(Received for publication, October 6, 1994; and in revised form, January 19, 1995)
From the
The cytochrome b heavy chain (gp91-phox)
is expressed nearly exclusively in terminally differentiating
myelomonocytic cells, thereby providing a model to study the events of
late myeloid differentiation. We describe a tissue culture assay for
studying interferon
induction of gp91-phox expression and a
cis-element in the gp91-phox promoter that is necessary but not
sufficient for this activity. In vitro assays reveal two
DNA-binding proteins that interact with this cis-element. One factor is
restricted to hematopoietic cells, is required for an interferon gamma
response, and binds to an element similar to the Ets protein family
consensus, although it does not correspond to known family members. The
second factor is the ubiquitous CCAAT-binding protein CP1, which is
dispensable for an interferon
response. Single base pair
mutations in the gp91-phox promoter that specifically abolish the
binding of the hematopoietic-associated factor have previously been
identified in chronic granulomatous disease patients (Newburger, P. E.,
Skalnik, D. G., Hopkins, P. J., Eklund, E. A., and Curnutte, J.
T.(1994) J. Clin. Invest. 94, 1205-1211). The data
reported here directly demonstrate the functional significance of the
hematopoietic-associated factor for gp91-phox promoter activity and
reveal the binding properties and tissue distribution of this novel
DNA-binding protein.
Pluripotent bone marrow stem cells can differentiate into
members of various hematopoietic lineages. This process includes
tissue-specific transcriptional regulation of a complement of genes
that characterizes each lineage. Granulocytes and monocyte/macrophages
comprise the category of mature, terminally differentiated phagocytic
cells and are characterized by the ability to generate toxic
free-radicals via the respiratory burst. Respiratory burst activity
requires a group of signaling components and the catalytic unit, the
NADPH-dependent oxidase(1, 2, 3) . The
expression of some of the components of the respiratory burst apparatus
is confined to terminally differentiated phagocytic cells. The
phagocyte-specific cytosolic components p47-phox(4) ,
p67-phox(5) , and the heavy chain of the membrane bound
cytochrome b (gp91-phox) (6) are
transcriptionally inactive until myeloid maturation progresses beyond
the promyelocyte stage and then are actively transcribed until cell
death. In addition, it has been observed that gp91-phox expression
increases in response to interferon
(IFN-
)(
)(7) . Absence of these gene products
leads to chronic granulomatous disease (CGD), a disorder of host
defense(8) . The transcriptional regulation of these
myeloid-specific genes provides a model for the events of terminal
myeloid differentiation.
Recent work has provided insight into the tissue-specific expression of the gp91-phox gene(9, 10) . We have previously demonstrated that 450 base pairs (bp) of the 5`-flank of the human gp91-phox gene is adequate to direct expression of a reporter gene specifically in a subset of monocyte/macrophages in transgenic mice(10) . These data suggest that important regulatory elements that control lineage and differentiation-specific expression of gp91-phox are present within the proximal 450 bp of the gp91-phox promoter. Similar tissue distribution of transgene transcription is observed with constructs containing up to 2.6 kilobase pairs of the proximal gp91-phox promoter(10) . The absence of detectable reporter gene expression in the majority of myelomonocytic cells suggests that additional regulatory elements are required to direct appropriate transcription in the full complement of phagocytic cells.
The study of two kindreds with a novel form of X-linked CGD also suggests the existence of important cis-elements in the proximal promoter region of the gp91-phox gene. gp91-phox protein is absent in the majority of phagocytic cells, although a subset of phagocytes express gp91-phox normally in this variant form of the disease(11) . Both of these kindreds carry a point mutation in the proximal promoter region of the gp91-phox gene(12) . The point mutations in these kindreds are at -55 and -57 bp relative to the published transcription start site(13) , and each specifically disrupts an interaction between the gp91-phox promoter and a DNA-binding protein (12) .
In this study, we
have examined in more detail the proximal gp91-phox promoter in an
attempt to identify cis-elements and the DNA-binding proteins that
interact with these elements, which mediate tissue-specific expression
of gp91-phox in myelomonocytic cells. Previous studies relied on
transgenic mice for functional analysis of gp91-phox promoter
fragments. While this technique has the advantage of permitting
promoter characterization in an authentic in vivo setting, the
time and expense of this approach are disadvantages. We report here
that IFN- induction of the gp91-phox gene can be studied using
stable transfectants of the myeloid cell line PLB985(14) , thus
providing a more rapid and convenient assay system with which to study
this aspect of gp91-phox promoter function.
