From the Lurleen B. Wallace Tumor Institute, Department of Hematology and Oncology and the Comprehensive Cancer Center, University of Alabama at Birmingham and the Birmingham Veterans Administration Hospital, Birmingham, Alabama 35294
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ABSTRACT |
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gp91phox is a subunit of the phagocyte
respiratory burst oxidase catalytic unit. Transcription of
CYBB, the gene encoding gp91phox, is restricted to
terminally differentiated phagocytic cells. An element in the proximal
CYBB promoter binds a protein complex, referred to as
hematopoiesis-associated factor (HAF1), that is necessary for
interferon- (IFN
)-induced gp91phox expression. In these
investigations, we determined that HAF1 was a multiprotein complex,
cross-immunoreactive with the transcription factors PU.1, interferon
regulatory factor 1 (IRF-1), and interferon consensus sequence-binding
protein (ICSBP). In electrophoretic mobility shift assay, the HAF1
complex was reconstituted by either in vitro translated
PU.1 with IRF-1 or PU.1 with ICSBP, but not by IRF-1 with ICSBP. HAF1a,
a slower mobility complex with the same binding site specificity as
HAF1, was also investigated. Similar to the HAF1 complex, the HAF1a
complex was cross-immunoreactive with PU.1, IRF-1, and ICSBP. Unlike
the HAF1 complex, reconstitution of the HAF1a complex required in
vitro translated PU.1 with both IRF-1 and ICSBP. An artificial
promoter construct containing the HAF1/HAF1a binding site was modestly
activated in the myelomonocytic cell line U937 by co-transfection
either with PU.1 and IRF-1 or with PU.1 and ICSBP, but it was strongly
activated by co-transfection with PU.1, IRF-1, and ICSBP. This
activation required serine 148-phosphorylated PU.1. These studies
describe a novel mechanism for PU.1 transcriptional activation via
interaction with both IRF-1 and ICSBP, a target gene for the
interaction of IRF-1 with ICSBP, and a novel activation function for
ICSBP as a component of a multiprotein complex.
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INTRODUCTION |
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During hematopoiesis, transcription of lineage-restricted genes
contributes to development of the various blood cell lineages. Although
erythropoiesis is regulated by lineage-restricted transcription factors
(1), many of the transcription factors that regulate myelopoiesis, such
as ETS proteins and interferon regulatory factor (IRF)1 proteins, are more
broadly expressed (2-4). The ETS transcription factor PU.1, which is
expressed exclusively in myeloid and B-cells, regulates the expression
of a number of genes common to both of these lineages, including the
genes encoding CD18 (2), and the major histocompatibility complex
I-A (5). However, PU.1 is also essential for transcription of genes
expressed in myeloid cells but not B-cells, including the gene encoding
the macrophage scavenger receptor (3), the gene encoding CD11b (6), and NCF1, which encodes p47phox (7). Also, PU.1 has been
implicated in transcription of the B-cell-specific genes encoding the
immunoglobulin
light chain (8), J chain (9),
2-4 enhancer
(10), and µ heavy chain (11). Therefore, myeloid versus
B-cell-specific regulation of genes by PU.1 must involve additional
mechanisms such as lineage-specific partners and/or lineage-specific
protein modification.
The IRF proteins have been postulated to be involved in transcriptional regulation of genes involved in the immune response. Post-translational modification and partnering have been postulated as mechanisms involved in regulation by IRF proteins (12, 13). The ubiquitously expressed interferon regulatory factor 1 (IRF-1) (12) has been demonstrated to physically interact with the myeloid and B-cell-specific interferon consensus sequence-binding protein (ICSBP) (12, 13). This interaction is governed by interferon-induced phosphorylation of both proteins and is postulated to provide a mechanism for activation of genes during the inflammatory response (13). However, the significance of this interaction in lineage-specific gene expression is hypothetical, since no target genes have been described.
