrating complex. A shorter exposure is presented below to
resolve the HAF-1/CP1 doublet. None, no competitor added; Homo,
competitor oligonucleotide homologous to probe; 55 CGD, same as
Homo, except containing a T to C mutation at position 55 bp; 57
CGD, same as Homo, except containing an A to C mutation at 57 bp;
PU.1, competitor oligonucleotide containing a binding site for
PU.127; Elf-1, competitor oligonucleotide containing a
binding site for Elf-126; Heter, heterologous
oligonucleotide corresponding to E36, a high-affinity binding site for
CDP.28 The positions of the HAF-1 and PU.1 complexes are
indicated by arrows.
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During the course of experiments to further characterize the HAF-1
DNA-binding activity, an additional EMSA complex of faster mobility was
observed interacting with the 68 to 30 bp
gp91phox promoter (Fig 2). This complex exhibits
sequence-specific DNA-binding properties, because it is disrupted by
homologous competition but not by an oligonucleotide containing an
Elf-1 binding site. Importantly, this complex is also efficiently
disrupted by an oligonucleotide containing a binding site for PU.1,
indicating that this complex exhibits a binding specificity similar to
PU.1. However, this complex is not disrupted by the 55 bp CGD
oligonucleotide, which disrupts the core ets binding site.
Similar to the HAF-1 complex, the putative PU.1 complex retains partial
binding affinity for a binding site containing the 57 bp CGD
mutation (57 CGD). These results indicate that the formation of
the putative PU.1 complex may also be required for normal
gp91phox promoter activity. Consistent with the
tissue-distribution of PU.1,27 the faster-migrating complex
is not apparent when nuclear extract isolated from Jurkat cells is
analyzed (Fig 2, right-hand panel).
Additional EMSA experiments were performed using antisera directed
against ets family members that are abundant in myeloid cells
to further characterize the HAF-1 and PU.1 complexes
(Fig 3). The HAF-1 complex formed when
nuclear extract isolated from PLB985 cells is analyzed is supershifted
by Elf-1 antiserum, whereas the putative PU.1 complex is disrupted by
PU.1 antiserum. In addition, the disruption/supershift effects on the
HAF-1 and PU.1 complexes are abolished by the addition of Elf-1 or PU.1
blocking peptides, demonstrating the specificity of the antiserum
effect on each complex. Similar results were obtained when the
supershift assays were performed using nuclear extract derived from
U937 and Jurkat cell lines, as well as primary human T cells (except
that the PU.1 complex is absent in Jurkat cells and primary T cells).
These results indicate that the HAF-1 complex contains Elf-1, a protein abundant in both myeloid and T cells.30,31 Interestingly,
the Elf-1 containing HAF-1 complex is approximately 25-fold more
intense than the PU.1 complex when nuclear extracts isolated from
PLB985 or U937 cells are analyzed.
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| Fig 3.
HAF-1 and PU.1 complexes are disrupted/supershifted
by antisera raised against ets family members. EMSA was
performed as described in Materials and Methods. Elf-1 or PU.1 antisera
and corresponding blocking peptides were added where indicated.
Antisera were added after preincubation with or without 0.4 µg of
blocking peptide. Similar to Fig 2, the bottom left-hand image is a
shorter exposure of the upper autoradiogram to permit resolution of the
HAF-1/CP1 doublet.
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To further assess the nature of the putative Elf-1 and PU.1-containing
complexes, EMSA was performed using total cellular protein isolated
from K562 cells after transient transfection with Elf-1 or PU.1
expression vectors (Fig 4). Overexpression of Elf-1 leads to an increase in the HAF-1 complex, which is
supershifted by Elf-1 antiserum. Overexpression of PU.1 leads to a
similar increase in the intensity of the PU.1 EMSA complex. A new
faster-migrating complex is additionally detected in this extract. Both
of these PU.1 EMSA complexes are similarly disrupted by the addition of PU.1 antiserum. Antisera disruption/supershifting of both the HAF-1 and
PU.1 complexes is abolished by the addition of the corresponding blocking peptide to the EMSA binding reaction. These results confirm that Elf-1 and PU.1 interact with the 68 to 30 bp region
of the gp91phox promoter.
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| Fig 4.