The studies reported
here reveal a cis-element in the proximal gp91-phox promoter that is
necessary but not sufficient for IFN- induction of transcription
and that co-localizes with promoter mutations identified in CGD
patients. The -50 to -57 region of the gp91-phox promoter
(5`-GAGGAAAT-3`) contains on the lower strand a sequence similar to the
consensus binding sequence for the Ets protein family. The core motif
for binding of Ets family members is 5`-GGA(A/T)-3`, with binding
specificity of the various family members conferred by additional
flanking sequences(15) . Various Ets factors have recently been
demonstrated to be essential for the transcription of myeloid and
lymphoid-specific genes, including major histocompatibility class II
I-A
(16) , CD18(17) , perforin(18) ,
macrophage scavenger receptor(19) , and the immunoglobulin
heavy chain genes(20) . In vitro assays reveal that a
DNA-binding protein required for normal gp91-phox promoter function is
largely restricted to cells of hematopoietic origin, shares an
overlapping binding site with a ubiquitous CCAAT-binding protein, and
exhibits a binding specificity distinct from previously described
members of the Ets protein family.
Mutated gp91-phox promoter sequences were sub-cloned into the BamHI and XbaI sites of the hGH reporter gene plasmid vector (Nichols Institute, San Juan, Capistrano). A neomycin resistance gene driven by the thymidine kinase promoter was inserted into the SalI site (TKneo cassette generously provided by Stuart Orkin, Boston Children's Hospital) in both parallel and anti-parallel orientation relative to the reporter gene. The orientation of the selectable marker did not affect reporter gene activity (data not shown).
Plasmid for generating a riboprobe specific for the
gp91-phox promoter-hGH reporter transgene mRNA was constructed by
subcloning double-stranded synthetic oligonucleotides into the
Bluescript KS (Clontech, Palo Alto, California)
transcription vector. Complementary oligonucleotides were synthesized
that contain the proximal 38 bases of the gp91-phox promoter and the
first 33 bases of hGH sequence. The oligonucleotides were annealed and
cloned into the HindIII and PstI sites of Bluescript
KS
. The chicken
-actin probe was a generous gift
of Celeste Simon (University of Chicago).
Myeloid cell lines
were differentiated by treatment with 0.1 µM phorbol
12-myristate 13-acetate (PMA) (Sigma) for 48 h (macrophage
differentiation), with 1 µM all-trans-retinoic
acid (Sigma) for 24 h followed by 60 mM dimethylformamide for
an additional 24 h (granulocyte differentiation), or treated with 500
units/ml human recombinant IFN- (Boehringer Mannheim) for 48 h.
Stable transfectants carrying the neomycin resistance cassette were
treated with 1000 units/ml human IFN-
for 6 days with the cells
kept continuously in log phase.
Myeloid cell lines were transfected
by electroporation using a Bio-Rad gene pulser (Bio-Rad). PLB985 cells
growing in log phase were resuspended at a density of 4
10
cells/ml in RPMI 1640 supplemented as above plus 20%
fetal bovine serum. Fifty µg of circular plasmid DNA containing a
gp91-phox promoter-hGH reporter gene construct and the TKneo cassette
was added to 800 µl of cells in an electroporation cuvette and
pulsed at 0.24 mV and 960 microfarads. The cells were incubated for 20
min on ice, expanded into 10 ml of RPMI 1640 supplemented as above plus
15% fetal bovine serum, and incubated overnight at 37 °C and 5%
CO
. The cells were selected for G418 resistance in the
usual RPMI 1640 media supplemented with geneticin at an initial
concentration of 0.5 mg of active drug/ml, increasing to 1.5 mg/ml over
2 weeks to select for high transgene copy number.
Stable
transfection was confirmed by Southern blot analysis. Genomic DNA was
isolated from 5-8 10
tissue culture cells
using the Elu-Quick system (Schleicher and Scheull) and probed for both
the TKneo cassette and the promoter-reporter gene construct using
formamide hybridization and random primer labeled probes(22) .
Probes were labeled with 50 µCi of
[
-
P]dCTP (10 mCi/ml, 3000 Ci/mmol, DuPont
NEN) per 100 ng of DNA template.