In these investigations, we explored the involvement of ETS and IRF
proteins in transcription of the myeloid-specific CYBB gene,
which encodes gp91phox, the heavy chain of the phagocyte
respiratory burst oxidase (14-16). The expression of gp91phox
is limited to phagocytic cells that have matured beyond the
promyelocyte stage and continues until cell death (17). CYBB
transcription is therefore both lineage- and differentiation
state-specific. In mature phagocytes, the expression of
gp91phox is further augmented by the inflammatory mediators,
including interferon gamma (IFN) (17), tumor necrosis factor-
(18), and lipopolysaccharide (18). The absence of gp91phox
protein leads to chronic granulomatous disease (CGD), a disorder of
host defense (19).
Investigation of two kindreds with CGD revealed a cis element in the
proximal CYBB promoter that was necessary for
lineage-specific, IFN-induced gp91phox expression (20, 21).
In the affected individuals of these two kindreds, normal
gp91phox protein was absent (21), and genomic DNA analysis
revealed a single bp mutation at either
55 or
57 bp of the
CYBB gene (21). By EMSA, either of these mutations disrupted
binding of a specific protein complex to the CYBB 5'-flank
(20, 21). Since binding of this complex was manifest only with nuclear
proteins isolated from cells of hematopoietic lineages (20), it was
referred to as the hematopoiesis-associated factor 1 (HAF1) complex.
Linked CYBB promoter-reporter constructs containing the
proximal 450 bp of the CYBB 5'-flank were transfected into
myeloid cell lines, and the transfectants were differentiated with
IFN
. Wild type promoter constructs, but not CGD mutant promoter
constructs, had IFN
-inducible reporter gene expression (20). No
IFN
-inducible reporter gene expression was demonstrated when the
450-bp CYBB promoter constructs were transfected into the
epithelial cell line, HeLa (20).
In this study, we investigated the components of the HAF1 complex. The
sequence of the HAF1 binding site (20) included an ETS binding
consensus sequence (50 to
55 bp; 5'-GAGGAA-3') (4, 22), immediately
adjacent to a sequence with homology to the IRF binding consensus
sequence (
53 to
63 bp; 5'-GAAATGAAAAC-3') (4, 23) (both on the
noncoding strand). This combination is reminiscent of the
immunoglobulin
3'-enhancer element (
E3') (8). The
E3' is a
positive cis element that binds PU.1 as a heterodimer with a
lymphoid-specific, IRF protein referred to as PU.1-interacting protein
(PIP) (8, 24). However, no similar interactions between PU.1 and IRF
family members have been documented in myeloid gene regulation. In
these studies, we investigated the presence of PU.1 and IRF family
members in the HAF1 complex and the action of PU.1 and IRF family
members on the CYBB HAF1 binding element.
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EXPERIMENTAL PROCEDURES |
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Plasmids
Protein Expression Constructs and cDNAs--
The cDNA
for human PU.1 was obtained from M. Klemsz (Indiana University,
Indianapolis, IN) and subcloned into the mammalian expression pSR
(25) (obtained from T. Gabig, Indiana University), the pBluescript
vector for in vitro transcription and translation (Stratagene, La Jolla, CA), and the pGEX2 vector for fusion protein expression (Amersham Pharmacia Biotech). The cDNA for murine PU.1 and the serine to alanine mutants of murine PU.1 at serines 148, serines 132 and 133, and serines 41 and 45 were obtained from M. Atchison (University of Pennsylvania, Philadelphia, PA) and subcloned
into pSR
. The human ICSBP cDNA was obtained from B-Z. Levi
(Technicon, Haifa, Israel) and subcloned into the mammalian expression
vector pcDNAamp (Invitrogen, San Diego, CA). The human IRF-1
cDNA was obtained from R. Pine (New York University Medical Center,
New York, NY) and subcloned into the mammalian expression vector
pcDNAamp. The human FLI-1 cDNA was obtained from R. Hromas (Indiana University) and subcloned into the pBluescript vector. The
human IRF-2 cDNA was obtained from G. Stein (University of Massachusetts, Worcester, MA). The human STAT91 cDNA was obtained from A. Kraft (University of Colorado, Denver, CO). The murine PIP
cDNA was obtained from H. Singh (University of Chicago).