HAF-1 and PU.1 complexes increase in intensity after
overexpression of Elf-1 or PU.1. EMSA was performed as described in
Materials and Methods. Probe corresponding to the 68 to 30 bp
region of the gp91phox promoter was incubated with
8 µg of total cellular protein isolated from K562 cells 15 hours
after electroporation with Elf-1 or PU.1 expression vectors. K562 cells
were transfected similarly to that described in Materials and Methods
for PLB985 cells. The lane containing nuclear extract isolated from
PLB985 cells provides a standard for the mobilities of the HAF-1 and
PU.1 complexes. Antisera and blocking peptides were added where
indicated. The relative intensity of EMSA complexes produced by whole
cell protein extracts cannot be directly compared with that produced by
the nuclear extract.
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Furguson plots were performed to estimate the mass of the HAF-1 EMSA
complex. This method relies on the relationship between the mass of a
complex and how the mobility of the complex alters as a function of
acrylamide concentration. This approach has been demonstrated to
provide an accurate measure of the mass of the protein components of
EMSA complexes.24,25 As shown in
Fig 5A, protein standards of various mass
exhibit characteristic mobility responses upon alterations of the
native gel acrylamide concentration. A plot of the mobility response
slopes versus the known mass of each protein standard produces a linear
Furguson plot (Fig 5B). Similar experiments were performed for the
HAF-1 complex. The determined mobility response slope for HAF-1
corresponds to a mass of 172 kD. Subtraction of 24 kD contributed by
the DNA probe produces an estimate of 148 kD for the mass of the
protein components of the HAF-1 complex. Hence, the HAF-1 complex is
significantly larger than the previously observed mass of Elf-1 (94 kD)26 and likely contains multiple protein species.
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| Fig 5.
Estimation of the mass of the HAF-1 complex. EMSA was
performed as described in Materials and Methods, using the 68 to
30 bp gp91phox promoter probe and nuclear
extract isolated from PLB985 cells. Furguson plot calculations were
performed as described in Materials and Methods. (A) The mobility
response slopes for each species, plotted as relative mobility (Rf)
versus acrylamide concentration. Protein standards are as follows:
( ) carbonic anhydrase (29 kD); ( ) ovalbumin (45 kD); (×) bovine
serum albumin (66 kD); ( ) bovine serum albumin dimer (132 kD); ( )
alcohol dehydrogenase (150 kD); ( )  -amylase (200 kD). ( )
denotes the behavior of the HAF-1 complex. (B) The Furguson plot of the
mobility response slopes presented in (A) versus mass. () The
protein standards; () the HAF-1 complex.
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Elf-1 and PU.1 each trans-activate the
gp91phox promoter.
To assess the effect of Elf-1 and PU.1 on gp91phox
promoter activity, cotransfection experiments were performed using the
102 to +12 bp gp91phox promoter/luciferase
reporter plasmid and expression vectors containing the Elf-1 or PU.1
cDNAs (or empty expression vector). These plasmids were cotransfected
into HeLa cells that lack endogenous Elf-1 and PU.132-34 or
into the PLB985 myeloid cell line. Cotransfection of the Elf-1
expression construct into HeLa cells produces a fivefold trans-activation of the gp91phox promoter, whereas
cotransfection with the PU.1 expression construct produces a sixfold
induction (Fig 6A). No significant synergy of trans-activation occurs after cotransfection of both PU.1 and Elf-1
expression constructs. No induction of luciferase expression was
detected after cotransfection of the parental pXP2 luciferase vector
with Elf-1 or PU.1 expression vectors (data not shown), indicating that
the trans-activation produced by Elf-1 and PU.1 requires the presence
of the gp91phox promoter fragment.
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| Fig 6.
Elf-1 and PU.1 each transactivate the
gp91phox promoter. HeLa cells (A) or PLB985 cells
(B) were cotransfected with gp91phox
promoter/luciferase reporter and expression vectors as described in
Materials and Methods. Luciferase vectors contained either the
wild-type 102 to +12 bp gp91phox promoter or
the same promoter fragment into which the 57 bp or 55 bp CGD
mutations were introduced. The expression vector that was cotransfected
with the luciferase vector is indicated below the graph. Data are
presented as the mean ± standard deviation. The number of independent
experiments (N) performed for each cotransfection is indicated below,
and three different plasmid preparations of each construct were
tested.