Stably transfected cells treated
with IFN- were compared with nontreated cells for expression of
hGH. Stable pools were tested with IFN-
stimulation over
increasing time periods to determine an optimal time for stimulation of
log phase cells. The IFN-
response was maximal after 6 days of
stimulation (data not shown). Titration of the dose of IFN-
demonstrated a maximal effect (with a variety of lots) at
1000-1200 units/ml (data not shown). Multiple independent pools
of cells were analyzed for each promoter construct.
Assays for hGH were performed using a radio-immunoassay system from Nichols Laboratories (San Juan Capestrano, CA). Results of hGH assays were normalized for the number of trypan blue-excluding cells/ml of culture.
Electrophoretic
mobility shift assays (40) (EMSA) were performed in a 20-µl
total volume with 2-4 µg of nuclear extract protein or
0.5-1 µl of rabbit reticulocyte lysate in a solution
containing 10 mM Tris-HCl (pH 7.5), 50 mM potassium
glutamate, 5 mM magnesium chloride, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and 50 µg/ml
poly(dIdC) (Sigma). Nuclear extracts were preincubated for 15 min
on ice with approximately a 200-fold molar excess of unlabeled
double-stranded oligonucleotide competitor (where indicated) followed
by an additional 15 min on ice after the addition of 1-2
10
cpm of probe. Bound and free probe were separated by
electrophoresis at 25 mA (constant current) on native 4% polyacrylamide
gels (19:1) in 45 mM Tris-HCl (pH 8.3), 45 mM borate,
1.25 mM EDTA at 4 °C. Gels were dried onto Whatman No. 3MM
paper (Whatman), and autoradiograms were exposed at -70 °C
using intensifying screens.
For antibody binding assays, nuclear extracts or rabbit reticulocyte lysate were incubated on ice in a 20-µl binding assay with 1 µg of an anti-ETS domain antibody raised against peptide corresponding to residues 362-374 of ets-1 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h either before or after the addition of oligonucleotide probe. Blocking assays were performed as above, but in the presence of 5-fold excess by weight of synthetic peptide, as per manufacturer's instructions. Antibodies directed against NF-Y (41) were preincubated with nuclear extract for 15 min prior to the addition of probe.
DNase I
protection assays were performed using a binding reaction that was
identical to that used for EMSA with the following exceptions. The
total volume was 50 µl, the amount of nuclear extract protein was
8-20 µg, and the amount of probe was 1-3
10
cpm/sample. After the binding reaction was complete, the
sample was incubated at room temperature for 1 min and supplemented to
10 mM magnesium chloride and 5 mM calcium chloride.
The reaction was incubated for 1 min at room temperature and
supplemented to 0.2-0.8 µg/ml with DNase I (Sigma) and
incubated for 1 min at room temperature. The DNase I digestion was
stopped by supplementing to 10 mM EDTA and 5% glycerol. Free
and bound probe were separated on a 5% native gel as above. After
electrophoresis, a wet autoradiogram was performed for 4 h at room
temperature. Free and shifted probe bands were eluted from the gel (22) and ethanol precipitated. The probes were resuspended in
80% formamide loading buffer and subjected to electrophoresis on a
denaturing polyacrylamide sequencing gel with a Maxam and Gilbert
G+A sequencing ladder(42) . The gel was fixed in 10%
methanol, 10% acetic acid and dried, and autoradiography was performed
at -70 °C with intensifying screens. Sequencing ladders for
each strand were generated by piperidine (Fisher) cleavage of dimethyl
sulfate (Aldrich)-methylated probe as described
previously(43) .
Methylation interference assays were performed with partially methylated end-labeled probe(43) . Binding reactions were performed as for DNase I protection assays, and free and bound probe were separated on 3.5% native polyacrylamide gels, recovered, and precipitated as above. Piperidine cleavage at G and A residues was performed as described(42) , and the probes analyzed on denaturing polyacrylamide sequencing gels as described for DNase I protection assays.
Total RNA was extracted from tissue
culture cells by the single-step extraction method as described
previously (45) and resuspended in 100% formamide(46) .
