(Received for publication, August 14, 1996, and in revised form, January 22, 1997)
From the Lurleen B. Wallace Tumor Institute, Department of Hematology and Oncology, and the Comprehensive Cancer Center, University of Alabama, Birmingham School of Medicine, Birmingham, Alabama 35294
The CYBB gene encodes
gp91phox, the heavy chain of the phagocyte-specific NADPH
oxidase. CYBB is transcriptionally inactive until the
promyelocyte stage of myelopoiesis, and in mature phagocytes, expression of gp91phox is further increased by
interferon- (IFN-
) and other inflammatory mediators. The
CYBB promoter region contains several lineage-specific cis-elements involved in the IFN-
response. We screened
a leukocyte cDNA expression library for proteins able to bind to
one of these cis-elements (
214 to
262 base pairs) and
identified TF1phox, a protein with sequence-specific
binding to the CYBB promoter. Electrophoretic mobility
shift assay with nuclear proteins from a variety of cell lines
demonstrated binding of a protein to the CYBB promoter that
was cross-immunoreactive with TF1phox. DNA binding of this
protein was increased by IFN-
treatment in the myeloid cell line
PLB985, but not in the non-myeloid cell line HeLa. Overexpression of
recombinant TF1phox in PLB985 cells increased endogenous
gp91phox message abundance, but did not lead to cellular
differentiation. Overexpression of TF1phox in myeloid
leukemia cell lines increased reporter gene expression from artificial
promoter constructs containing CYBB promoter sequence. These data suggested that TF1phox increased expression of
gp91phox.
Mature phagocytes are characterized by the ability to generate
superoxide and other toxic free radicals via the respiratory burst
(1-3). The catalytic unit of the respiratory burst oxidase is a
membrane-associated b cytochrome with heavy and light chain protein subunits that are referred to as gp91phox (4) and
p22phox (5). Congenital absence of either subunit leads to
chronic granulomatous disease, a disorder of host defense (6).
gp91phox is expressed nearly exclusively in phagocytic
cells that have progressed beyond the promyelocyte stage of
myelopoiesis (7). Therefore, expression of gp91phox is both
lineage- and differentiation state-specific. Transcription of the
CYBB gene, which encodes gp91phox, is augmented
in mature phagocytes by inflammatory mediators including interferon-
(IFN-
)1 (8), tumor necrosis factor-
,
and lipopolysaccharide (9), although transcription is decreased by
IFN-
(8). Regulation of gp91phox expression is therefore
important in modulation of the inflammatory response.
Previous investigations indicated that gp91phox expression
in immature myeloid cells was repressed by the CCAAT displacement
protein (CDP) (10). CDP, a homeodomain protein, was purified from HeLa cells by affinity to the 76 to
138 base pair (bp) sequence of the
CYBB promoter and cloned from an endothelial cell cDNA
library (11). Overexpression of CDP in the myeloid leukemia cell line HL-60 blunted PMA induction of gp91phox expression (12).
Recombinant CDP bound in vitro to the promoters of a number
of developmentally regulated genes and was postulated to function as a
differentiation state-specific transcriptional repressor (13, 14).
A positive regulatory element in the CYBB promoter was
identified by analysis of two chronic granulomatous disease kindreds (15) with promoter mutations at 55 or
57 bp (16, 17). Either of
these mutations disrupted in vitro binding of a specific protein complex, referred to as HAF1
(ematopoiesis-ssociated actor
) (15). 450 bp of proximal promoter sequence directed IFN-
-inducible reporter gene expression in the promyelocytic leukemia cell line PLB985 (18), and this inducible expression was
abolished by introduction of the
55 bp chronic granulomatous disease
promoter mutation (15). HAF1 binding was necessary, but not sufficient,
for IFN-
-stimulated gp91phox transcription since PLB985
transfectants with constructs containing 100 bp of proximal promoter
sequence did not respond to IFN-
. These results suggested that
elements between
100 and
450 bp must be involved in IFN-
-induced
transcription.
Further evaluation of the 100 to
450 bp promoter region revealed
the presence of three IFN-
response elements (19). Successive promoter truncations located the IFN-
response elements between
100 and
210 bp,
210 and
310 bp, and
310 and
450 bp (19). Electrophoretic mobility shift assay (EMSA) demonstrated
sequence-specific binding of protein complexes to probes representing
114 to
184 bp,
213 to
262 bp, and
331 to
381 bp (19).