Minimal Promoter/Reporter Constructs--
Concatenated
CYBB promoter sequences were subcloned into the minimal
promoter/reporter vector, p-TATACAT (26) (obtained from Dr. A. Kraft,
University of Colorado, Denver, CO). Synthetic oligonucleotides with
the wild type or 57 bp CGD mutant sense and antisense CYBB
promoter sequences
32 to
69 bp were annealed, phosphorylated with
polynucleotide kinase, and ligated with T4 DNA ligase (Boehringer
Mannheim). The concatenated oligonucleotides were subcloned into the
p-TATACAT vector. Clones that were confirmed by dideoxy sequencing to
contain either one or four correct copies of the
32 to
69 bp
promoter sequence in the sense orientation were selected.
Plasmids Used to Generate EMSA Probes--
Duplex synthetic
oligonucleotides with the wild type 32 to
69 bp promoter sequence
and the
55 or
57 bp mutant sequences (20, 21) were subcloned into
the plasmid vector pUC19, and correct sequence was confirmed by dideoxy
sequencing.
Oligonucleotides
Oligonucleotides were synthesized by the core facility at the
Herman B. Wells Center for Pediatric Research at Indiana University, in
the Oligonucleotide Core Facility of the Comprehensive Cancer Center of
the University of Alabama at Birmingham, or in the laboratory of Dr.
Donald Miller at the University of Alabama at Birmingham. CYBB promoter sequence (4) oligonucleotides used were
as follows: CYBB promoter 32 to
69 bp,
5'-CTGCTGTTTTCATTTCCTCATTGGAAGAAGAAGCATAG-3'; CGD mutant promoter
32
to
69 bp (
57) (21),
5'-CTGCTGTTTTCCTTTCCTCATTGGAAGAAGAAGCATAG-3'; CGD mutant
promoter
32 to
69 (
55) (21),
5'-CTGCTGTTTTCATGTCATTGGAAGAAGAAGCATAG-3'; E74 (22),
5'-AATAACCGGAAGTAACTC-3'; PU.1 binding site from the major
histocompatibility complex class II gene (27),
5'-TGAAATAACCTCTGAAAGAGGAACTTGGTTAGGTA-3'; Pu.1/PIP binding site from
the immunoglobin
E3' (8), 5'-CTTTGAGGAACTGAAAACAGAACCTA-3'; GASGBP
(
-interferon activation sequence of the guanylate-binding protein)
(28), 5'-AGTTTCATATTACTCTAAATC-3'; CYBB promoter
214 to
243 bp (IRF-2 binding site) (29),
5'-AGAAATTGGTTTCATTTTCCACTATGTTTAATTGTGACTGGATCATTAT-3'.
Cell Culture and Stable Transfections
All cell lines were of human origin. The epithelial carcinoma
line HeLa (30) was obtained from ATCC (Rockville, MD). The promyelocytic leukemia cell line PLB985 (31) was provided by Thomas
Rado (University of Alabama at Birmingham). The promyelocytic leukemia
cell line U937 (32) was obtained from Andrew Kraft (University of
Colorado, Denver, CO). Cell lines were maintained and differentiated as
described previously (20). PLB985 and U937 cells were treated with
human recombinant IFN (Boehringer Mannheim) at 100 units/ml for
various times. HeLa cells were treated with IFN
at 50 units/ml for
24 h.
In Vitro Translation of Proteins
In vitro transcription and translation of human and murine PU.1, ICSBP, IRF-1, IRF-2, STAT91, and TF1phox sequences cloned into pBluescript or pcDNAamp was performed using the in vitro transcription and rabbit reticulocyte lysate in vitro translation systems by Promega according to the manufacturer's instructions (Promega, Madison, WI).
Electrophoretic Mobility Shift Assays
Nuclear extract proteins were prepared from tissue culture cells growing at log phase by the method of Dignam (33) with protease inhibitors as described (20). Protein assays were performed by the method of Lowry (34).