Annealing reactions were performed in hybridization buffer with
2-20 µg of total RNA (or yeast tRNA as a control) and
10 to 2
10
cpm of probe in a final
volume of 30 µl and incubated for 16 h at 55 °C. Elevation
above this annealing temperature (up to 75 °C) did not further
reduce background. RNase digestion was performed at 37 °C for 15
min in a total volume of 400 µl containing 40 µg/ml of RNase A
(Boehringer Mannheim) and 700 units/ml RNase T1 (Boehringer Mannheim)
in 10 mM Tris-HCl (pH 7.5), 0.3 M NaCl, and 5 mM EDTA. RNase digestion was terminated, and the protected fragments
were analyzed on a denaturing polyacrylamide sequencing gel as
described(47) . The gel was fixed, and autoradiography
performed as described above.
Reporter gene expression was analyzed following
IFN- stimulation and compared with unstimulated transfectants.
IFN-
stimulation of myeloid cell lines has previously been
demonstrated to increase transcription of the gp91-phox
gene(48) . No induction of reporter gene expression occurs upon
IFN-
stimulation of transfectants that contain a promoterless
reporter gene construct (0.4 ± 9.6%) (Fig. 1). However,
stable transfectants carrying a 450-bp gp91-phox promoter construct
demonstrate a statistically significant increase in hGH reporter gene
expression upon stimulation with IFN-
(117.1 ± 20.9%) (Fig. 1). Analysis of a construct containing 1.5 kilobase pairs
of gp91-phox promoter gave similar results (data not shown).
Importantly, no increase in promoter activity occurs following
IFN-
stimulation of HeLa cells carrying the 450-bp gp91-phox
promoter-hGH construct (data not shown). Thus, the stimulation observed
with the PLB985 transfectants reflects a myeloid-specific regulatory
process and not a more general IFN response.
Figure 1:
Effect of IFN- treatment on
reporter gene expression in stably transfected PLB985 cells. Expression
levels are presented as the percent change in reporter gene expression
in response to IFN-
stimulation. No promoter/hGH, human
growth hormone vector alone; 450 bp promoter/hGH, proximal 450
bp of the gp91-phox promoter; 100 bp promoter/hGH, proximal
100 bp of gp91-phox promoter; 450 bp (-57 A to C)
promoter/hGH, CGD mutation at -57 bp; 450 bp (CCGGT)
promoter/hGH, mutation of the inverted CCAAT box region. NS, not statistically significant.
No statistically
significant change in reporter gene expression is observed, however,
upon IFN- stimulation of transfectants that contain the proximal
100 bp of gp91-phox promoter (6.5 ± 10.8%). Although not
sufficient for reporter gene expression, the proximal 100 bp of
gp91-phox promoter contains a cis-element found to be mutated in
kindreds with variant CGD(12) . A -57 bp A to C mutation
that mimics one of the CGD lesions was introduced into the 450 bp
gp91-phox promoter sequence, transfected into PLB985 cells, and assayed
for reporter gene expression. No statistically significant increase in
reporter gene expression is demonstrable with the mutant construct (3.1
± 15.3%) (Fig. 1), suggesting that the CGD kindred
mutations disrupt a cis-element that is necessary for IFN-
-induced
expression of gp91-phox.
The proximal gp91-phox promoter also
contains an inverted CCAAT-box at -45 to -49 bp. To
investigate the functional contribution of this element, a mutation was
introduced into the 450-bp gp91-phox promoter sequence that changes the
element from CCAAT to CCGGT. This mutation has been previously reported
to disrupt the binding of CCAAT box binding factors to other CCAAT box
elements(9) . Assay of the mutant construct in PLB985
transfectants demonstrates an IFN--stimulated increase in reporter
gene expression (127.4 ± 27.5%) similar to that observed with
the wild-type 450-bp sequence (Fig. 1). This result indicates
that the inverted CCAAT box element is not necessary for
IFN-
-stimulated gp91-phox expression. As expected, a promoter that
contains both the CGD and CCAAT box mutations fails to respond to
IFN-
(data not shown).
The transcription start site for the
gp91-phox promoter-hGH transgene was determined by RNase protection
assay (Fig. 2). Following IFN- stimulation, two protected
bands are detected with RNA isolated from transfectants carrying the
wild-type promoter sequence (Fig. 2, lane2)
but are absent when RNA isolated from transfectants carrying the
CGD-mutant promoter sequence is analyzed (Fig. 2, lane4). The position of the lower band corresponds to the
published transcription initiation site of the gp91-phox
gene(13) . Transfectants carrying the CCGGT-mutant promoter
sequence yield an RNase protection pattern similar to that illustrated
for the wild-type promoter (data not shown). These results are
consistent with the observed IFN-
-induced increase in reporter
gene product activity and indicate that increased reporter gene
expression in response to IFN-
is due to authentic gp91-phox
promoter activity. RNase protection assays using a riboprobe for
-actin were performed as an internal standard for the quantity of
RNA in the hybridization reactions (Fig. 2, bottompanel).