Binding of the complexes was increased by IFN-
, retinoic acid, or
PMA treatment of PLB985 cells; therefore, the complexes were referred
to as BID1, BID2, and BID3, respectively, because inding
ncreased during ifferentiation (19).
Treatment of the epithelial cell line HeLa with IFN-
did not
increase BID1, BID2, or BID3 binding (19).
The BID2 complex bound to 224 to
236 bp of the CYBB
promoter (19). Although this sequence was homologous to both the
interferon-stimulated response element (ISRE) (20) and the common
binding site consensus sequence for the interferon response factors
IRF-1 and IRF-2 (21), the BID2 complex was not recognized by antibodies
to Stat1
, IRF-1, IRF-2, ICSBP, or interferon-stimulated gene factor
3
(ISGF3
) (19). IRF-2 bound to this region of the promoter in
EMSA using nuclear proteins from undifferentiated PLB985 cells and was
distinct from BID2 binding (19). However, IRF-2 binding was obscured by
BID2 complex binding in nuclear proteins of differentiated PLB985 cells
(19). Immediately 3
to the BID2-binding site was a homeodomain-binding
consensus sequence (ATTA). This suggested the presence of a second
CDP-binding site adjacent to the BID2 site since CDP preferentially
bound ATTA-containing sequences in binding site selection assays (22,
23).
Introduction of a mutation into the 450-bp proximal promoter sequence
that disrupted in vitro binding of the BID2 complex attenuated IFN--induced expression of a linked reporter gene in
PLB985 transfectants (19). Therefore, the BID2 complex might represent
a previously unidentified protein(s) that bound to a positive
regulatory element, interacted with IRF-2 and/or CDP, and contributed
to lineage-specific gp91phox expression. The identity of
BID2 component proteins was sought by molecular cloning techniques.
We previously reported cloning a cDNA by screening a human
leukocyte library for proteins that bound a DNA probe representing the
214 to
262 bp sequence of the CYBB promoter (24).
Although this promoter sequence included the BID2-binding site, the
predicted amino acid sequence of the cloned cDNA was not homologous
to previously described interferon response factors. However, the
cloned cDNA contained a sequence homologous to the S1-RM domain of
ribosomal protein S1 (24). The S1-RM domain has been postulated to bind single-stranded RNA (25). The cloned S1-like protein had high affinity
for binding double- and single-stranded DNAs, but low affinity for
binding single-stranded RNA (24). Since the S1-like protein was of low
abundance and immunoreactive S1-like protein was not found in ribosomal
preparations, we concluded that the cloned cDNA was unlikely to
represent human ribosomal S1 protein (24). The S1-like protein was
referred to as TF1phox and is further characterized in this
study.
Plasmids
TF1phox Expression ConstructsThe 1.5-kb
sequence clone (includes the entire coding sequence) was excised from
gt11 by digestion with EcoRI and subcloned into the
plasmid vector pUC19 as described (24). A 5
-clone sequence (+1 to
+1866 bp of the cDNA) was similarly isolated from
DNA and
subcloned into pUC19. The 2.69-kb sequence (+1 to +2690 bp, the entire
cDNA) was constructed by ligation of the 5
-clone and the original
1.5-kb clone sequences at a unique HpaI site found in the
sequences common to both clones. For in vitro translation, the TF1phox sequences were subcloned into the pBluescript
plasmid (CLONTECH, Palo Alto, CA).
For expression in Escherichia coli, the 1.5-kb clone
sequence was subcloned into the EcoRI site of the plasmid
pGEX1 (Pharmacia Biotech Inc.) (26) in the sense and antisense
orientations (TF1phox/pGEX1 and
TF1phox(R)/pGEX1, respectively). For expression in
mammalian cells, the 1.5-kb clone sequence was subcloned into the
EcoRI site of the plasmid pSR (obtained from T. Gabig,
Indiana University, Indianapolis, IN) (27) or pRc(CMV) (Invitrogen,
Sorrento, CA). TF1phox cDNA sequences were
epitope-tagged at the C terminus of the coding sequence with the
influenza hemagglutinin epitope (HAI; amino acid sequence IDYPYDVPDYAE
(28)). Complementary synthetic oligonucleotides encoding the epitope
tag were annealed, phosphorylated with polynucleotide kinase
(Boehringer Mannheim), and ligated in frame into an EcoRV site found 20 amino acids from the end of the TF1phox
coding sequence. Correct orientation of the cDNA was confirmed for
all constructs by dideoxy sequencing (Sequenase system, Amersham Corp.).