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To determine if trans-activation by Elf-1 and PU.1 is affected by the
gp91phox promoter mutations identified in CGD
patients, similar experiments were performed using a luciferase
reporter construct carrying either the 57 bp or 55 bp CGD
mutation in the context of the 102 to +12 bp
gp91phox promoter. Both Elf-1 and PU.1
trans-activation of the gp91phox promoter are
significantly reduced (P < .05) after introduction of either
of the CGD mutations (Fig 6A). The 57 bp CGD mutation has a
smaller effect than the 55 bp mutation on the trans-activation capacity of both Elf-1 and PU.1, consistent with the finding that the
57 bp CGD mutation has a less dramatic effect on the binding affinity of Elf-1 and PU.1 to the gp91phox promoter
(Fig 2).
Cotransfection experiments were also performed with PLB985
cells.21 Again, overexpression of either Elf-1 or PU.1
leads to trans-activation of the 102 to +12 bp
gp91phox promoter (3-fold and 16-fold,
respectively; Fig 6B), and trans-activation is significantly reduced
after introduction of the 57 bp or 55 bp CGD mutations
into the 102 to +12 bp gp91phox
promoter/luciferase reporter construct (Fig 6B). Similar to HeLa cells,
introduction of the 55 bp CGD mutation leads to a more dramatic
decrease in trans-activation by either Elf-1 or PU.1 than occurs after
introduction of the 57 bp CGD mutation, and no synergy is
observed after the cointroduction of both Elf-1 and PU.1 expression vectors.
| |
DISCUSSION |
The results reported here demonstrate that the ets family
transcription factors Elf-1 and PU.1 function as activators of the gp91phox promoter. Both Elf-1 and PU.1 bind
specifically to the 68 to 30 bp region of the
gp91phox promoter. Mutations at 57 bp or
55 bp of the gp91phox promoter, which cause
CGD, significantly reduce Elf-1 and PU.1 DNA-binding activity,
indicating that inhibition of ets factor interactions with this
element is causally related to the development of CGD. Furthermore,
transient overexpression of Elf-1 and PU.1 in the nonhematopoietic cell
line HeLa and the myeloid cell line PLB985 trans-activates the
102 to +12 bp gp91phox promoter, and this
effect is dependent on an intact ets factor binding site.
Interestingly, the 55 bp gp91phox promoter
mutation identified in a CGD patient disrupts the GGAA ets
factor consensus binding site and causes a more dramatic reduction in
the binding affinity and trans-activation activity of Elf-1 and PU.1
compared with the 57 bp CGD mutation. The fact that both the
55 bp and 57 bp mutations produce CGD may indicate a
requirement for a threshold level of ets site occupancy for
efficient gp91phox transcription, a level that
neither of these two CGD promoter mutations satisfies.
Because both Elf-1 and PU.1 binding affinity is decreased by each of
the four gp91phox promoter mutations identified in
CGD patients,17,18 it is difficult to assess the relative
function of each ets factor for normal
gp91phox expression in vivo. PU.1 exhibits a more
potent trans-activation activity in PLB985 cells, but the Elf-1
containing HAF-1 complex is much more abundant in nuclear extract
isolated from these cells. Hence, the HAF-1 complex may be functioning
near a maximal level in these cells before overexpression of Elf-1. A
similar juxtaposition of ets factors was previously found for
the myeloid cell-specific c-fes promoter, in which Elf-1 and PU.1 both
bind to an ets binding site.35 However, in this
case, the intensity of the PU.1 and Elf-1 EMSA complexes are
comparable, and only PU.1 (not Elf-1) was able to trans-activate the
c-fes promoter in cotransfection assays.