Figure 2:
RNase protection analysis of the
transcription start site for the gp91-phox promoter/hGH-reporter gene
in PLB985 transfectants. Lane1, 450-bp promoter/hGH; lane2, IFN--treated 450-bp promoter/hGH; lane3, -57 bp (A to C) CGD mutation 450- bp
promoter/hGH; lane4, IFN-
-treated -57 bp
(A to C) CGD mutation 450 bp promoter/hGH; lane5,
yeast t-RNA. Toppanel, total RNA (20 µg) probed
for the gp91-phox promoter/hGH transcript (see ``Materials and
Methods''). Bottompanel, total RNA (1 µg)
probed for the
-actin transcript. Arrows indicate
positions of protected bands.
Figure 3:
Tissue distribution of DNA-binding
proteins that interact with the -30 to -68 bp region of the
gp91-phox promoter. EMSA was performed as described under
``Materials and Methods'' using 2 µg of nuclear extract
from a variety of cell lines. Lane1,
undifferentiated PLB985 cells; lane2, PLB985 cells
treated with retinoic acid and dimethylformamide; lane3, PLB985 cells treated with PMA; lane4, PLB985 cells treated with IFN-; lane5, CESS cells (B-cell); lane6, MOLT-4
cells (T-cell); lane7, K562 cells (chronic
myelocytic leukemia in erythroleukemia blast crisis); lane8, HEL cells (acute myeloid leukemia, erythroleukemia
type); lane9, HeLa cells (epithelial carcinoma); lane10, HepG2 cells (hepatocellular carcinoma). Arrows indicate positions of two specific
complexes.
The tissue distribution of each binding activity was also examined. EMSA using nuclear extracts from the human hematopoietic cell lines CESS, MOLT-4, K562, and HEL demonstrate protein complexes of the same mobilities (Fig. 3, lanes5-8), indicating that both activities are present in a wide variety of hematopoietic cells. However, the faster mobility complex is much less abundant in nuclear extracts from the nonhematopoietic cell lines HeLa and HepG2 (Fig. 3, lanes9 and 10). We conclude that the slower mobility complex is ubiquitous, while the faster mobility complex is largely restricted to cells of hematopoietic origin, hereafter denoted hematopoietic associated factor 1 (HAF-1). Faint bands above the indicated doublet are not reproducible and may reflect multiple proteins bound to the probe.
The sequence specificity of each DNA-binding activity was examined in more detail. Formation of the two dominant complexes is disrupted by an excess of homologous unlabeled double-stranded oligonucleotide competitor (Fig. 4A, lane2), while a competitor containing the -57 bp CGD mutation fails to disrupt the HAF-1 complex (Fig. 4A, lane3). Similar results were obtained using a competitor containing the -55-bp CGD promoter mutation (data not shown). Conversely, an oligonucleotide containing a mutation of the inverted CCAAT box fails to disrupt the slower mobility complex (Fig. 4A, lane4), suggesting that a CCAAT-binding factor is present in this complex. An isoform of C/EBP is highly expressed in myelomonocytic cells(49) . However, competition with C/EBP binding sites fails to disrupt the CCAAT factor complex (Fig. 4A, lane5, and data not shown). Studies with additional oligonucleotide competitors and antibodies for CCAAT-binding factors identify this activity as the ubiquitous factor CP1 (data not shown).