Concatenated
CYBB promoter sequences were subcloned into the minimal
promoter/reporter vector p-TATA/CAT (obtained from Dr. A. Kraft,
University of Alabama at Birmingham) (29). Synthetic oligonucleotides
with the sense and antisense 194 to
244 bp gp91phox
promoter sequences were annealed, phosphorylated with polynucleotide kinase, and ligated with T4 DNA ligase (Boehringer Mannheim). The
concatenated oligonucleotides were subcloned into the p-TATA/CAT vector. Clones that were confirmed by dideoxy sequencing to contain four correct copies of the
194 to
244 bp promoter sequence in the
sense or antisense orientation were selected.
Duplex synthetic
oligonucleotides with the 194 to
244 bp,
213 to
262 bp, or
76
to
138 bp promoter sequence were subcloned into the plasmid vector
pUC19, and correct sequence was confirmed by dideoxy sequencing.
Human gp91phox cDNA sequence was obtained
from Dr. Peter Newburger (University of Massachusetts, Worcester, MA).
Human p47phox (30) and p67phox (31) cDNA
sequences were obtained from Dr. Thomas Leto (National Institutes of
Health, Bethesda, MD). -Actin cDNA sequence was obtained from
Dr. D. Skalnik (Indiana University, Indianapolis, IN). Human CD18
cDNA (32) was obtained from Dr. Kent Robertson (Indiana
University).
Oligonucleotides
Oligonucleotides were synthesized either by the core facility at
the Herman B. Wells Center for Pediatric Research or in the laboratory
of Dr. Donald Miller at the University of Alabama at Birmingham.
CYBB promoter sequence (10) oligonucleotides used were as follows: 194 to
244 bp gp91phox promoter,
5
-agaaattggtttcattttccactatgtttaattgtgactggatcatta-3
;
213 to
262
bp gp91phox promoter,
5
-gttatttatctcttagttgtagaaattggtttcattttccactatgttta-3
;
215 to
240 bp gp91phox promoter,
5
-gaaattggtttcattttccactatgt-3
; gp91phox BID2-binding
site mutant (
194 to
244 bp),
5
-agaaattggtcctgccttccactatgtttaattgtgactggatcatta-3
; gp91phox BID1-binding site (
113 to
182 bp),
5
-tttgtagttgttgaggtttaaagatttaagtttgttatggatgcaagcttttcagttgaccaatgattat-3
; gp91phox BID1-binding site mutant (
113 to
182 bp),
5
-tttgtagttgttgaagctcaaggactcagacctgttatggatacaagcccccaagtcgaccaatgattat-3
;
76 to
137 bp gp91phox CDP-binding site,
5
-gcttttcagttgaccaatgattattagccaatttctgataaaagaaaaggaaaccgattgc-3
; and also including the
93 to
165 bp gp91phox
CDP-binding site,
5
-aaagatttaagtttgttatggatgcaagcttttcagttgaccaatgattattagccaatttctgataaaaga-3
. Other oligonucleotide sequences included the following: CCAAT box
from the MHC class II gene (15),
5
-gtctgaaacatttttctgattggttaaaagttgagtgct-3
; GASGBP
(-interferon ctivation equence
of the uanylate-inding rotein)
(33), 5
-agtttcatattactctaaatc-3
; PU.1-binding site from the MHC class
II gene (15), 5
-tgaaataacctctgaaagaggaacttggttaggta-3
; and binding
site for Ets-1 (15), 5
-ttccagaggatgtggcttctgcgggagagctt-3
.