Oligonucleotide probes were prepared from plasmid constructs by
restriction digestion and filled in by Klenow fragment with a
nucleotide mix containing dATP, dGTP, dTTP (Boehringer Mannheim) and
[-32P]dCTP (Amersham Pharmacia Biotech). Specific
activity of the probes was ~2 × 106 cpm/pmol of
DNA.
EMSA and antibody supershift assays were performed as described (20). Antiserum to PU.1 was obtained from M. Klemsz (Indiana University) (27) and antiserum to FLI-1 was obtained from Alan Bernstein (Mount Sinai Hospital, Ontario, Canada) (22). Anti-IRF-2 and anti-ISGF3 (STAT91/84) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-IRF-1 antiserum (whole protein) was obtained from Richard Pine (Public Health Research Institute, New York, NY) (35). Anti-IRF-1 serum directed against amino acids 306-325 (nonconserved among IRF family members) was obtained from Santa Cruz Biotechnology. Anti-ICSBP serum (whole protein) was obtained from Keiko Ozato (National Institutes of Health, Bethesda, MD) (36). Antisera to peptides in ICSBP nonconserved with other IRF family members (anti-310, anti-311, and anti-312) was obtained from S. Vogel (Armed Forces Institute, Bethesda, MD) (37). Anti-PIP serum was a very generous gift from M. Atchison (University of Pennsylvania, Philadelphia, PA).
Co-precipitation Assays
PU.1pGEX2 and control pGEX2 in JM109 Escherichia coli
were grown to log phase, supplemented to 0.1 mM
isopropyl-1-thio--D-galactopyranoside, and incubated for
3 h at 37 °C with shaking. The cells were harvested and
resuspended in HN buffer (20 mM HEPES (pH 7.4), 0.1 M NaCl, 2 mM MgCl2, 0.1 mM EDTA, 0.5% Nonidet P40, 0.1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 5 mM NaF) and
sonicated on ice. Debris was removed by centrifugation, and the lysate
was incubated 30 min at 4 °C with glutathione-agarose beads (Sigma) and washed extensively with HN buffer. The beads were preincubated for
30 min at 4 °C with 5 µl of control rabbit reticulocyte lysate and
then for 1 h with 20 µl of [35S]methionine-labeled
in vitro translated protein and washed extensively in HN
buffer. Proteins were eluted with SDS-PAGE sample buffer and separated
on 12% SDS-PAGE, and an autoradiograph was performed.
Immunoprecipitation experiments were performed with 200 µg of nuclear proteins at 0.2 µg/ml in HN buffer with protease inhibitors as above. Nuclear proteins were incubated with 5 µl of either preimmune serum or anti-ICSBP serum (310 and 311 at 1:1) or IRF-1 antibody (Santa Cruz, 1 mg/ml) for 4 h at 4 °C, followed by 1-h incubation with 50 µl of 50% Staphylococcus protein A-Sepharose bead slurry. Beads were washed five times with 1 ml of HN buffer, and proteins eluted by boiling in SDS sample buffer, separated on 12% SDS-PAGE, transferred to nitrocellulose, and Western blotted with 0.02 µg/ml anti-PU.1 polyclonal antibody (Santa Cruz Biotechnology) and detected by chemiluminescence (Amersham Pharmacia Biotech) as per the manufacturer's instructions.
Transient Transfection and Reporter Gene Assays
U937 cells were transfected by electroporation at 0.24 mV and
960 microfarads in a Bio-Rad Gene Pulsar, and HeLa cells were transfected at 0.15 mV and 960 microfarads. U937 cells were transfected with 70 µg of p-TATACAT, p-haf1TATACAT, or p-cgdTATACAT; 30 µg of
pSR or PU.1/pSR
; either 30 µg of pcDNAamp,
IRF-1/pcDNAamp, or ICSBP/pcDNAamp or 15 µg each of
IRF-1/pcDNAamp and ICSBP/pcDNAamp; and 20 µg of
p-CMV/
-galactosidase (CLONTECH, Palo Alto, CA). HeLa cells were transfected with 15 µg of p-TATACAT, p-haf1TATACAT, or p-cgdTATACAT; 20 µg of pSR
or PU.1/pSR
; 10 µg of
IRF-1/pcDNAamp and ICSBP/pcDNAamp; and 5 µg of
p-CMV/
-galactosidase. Cell extracts were made by freeze/thaw cycles
as described (38) in the presence of 2 mM
phenylmethylsulfonyl fluoride.