Figure 4:
Characterization of HAF-1 DNA-binding
properties. A, determination of HAF-1 binding site
specificity. EMSA was performed as described under ``Materials and
Methods'' using 2 µg of nuclear extract from undifferentiated
PLB985 cells and a probe corresponding to the -30 to -68 bp
region of the gp91-phox promoter. Oligonucleotide competitions were
performed using a 200-fold molar excess of double-stranded unlabeled
oligonucleotides. The arrows indicate the two specific
complexes. Competitors are as follows: lane1, none; lane2, homologous; lane3,
-57 A to C CGD mutation; lane4, CCAAT-box
mutated to CCGGT; lane5, C/EBP binding
site(23) ; lane6, Ets-1 binding
site(24) ; lane7, PEA3 binding site (25) ; lane8, Elf binding site (IL2
gene)(26) ; lane9, E74 binding
site(27) ; lane10, GABP binding
site(28) ; lane11, PU.1 binding
site(14) . Arrows indicate positions of two specific
complexes. B, determination of whether Fli-1 binds to the
-30 to -68 bp region of the gp91-phox promoter. Fli-1 protein was synthesized and EMSA was performed as described under
``Materials and Methods.'' Lanes1-5,
Fli-1 binding site probe; lane1, control lysate; lane2, 2 µl of Fli-1 protein; lane3, 0.5 µl of Fli-1 protein; lane4,
0.5 µl of Fli-1 protein plus anti-ETS domain antibody; lane5, same as lane4 with addition of
blocking ETS-domain peptide; lanes6 and 7,
-30 to -68 bp gp91-phox promoter probe. Lane6, 2 µl of Fli-1 protein; lane7,
control lysate. C, determination of whether HAF-1 contains an
ETS-domain. Nuclear extract was isolated from PLB985 cells stimulated
with IFN-, preincubated with the anti-ETS domain monoclonal
antibody and then incubated with a -30 to -68 bp gp91-phox
promoter probe. Lane1, PLB985 nuclear extract plus
10-fold excess blocking peptide; lane2, same as 1,
but without blocking peptide. Arrow indicates position of
HAF-1 complex.
Since a consensus binding site for the Ets protein family is found within the proximal gp91-phox promoter, and a subset of Ets proteins is expressed in hematopoietic cells, competition studies were performed with high affinity binding sites to known Ets proteins. No significant competition of the HAF-1 complex is detected using binding sites for the Ets family members Ets-1, Elf-1 (IL-2 gene), or PU.1 (Fig. 4A, lanes6, 8, and 11). In addition, no competition is observed with Elf-1 binding sites derived from the CD4 and HIV-2 promoters (data not shown). Only slight disruption of the HAF-1 complex is demonstrated with both a GABP binding site and a PEA3 element that is a binding site for many Ets proteins, including Ets-1 and Ets-2 (25) (Fig. 4A, lanes10 and 7). However, high affinity competition of the HAF-1 complex is observed with a binding site for Drosophila E74.
Ets proteins known to interact with the E74 site, such as Erg-1, Erg-2, Elk-1, Fli-1, Ets-1, Ets-2, SAP-1, Erp, PU.1, and Elf-1, are therefore candidates for HAF-1(15, 26, 29, 50, 51) . However, as described above, high affinity binding sites for PU.1, Ets-1, and Elf-1 fail to compete efficiently for HAF-1 binding. Elk-1 and SAP-1 are expressed poorly in spleen but to high levels in liver(52) , Erg-1 is expressed highly in some epithelial lines but at low levels in MOLT-4 cells(53) , Erg-2 is not present in HL60, HEL, and K562 cell lines(54) , and Erp is scarce in B-cell lines(29) . These distributions are inconsistent with that observed for HAF-1. Therefore, only Fli-1 was investigated in further detail. In vitro synthesized Fli-1 protein binds to an authentic Fli-1 binding site (15) (Fig. 4B, lanes2 and 3) but is unable to bind to the -30 to -68 bp gp91-phox promoter probe (Fig. 4B, lanes6 and 7). In summary, these data indicate that HAF-1 is not Fli-1.
Members of the Ets family of transcription factors are characterized by homology in a highly conserved ETS-domain that is involved in binding site recognition(55) . Binding of Fli-1 is disrupted by the addition of an anti-ETS domain antibody (Fig. 4B, lane4), an effect that is blocked by the addition of an excess of ETS-domain peptide (Fig. 4B, lane5). However, no disruption of the HAF-1 complex occurs following preincubation of nuclear proteins with the ETS-domain antibody (Fig. 4C). Similar results were obtained using an antibody raised against Fli-1 (15) (data not shown). In additional control experiments, the anti-ETS domain antibody disrupted complexes formed following incubation of PLB985 nuclear extract with consensus binding sites for Ets-1 and PU.1 (data not shown). Hence, we are unable to demonstrate that HAF-1 contains an ETS-domain.