Cell Culture and Stable Transfections
All cell lines were of human origin. The promyelocytic cell line HL-60 (34), the chronic myelogenous leukemia K562 (35), the epithelial carcinoma line HeLa (36), and the T-cell leukemia line MoLT-4 (37) were obtained from American Type Culture Collection (Rockville, MD). The B-cell line CESS (38) was provided by Yu Chung Yang (Indiana University). The myelomonoblastic line PLB985 (18) was provided by Thomas Rado (University of Alabama at Birmingham). The promyelocytic leukemia cell line U937 (39) was obtained from Dr. A. Kraft (University of Alabama at Birmingham).
Cell lines were maintained and differentiated as described previously
(15). HeLa cells were treated for various times with human recombinant
interferon- at 50 units/ml. Stably transfected PLB985 cell lines
carrying the neomycin resistance gene were supplemented with 1.0 mg of
Geneticin/ml (G418, Life Technologies, Inc.).
PLB985, U937, or HeLa cells were transfected by electroporation in phosphate-buffered saline at 960 microfarads and 0.24 V using a Gene Pulser (Bio-Rad). Stable transfection of vector or insert sequences was demonstrated by Southern blotting as described previously (15).
Library Screening and cDNA Sequencing
A human leukocyte gt11 library (CLONTECH) was screened with
the 1.5-kb TF1phox clone sequence by DNA-DNA hybridization
as described (40). The TF1phox sequence was labeled with
32P by the random primer method (41) to a specific activity
of 1 × 108 cpm/µg. Positive plaques were purified
through six iterations as described (24).
Expression of TF1phox as a GST Fusion Protein, Generation of TF1phox Antiserum, and Western Blotting
E. coli JM109 cells were transformed with pGEX1,
TF1phox/pGEX1, or TF1phox(R)/pGEX1, and
positive clones were selected. Transformed E. coli cells
were grown and stimulated with
isopropyl-1-thio--D-galactopyranoside at log phase as
described (26). Fusion proteins were affinity-purified by binding to
glutathione-Sepharose (Pharmacia Biotech Inc.) as described by the
manufacturer. Purified fusion protein was used to generate rabbit
antiserum by Hazelton Research Labs (Denver, PA). Purified fusion
proteins used in EMSA were dialyzed into 20 mM Hepes (pH
7.9), 0.1 M KCl, 0.2 mM EDTA, and 20%
glycerol. Western blotting and detection were performed using the ECL
system (Amersham Corp.) according to the manufacturer's
instructions.
In Vitro Translation of Proteins
In vitro transcription and translation of TF1phox sequences cloned into pBluescript were performed using the in vitro transcription and rabbit reticulocyte lysate in vitro translation systems (Promega) according to manufacturer's instructions.
Electrophoretic Mobility Shift Assays
Nuclear extract proteins were prepared from tissue culture cells growing at log phase by the method of Dignam et al. (43) with protease inhibitors as described (15). Protein assays were performed by the method of Lowry et al. (44).
Oligonucleotides probes were prepared from plasmid constructs by
restriction digestion with EcoRI and XbaI and
filled in by Klenow fragment with a nucleotide mixture containing
unlabeled dATP, dGTP, and dTTP (Boehringer Mannheim) and
[-32P]dCTP (Amersham Corp.). The specific activity of
the probes was ~2 × 106 cpm/pmol DNA.
EMSA and antibody supershift assays were performed as described (15). Anti-CDP serum was obtained from Dr. Elis Neufeld (Boston Children's Hospital, Boston). Anti-IRF-2 and anti-Stat91/84 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-IRF-1 antiserum was obtained from Dr. Richard Pine (Research Institute, New York) (45). Anti-ICSBP serum was obtained from Dr. Keiko Ozato (National Institutes of Health) (46). Anti-HAI tag antibody was obtained from Boehringer Mannheim.
RNA Preparation and Analysis
Total cellular RNA was isolated from log-phase cells by the single step method as described (47). The polyadenylated RNA fraction was isolated by two rounds of affinity chromatography with oligo(dT)-cellulose (Pharmacia Biotech Inc.) as described (48). RNA was electrophoresed on denaturing formaldehyde gels and transferred to Magnagraph membranes (Micron Separations, Westborough, MA) according to the manufacturer's instructions. Blots were hybridized with random primer-labeled DNA probes and stripped for reprobing according to the manufacturer's instructions.