Although PU.1-deficient neutrophils lack both
gp91phox expression and a respiratory
burst,36 consistent with a requirement of PU.1 for
gp91phox expression, this result should be
interpreted with caution because PU.1-deficient neutrophils do not
mature fully. We have previously reported the binding of the
transcriptional repressor CDP to multiple sites in the
gp91phox promoter.10,12 CDP is
downregulated during myeloid differentiation, coincident with induction
of gp91phox expression. Constitutive expression of
CDP prevents gp91phox induction during terminal
myeloid differentiation,37 and ablation of CDP binding
sites results in increased gp91phox promoter
activity in HEL and K562 cells that normally do not express
gp91phox.10,12 Persistence of CDP
DNA-binding activity in PU.1-deficient neutrophils could also explain
the lack of gp91phox expression in these cells. In
this context, it is interesting that the other components of the
respiratory burst oxidase complex are expressed in neutrophils lacking
PU.1, including p47phox, whose promoter has been
previously shown to be regulated by PU.1.38 In contrast,
CDP appears to specifically regulate the gp91phox
component of the oxidase, because other oxidase subunits such as
p47phox are expressed normally during myeloid
differentiation in the presence of constitutive CDP
expression.39
During the preparation of this manuscript, two other reports described
the binding of PU.1 to the gp91phox
promoter.18,39 However, these reports differed regarding
which EMSA complexes contain PU.1. Consistent with our data, Suzuki et
al18 identified PU.1 in the faster-migrating complex, but not in the predominant HAF-1 complex. In contrast, Eklund et
al39 additionally identified PU.1 as a component of the
HAF-1 complex. We performed EMSA supershift assays using two additional
antisera directed against full-length PU.1, similar to what was used by Eklund et al,39 but were unable to reproduce their result
(data not shown). We speculate that the findings of Eklund et
al39 may represent a nonspecific effect, because multiple
EMSA complexes were disrupted by the addition of PU.1 antiserum in that
report. The presence of the HAF-1 complex in nuclear extract derived
from primary human T cells and the Jurkat and Molt-48
T-cell lines also argues against PU.1 being a component of the HAF-1
complex, because PU.1 is absent in these cells.32,33,40 Instead, our data indicate that Elf-1 is the ets family member present in the HAF-1 complex, which is the dominant complex that forms
with the gp91phox promoter element that is mutated
in a subset of CGD patients.
Eklund et al39 recently reported that the HAF-1 complex
also contains IRF-1 and IFN consensus sequence-binding protein (ICSBP), consistent with the large mass of the HAF-1 complex as deduced by
Furguson plot analysis (Fig 5). We have confirmed by antiserum supershift EMSA analysis that the HAF-1 complex contains ICSBP (data
not shown). However, Elf-1 appears to be the rate-limiting component
for the formation of the multimeric HAF-1 complex, because this EMSA
complex becomes more intense after transient overexpression of Elf-1
(Fig 4). Furthermore, the disruption of the HAF-1 complex by Elf-1
antiserum indicates that Elf-1 is a necessary component of the HAF-1
DNA-binding complex. The multimeric nature of the HAF-1 complex may
explain why an antiserum directed against the highly conserved ETS
DNA-binding domain of ets factors failed to disrupt the complex
in EMSA.8 We speculate that the ETS domain epitopes of
Elf-1 may be masked by other components of the HAF-1 complex. Eklund et
al39 also found that a minimal promoter linked to multiple
copies of the gp91phox promoter ets binding
site is slightly trans-activated by PU.1 in U937 myeloid cells, but not
in HeLa cells. In contrast, we find that the 102 to +12 bp
gp91phox promoter is significantly trans-activated
by overexpression of either Elf-1 or PU.1, and that this effect is
observed in either a myeloid cell line (PLB985) or a nonhematopoietic
cell line (HeLa). This indicates that these ets factors
trans-activate the intact proximal gp91phox
promoter in the absence of other myeloid cell-restricted factors and
thus may play a major role in directing lineage-restricted expression
of the gp91phox gene in mature phagocytes.
Regulated CDP-mediated repression also plays an important function in
restricting gp91phox expression to mature
phagocytes. However, gp91phox is not expressed in
all tissues lacking CDP DNA-binding activity. The results presented
here suggest a model in which induction of gp91phox
promoter activity in myeloid cells is achieved through the
downregulation of CDP in the context of the lineage-restricted
transcriptional activating factors Elf-1 and/or PU.1.
| |
ACKNOWLEDGMENT |
The authors are grateful to Jeffrey Leiden and Michael Klemsz for
providing Elf-1 and PU.1 expression vectors and to Richard Maki and
David Kabat for providing PU.1 polyclonal antisera. We also thank Karen
Pollok for providing primary human T cells, Diana Catt for helpful
discussions regarding Furguson plots, and Wen Luo for his assistance in
constructing the CGD mutation promoter vectors.
| |
FOOTNOTES |
Submitted October 12, 1998; accepted January 13, 1999.
Supported by National Institutes of Health Grant No. CA58947 awarded to
D.G.S. and by an Arthritis Foundation Postdoctoral Fellowship awarded
to K.S.V.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to David G. Skalnik, PhD, Wells Center for
Pediatric Research, Cancer Research Building, Room 472, 1044 W Walnut
St, Indianapolis, IN 46202; e-mail: dskalnik{at}iupui.edu.
| |
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TOP
ABSTRACT
INTRODUCTION