The binding characteristics of HAF-1 were further studied by DNase I footprinting and methylation interference assays. DNase I footprinting reveals that the HAF-1 complex protects a region between -45 and -62 bp of the gp91-phox promoter (Fig. 5A). Methylation interference analysis of the HAF-1 complex demonstrates contact bases consistent with the Ets family of DNA binding proteins (Fig. 5B)(54) . Both the DNase I footprinting and methylation interference data indicate that CP1 and HAF-1 exhibit overlapping but distinct binding sites in the proximal gp91-phox promoter (data not shown).
Figure 5: Characterization of the HAF-1 binding site. A, DNase I footprint analysis of HAF-1 interacting with the -30 to -68 bp region of the gp91-phox promoter. Nuclear extract was isolated from undifferentiated PLB985 cells as described under ``Materials and Methods.'' Lane1, G+A ladder of the coding strand; lane2, DNase I footprint of HAF-1 for the coding strand; lane3, DNase I digestion ladder of the coding strand; lane4, DNase I footprint of HAF-1 for the non-coding strand; lane5, DNase I digestion ladder of the non-coding strand; lane6, G+A ladder of the noncoding strand. B, methylation interference analysis of HAF-1 interacting with the -30 to -68 bp region of the gp91-phox promoter using nuclear extract from undifferentiated PLB985 cells. Lane1, coding strand probe shifted in the HAF-1 complex; lane2, unshifted coding strand probe; lane3, noncoding strand probe shifted in the HAF-1 complex; lane4, unshifted noncoding strand probe. Summary of HAF-1 binding characteristics is illustrated below. Bars indicate position of footprints, hatchmarks denote hypersensitive sites. Arrows indicate position of promoter mutations identified in CGD patients(12) . Weak contact bases are indicated with an opencircle, and strong contact bases are indicated with a filledcircle.
During terminal myeloid hematopoiesis, a number of
phagocyte-specific genes, including the gene for gp91-phox, become
transcriptionally active(6) . To understand the events involved
in the tight lineage specificity of this process, we analyzed the
proximal gp91-phox promoter for cis-elements important in
IFN--induced transcription of this gene. Stable transfectants of
PLB985 cells containing gp91-phox promoter-hGH constructs exhibit
IFN-
-inducible transgene expression, thus permitting study of
IFN-
response elements in the gp91-phox promoter. Using this
approach, we demonstrate that elements necessary for IFN-
-induced
transcription reside between -100 and -450 bp of the
gp91-phox promoter. We also demonstrate that, although the proximal 100
bp of promoter is not sufficient for the IFN-
transcription
response, there is a cis-element within the proximal 100 bp that is
necessary for the IFN-
response. RNase protection assays
demonstrate a stimulation of transgene transcription from the authentic
gp91-phox initiation site in response to IFN-
treatment.
Study
of phagocyte function has been facilitated by the existence of CGD
kindreds with various respiratory burst
defects(4, 5, 7) . We demonstrate that
gp91-phox promoter mutations previously described in two kindreds with
variant CGD (12) disrupt a DNA-protein interaction that is
required, but is not sufficient, for IFN--induced gp91-phox
transcription. This binding activity is largely restricted to cells of
hematopoietic origin, and hence is denoted HAF-1.
The presence of
HAF-1 in nuclear extracts in cell lines not transcribing gp91-phox
further suggests that binding of HAF-1 is not sufficient to activate
transcription of the gp91-phox gene in response to IFN-. However,
the abundance of HAF-1 activity is not increased in nuclear extract
isolated from IFN-
-stimulated myeloid cells and hence is not
directly involved in the signal-transduction process. A distinct
IFN-
responsive element presumably resides elsewhere in the
proximal gp91-phox promoter construct, consistent with the finding that
elements between -100 bp and -450 bp are required for an
IFN-
response. Inspection of this DNA sequence, however, did not
reveal consensus sequences for IFN-
responsive
elements(56, 57, 58) . Additional studies
utilizing the functional assay system described in this report should
permit an identification of the cis-elements that are necessary and
sufficient for IFN-
-induced transcription of the gp91-phox gene.
Although not myelomonocytic-specific, HAF-1 binding may contribute to phagocyte-specific expression of gp91-phox through cooperation with other cis-elements and trans-factors. For example, we have previously demonstrated that binding of CCAAT-displacement protein (CDP) to an element at -120 bp of the gp91-phox promoter serves to repress promoter activity. We proposed that down-regulation of CCAAT-displacement protein DNA-binding activity during terminal differentiation serves to ``poise'' the gp91-phox promoter for activation upon binding of a number of ubiquitous or lineage-restricted transcriptional activating proteins(9) .