Polymerase chain reaction-5-rapid amplification of cDNA ends was
performed as described (49). Poly(A) RNA isolated from PLB985 cells
stimulated for 3 h with PMA (2 µg) was used as a template. Two
nested primers complementary to sequences within the first 100 bp of
the coding sequence of TF1phox were used. Products were
analyzed by Southern blotting and hybridization to TF1phox
sequences.
Transient Transfection and Reporter Gene Assays
PLB985 cells were transfected by electroporation as described
above. PLB985 stable transfectants with either control pSR vector or
TF1phox/pSR
were transfected with 100 µg of p-TATA/CAT
or p-cybbTATA/CAT and 30 µg of pCMV/
-galactosidase (as an internal
control for transfection efficiency). Transfectants were incubated for
72 h at 37 °C in 5% CO2 in 10 ml of RPMI 1640 medium, 10% fetal calf serum, 1% penicillin/streptomycin, and 1 mg/ml
Geneticin. Cell extracts were made by hypotonic lysis as described (50)
in the presence of 2 mM phenylmethylsulfonyl fluoride.
-Galactosidase assays were performed as described (51).
Chloramphenicol acetyltransferase assays (CAT) were performed by the
extraction method using [3H]chloramphenicol (Amersham
Corp.) as described (50).
U937 cells were transfected with 70 µg of p-TATA/CAT or
p-cybbTATA/CAT, 30 µg of pRc or TF1phox/pRc, and 30 µg
of pCMV/-galactosidase as described above. Cell extracts were
analyzed as described above after 24 h of incubation at 37 °C
in 5% CO2 in 10 ml of Dulbecco's modified Eagle's
medium, 10% fetal calf serum, and 1% penicillin/streptomycin.
To identify DNA-binding proteins that might be
involved in gp91phox transcription, EMSA were performed
with a DNA probe representing the 194 to
244 bp sequence of the
promoter (Fig. 1). This sequence contained the BID2
complex-binding site (19), a sequence similar to the ISRE sequence (20)
and the IRF-1/IRF-2 protein recognition site (
224 to
236 bp) (21).
This sequence also contained a homeodomain protein recognition core, a
potential CDP-binding site (
212 to
215 bp) (Table I)
(10). To identify DNA-binding proteins that might be myeloid
lineage-specific, EMSA with nuclear proteins from the myeloid cell line
PLB985 before and after treatment with IFN-
were compared with EMSA
with nuclear proteins from the IFN-
-responsive epithelial cell line
HeLa.
|
EMSA with the 194 to
244 bp probe and nuclear proteins from PLB985
cells or HeLa cells demonstrated binding of a dominant low mobility
complex, similar to the mobility previously described for CDP (10, 11).
However, the PLB985 complex was a doublet, and the HeLa complex
migrated as a single band. These findings were consistently observed
with three sets of protein extracts from each of the two cell lines.
Homologous double-stranded oligonucleotide efficiently competed for
binding of the low mobility complex to the
194 to
244 bp probe, as
did competitor oligonucleotides containing the
76 to
137 bp and
93 to
165 bp CYBB promoter sequences (Fig. 1). The
latter two sequences included a previously described CDP-binding site
(10, 11). There was no competition for the low mobility complex with
several unrelated oligonucleotides (Fig. 2A
and data not shown).
In EMSA with nuclear proteins from HeLa cells, the low mobility complex
was disrupted by incubation with anti-CDP serum. However, incubation of
PLB985 nuclear proteins with anti-CDP serum did not result in
disruption of the low mobility complex doublet (Fig. 2B).
For comparison, control EMSA were performed using the 76 to
137 bp
promoter region. As described, in EMSA using nuclear proteins from both
HeLa and PLB985 cells, low mobility protein complexes bound to this
probe (10, 11). However, the complex generated with PLB985 nuclear
extract was slightly faster in mobility than the HeLa complex. These
protein complexes were demonstrated to represent specific protein
binding (data not shown; consistent with previous studies (10, 11)).
Anti-CDP serum disrupted binding of the slow mobility complex to the
76 to
137 bp probe in assays with HeLa nuclear proteins (as
described previously (11)); however, in EMSA using PLB985 nuclear
extract proteins, the dominant slow mobility complex was not disrupted
by anti-CDP serum (Fig. 2C).