-Galactosidase assays were performed
as described (39). Chloramphenicol acetyltransferase (CAT) assays were
performed by the extraction method using
[3H]chloramphenicol (Amersham Pharmacia Biotech) as
described (38).
Cell extracts were analyzed as above after 24 h of incubation at
37 °C and 5% CO2 in 10 ml of DME, 10% fetal calf
serum, 1% penicillin-streptomycin followed by 24 h with or
without IFN.
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RESULTS |
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The Transcription Factor PU.1 Interacts with the Proximal CYBB
Promoter--
To determine whether the ETS consensus sequence in the
HAF1 binding site (20) was able to interact with PU.1, EMSA was
performed with in vitro translated PU.1 and a
double-stranded oligonucleotide probe with the 32 to
69 bp
CYBB promoter sequence. In vitro translated PU.1
generated a complex not found with control lysate (Fig.
1). Binding of this complex was competed
for by unlabeled homologous oligonucleotide and oligonucleotides
containing the PU.1 binding sites from two other genes (8, 27) but not
by oligonucleotides representing either of the CYBB CGD
kindred mutations (
55 or
57 bp) or an unrelated oligonucleotide
(Fig. 1A). The complex generated by in vitro
translated PU.1 was recognized by PU.1 antiserum but not by rabbit
preimmune serum or antiserum to the ETS protein FLI-1 (Fig.
1B). EMSA with in vitro translated FLI-1 and the
32 to
69 bp CYBB promoter sequence probe demonstrated no
binding, although in vitro translated FLI-1 bound a high
affinity FLI-1 binding site (E74; Ref. 22).
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Interferon Regulatory Factors IRF-1 and ICSBP Interact with the
Proximal CYBB Promoter--
Similarly to the PU.1/PIP binding site in
the immunoglobulin gene 3'-enhancer element, the
32 to
69 bp
sequence of the CYBB promoter also contained a PU.1 binding
site overlapping an ISRE (23). Since factors of the IRF and STAT
families bind to ISRE sequences, the possibility that HAF1 might be
composed of PU.1 with PIP or another IRF, or else a STAT, was
explored.
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Interaction of PU.1 with a CYBB Promoter Element Increases
Transcription--
To determine the effect of PU.1 upon the
CYBB promoter, U937 cells were co-transfected with an
expression vector containing the PU.1 cDNA (PU.1/pSR) and an
artificial promoter construct. The artificial promoter constructs
included either four copies of the wild type
32 to
69 bp
CYBB promoter sequence or four copies of the
57 bp mutant
sequence, linked to a minimal promoter and a CAT reporter gene. A
comparison was made between CAT activity from the control plasmid
(p-TATACAT; Ref. 26) and each of the CYBB promoter
sequence-containing constructs (p-haf1TATACAT or p-cgdTATACAT for the
wild type and mutant promoter constructs, respectively). Results were
expressed as a percentage increase or decrease in CAT activity of the
CYBB sequence containing plasmid relative to CAT activity of
control p-TATACAT plasmid (therefore, p-TATACAT activity was 0%). Each
experiment was repeated six times.
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Interaction of IRF-1 and ICSBP with a CYBB Promoter Elements
Increase Transcription--
To investigate the effect of IRF-1 and
ICSBP on the 32 to
69 bp CYBB promoter sequence, U937
cells were co-transfected with an expression vector containing either
the cDNA for IRF-1 or ICSBP (IRF-1/pcDNAamp and
ICSBP/pcDNAamp, respectively) and either p-TATACAT or
p-haf1TATACAT. As above, results are expressed as the percentage increase in absolute CAT activity of p-haf1TATACAT transfectants over
the absolute CAT activity of p-TATACAT transfectants (p-TATACAT activity is 0%). Each experiment was repeated four times.