The DNase I footprint of HAF-1 covers a region of gp91-phox promoter
that contains the core binding site for the Ets family of transcription
factors(15) . The minimal six-bp recognition sequence for the
Ets family members Ets-1, Ets-2, and PU.1/Spi-1 is
5`-(A/C)G
G
A
(A/T)
(C/G)
-3` (15) . Binding of the Ets protein Fli-1 has additional
requirements outside of the Ets core motif
(5`-C
(C/A)
G
G
A
A
G
T
-3`),
binding poorly in the absence of either C
or T
and not at all in the absence of both(15) . Although the
gp91-phox promoter sequence is not identical to any of the defined Ets
family member high affinity binding sites, there are striking
similarities. The gp91-phox promoter sequence (antisense strand) from
-50 to -57 is
5`-G
A
G
G
A
A
A
T
-3`
(bases mutated in the two CGD kindreds are underlined(12) ).
Methylation interference analysis of the HAF-1 complex demonstrates
that the G
G
bases are important for HAF-1
binding, consistent with binding requirements demonstrated for several
Ets family members.
Several lines of evidence indicate that HAF-1
does not correspond to previously described Ets factors. Abundant HAF-1
binding activity is found in all of the hematopoietic cell nuclear
extracts tested (K562, HEL, CESS, MOLT-4, PLB985, and HL60), but not in
nonhematopoietic lines (HeLa and HepG2), and is not altered by
treatment of PLB985 or HL60 cells with PMA, retinoic acid, or
IFN-. This tissue distribution narrows the potential Ets proteins
to consider. Of the Ets proteins known to be expressed predominately in
hematopoietic cells, all but Fli-1 can be ruled out based on binding
site specificity. However, Fli-1 has a somewhat more limited tissue
distribution than that observed for HAF-1. Although mRNA for Fli-1 has
been detected in many immature and mature T- and B-cell lines and in
the erythroleukemia cell line HEL, mRNA levels are undetectable in K562
cells(59) . Also, although mouse bone marrow macrophages have
been demonstrated to express Fli-1 message, detectable Fli-1 mRNA is
abolished by stimulation of macrophages with PMA or
IFN-
(59) . In addition, in vitro synthesized
Fli-1 is unable to bind to the -30 to -68 bp gp91-phox
promoter element. These results indicate that HAF-1 is not Fli-1, and
we conclude that the tissue distribution and binding site specificity
of HAF-1 do not conform to known Ets proteins.
In addition, the HAF-1 complex is not disrupted by an antibody directed against the conserved ETS-domain. We cannot exclude the possibility that HAF-1 contains an ETS domain unavailable for interaction with the monoclonal antibody due to protein-protein interactions or stearic hindrance, or that HAF-1 contains a divergent ETS-domain that is not recognized by the antibody. Alternatively, HAF-1 may represent a novel transcription factor with a binding specificity similar to an Ets protein. Search of a transcription factor binding site data base (60) failed to identify additional consensus binding sites within this promoter element.
Binding studies indicate that the ubiquitous CCAAT-binding
factor CP1 interacts with a binding site that overlaps the HAF-1 site
within the proximal gp91-phox promoter. Functional analysis indicates
that CP1 binding at this site is not required for IFN--induced
gp91-phox transcription. It remains possible, however, that this
element is functionally relevant to gp91-phox promoter activity in
vivo, perhaps in cooperation with other cis-elements and
trans-factors in the full complement of myelomonocytic cells.
The
data presented in this study indicate that binding of HAF-1 to the
proximal gp91-phox promoter is necessary but not sufficient for
IFN- induction of gp91-phox expression. The finding that loss of
HAF-1 binding is associated with a variant form of CGD is suggestive of
an important role for HAF-1 in the regulation of gp91-phox expression.
The characterization of HAF-1 binding properties reported here should
aid in efforts to obtain molecular clones for this factor.
Identification of the HAF-1 protein(s) will allow detailed study of the
function and regulation of this factor, including the relationship of
HAF-1 to the Ets family, and may shed light on the molecular mechanisms
involved in terminal myeloid differentiation.