Co-transfection of p-TATACAT with PU.1, IRF-1, or ICSBP, in any
combination, had no significant effect on absolute CAT activity (with
or without IFN
).
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DISCUSSION |
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Several potential mechanisms have been implicated in lineage-specific gene regulation. In myeloid and B-cells, gene regulation appears to involve interaction between transcription factors of some lineage specificity, which confer greater lineage specificity by acting in combination. In these studies, we demonstrated that interaction between the ETS protein PU.1 and the IRF proteins ICSBP and IRF-1 was involved in lineage-specific expression of the CYBB gene, which encodes gp91phox. This interaction required the presence of all thee proteins simultaneously and increased gp91phox expression in myeloid cell lines.
Although the transcription factor PU.1 has been demonstrated to have a role in regulation of a number of myeloid genes (2, 6, 7), the present studies suggested a different mechanism for PU.1 than had been previously described. In regulation of some myeloid genes, PU.1 binding enhanced, but was not essential for, promoter function (2, 6). This was in contrast to the PU.1 binding elements in the CYBB gene and the NCF1 gene (encoding p47phox) (7). In both of these respiratory burst oxidase component genes, promoter mutation that abolished PU.1 binding also abolished promoter function. However, in contrast to functional studies of the NCF1 promoter element (7), our studies demonstrated that PU.1 alone was not adequate to confer lineage-inappropriate expression by the CYBB promoter element. Therefore, the NCF1 promoter element was functional in the absence of other lineage-specific factors in contrast to the requirement for IRF-1 and ICSBP with PU.1 for the CYBB promoter element.
B-cell-specific Ig gene transcription required PU.1 to partner with
the IRF protein PIP (8, 24), although ICSBP could substitute for PIP in
activation of a reporter gene construct with the Ig
element (42).
However, other functional partners for PU.1, with target genes for PU.1
partnering, had not previously been described. In these studies, we
demonstrated that ICSBP and IRF-1 were functional partners for PU.1
interacting with the CYBB promoter element. This interaction
required PU.1 serine 148, as had been described for PU.1-PIP partnering
(8), suggesting a general pattern to PU.1 interaction with IRF
proteins. However, in contrast to Ig
activation by PU.1-PIP, maximal
activation of the CYBB promoter element required PU.1
interaction with both IRF-1 and ICSBP in a heterotrimer. This was a
novel mechanism for PU.1 function. In the PU.1 interaction with PIP,
the transcriptional activation domain of PU.1 was dispensable (41).
Further investigations will determine if PU.1 participates in
transcriptional activation of the CYBB gene directly or
serves as an anchor for transcriptional activation by one or both of
the IRF proteins.
It had been postulated that interaction between IRF-1 and ICSBP was an
important mechanism for regulation of genes involved in the immune
response (43). In one model of this interaction, ICSBP bound an ISRE or
positive regulatory domain 1 element and functioned as a repressor
(43). In cells treated with IFN, this repression was antagonized by
IRF-1 binding to the same element and functioning as an activator (43).
In co-transfection experiments with HeLa cells, other investigators
demonstrated that overexpression of ICSBP repressed artificial promoter
constructs containing various ISRE and positive regulatory domain 1 elements (44). However, in co-transfection experiments with the
myelomonocytic cell line U937, these investigators also found that
overexpression of ICSBP repressed some of these artificial promoter
constructs but mildly activated others (44). These results suggested
that the interaction of ICSBP with promoter elements might be more
complex.
Recent work by another group of investigators provided additional
insight into the complexity of interaction between IRF-1 and ICSBP (12,
13). These investigators demonstrated that only nonphosphorylated ICSBP
was capable of direct DNA binding and that ICSBP was phosphorylated
constitutively in U937 cells (13). Phosphorylated ICSBP bound DNA
indirectly via interaction with IRF-1, which bound DNA in response to
IFN stimulation (13). This interaction required tyrosine
phosphorylation of IRF-1 (13). These investigators, however, did not
identify any target genes for this potential IRF-1 interaction with
ICSBP. Our investigations identified a target gene for the IRF-1
interaction with ICSBP and described a functional interaction in which
ICSBP is involved in activation, not repression of gene expression.
The current investigations suggested that the previously described HAF1 complex (20) was composed of PU.1 with either IRF-1 or ICSBP and that the newly described HAF1a complex was composed of PU.1 with both IRF-1 and ICSBP. According to this model, there were three possible interactions among PU.1, IRF-1, and ICSBP. In all of these interactions, PU.1 was postulated to function as the DNA-binding anchor due the inability of either IRF-1 or ICSBP alone, or IRF-1 together with ICSBP, to bind directly to the CYBB promoter sequence. Possible combinations consistent with the data were as follows: PU.1 bound IRF-1, which bound ICSBP; PU.1 bound ICSBP, which bound IRF-1; or PU.1 bound directly to both IRF-1 and ICSBP simultaneously. The third possibility seems to be the least likely due to the potential for stearic hindrance and the known ability of the two IRF proteins to physically interact (12, 13). Further investigations will clarify the interactions and determine the domains involved.
Overexpression of PU.1, IRF-1, and ICSBP in HeLa cells did not activate transcription from a co-transfected artificial promoter construct containing the HAF1/HAF1a binding CYBB promoter element. One possible explanation for this was that an additional, not yet identified, myeloid-specific transcription factor was involved. Another possible explanation was that the interaction of PU.1, IRF-1, and ICSBP required lineage-specific post-translational protein modification(s). According to this hypothesis, the essential protein modifications may have occurred when the proteins were overexpressed in the myeloid cell line, but not in the epithelial cell line.
Since serine 148 phosphorylation of PU.1 was necessary for functional interaction of PU.1 with IRF-1 and ICSBP, and tyrosine phosphorylation of IRF-1 and ICSBP was previously demonstrated to be necessary for interaction of IRF-1 and ICSBP with each other (13), it was possible that the mechanism of CYBB gene regulation required specific phosphorylation of the three proteins. Further, it was possible that this protein phosphorylation depended upon signal transduction pathways leading to kinase activity specific to myeloid cells. These possibilities are presently under investigation in our laboratory.
Regulation of gp91phox expression involves the coordinate action of a number of transcriptional repressors and activators (4, 29, 45). These factors have variable stringency of lineage restriction and are likely to confer lineage specificity by acting in combinations. Our investigations suggested that post-translational modifications, of variable lineage specificity, may also be involved. Further investigations of myeloid gene regulation will involve identification of the DNA-binding proteins, and also myeloid-specific signaling events that modify these proteins.
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FOOTNOTES |
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* This work was supported by the following grants (to E. A. E.): a Veterans Administration Merit Review, National Institutes of Health Clinical Investigator Career Development Award KO8HL0313, National Institutes of Health FIRST Award HL5400, and an American Medical Association Florence Carter Leukemia Fellowship.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M66390 (CYBB genomic sequence).
To whom correspondence and reprint requests should be addressed:
Wallace Tumor Institute, WTI554, University of Alabama, Birmingham, 1824 6th Ave. S., Birmingham, AL 35294. Tel.: 205-934-8630; Fax: 205-934-9573.
1
The abbreviations used are: IRF, interferon
regulatory factor; CGD, chronic granulomatous disease; IFN,
interferon-
; bp, base pair; EMSA, electrophoretic mobility shift
assay; PIP, PU.1-interacting protein; ICSBP, interferon consensus
sequence-binding protein; HAF1, hematopoiesis-associated factor 1;
ISRE, interferon-stimulated response element;
3'E, Ig
3'-enhancer
element; GST, glutathione S-transferase; PAGE,
polyacrylamide gel electrophoresis.
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REFERENCES |
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