Treatment of PLB985 and HeLa cells with IFN- resulted in the
disappearance of the dominant low mobility complex from the nuclear
proteins of both cell types at 24 h (Fig. 3). In
PLB985 cells (Fig. 3A), but not HeLa cells (Fig.
3B), IFN-
treatment also resulted in binding of a novel
protein complex of higher mobility. This higher mobility complex is the
BID2 complex that we have previously characterized (19). We previously
reported that the BID2 complex represented sequence-specific binding
and that BID2 binding increased upon differentiation of PLB985 cells with IFN-
, PMA, or retinoic acid (19). The BID2 complex appeared after 24 h of IFN-
treatment (Fig. 3A). These
results were consistently observed with at least three nuclear extract
preparations for each cell type, under each condition.
As we previously described, EMSA demonstrated binding of two additional
protein complexes, of similar mobility to the BID2 complex, to the
region of the promoter containing the BID2-binding site (seen in Fig.
3) (19). We previously demonstrated that the complex of slightly slower
mobility than BID2 (referred to as 2a) did not change in response to
IFN- or PMA; did not interact with antibodies to IRF-1, IRF-2,
ICSBP, ISGF3
, or Stat91/86; and represented nonsequence-specific
protein binding to the probe (19). We also demonstrated that the
complex of slightly faster mobility than BID2 had sequence-specific
binding (19) and was disrupted by an antibody to IRF-2, but not by
antibodies to IRF-1 (45), ICSBP (46), ISGF3
, or Stat91/86 (19). IRF2
binding became obscured by the BID2 complex in PLB985 nuclear proteins after treatment with IFN-
or PMA (19).
A human leukocyte
cDNA expression library was screened for proteins binding to the
214 to
262 bp sequence of the promoter of the gene coding for
gp91phox, and a 1.5-kb clone was identified (24). Since
there was an open reading frame to the 5
-end of this clone, the
library was rescreened under high stringency by DNA-DNA hybridization
(40) using the original clone as a random primer-labeled probe. Clones with overlapping sequences were obtained that identified a putative full-length cDNA of 2.69 kb. The 2.69-kb sequence coded for a 1.186-bp open reading frame with a 5
-untranslated region of 1.242 kb
and a 3
-untranslated region of 253 bp. This open reading frame was
entirely contained within the original 1.5-kb clone and coded for a
protein with a predicted molecular mass of 41.5 kDa. Only one clone
obtained by rescreening contained sequence that was 5
to the original
1.5-kb clone, and all of the clones but one had the same 3
-end as the
original 1.5-kb clone.
The 1.5-kb clone had a Kozak consensus sequence (52) immediately
upstream of the first methionine codon, and additional methionine
codons farther 5 in the sequence of the 2.69-kb clone all had an
in-frame stop codon before reaching the original 1.5-kb clone sequence.
As previously reported, the coding sequence of the 1.5-kb clone had a
predicted amino acid sequence with sequence similarity to S1-RM (24).
Additional analysis of the amino acid sequence determined that there
was a potential nuclear localization domain (KRVK) at amino acids
297-300 (53). A GeneBankTM search revealed that no
described ribosomal S1 protein included a nuclear localization domain.
The predicted amino acid sequence contained no domains traditionally
associated with transcription factors. Additionally, there was no
homology to either the Stat or IRF families of IFN response factors,
although the predicted amino acid sequence contained an excess of
charged residues, similar to IRF-like proteins (54). The protein was
referred to as TF1phox.
The TF1phox transcript size was determined by Northern
blotting and polymerase chain reaction-5-rapid amplification of
cDNA ends. The 1.5-kb sequence probe hybridized to an mRNA of
2.7 kb on PLB985 poly(A) Northern blots (Fig.
4A), as did a probe containing only sequence
from the 2.69-kb clone not present in the 1.5-kb clone (nonoverlapping
sequence). When adjusted for differences in RNA loading (by
hybridization to a
-actin probe), there was no significant change in
TF1phox mRNA levels in response to IFN-
(Fig.
4A) or PMA (data not shown). Polymerase chain
reaction-5
-rapid amplification of cDNA ends was performed on
poly(A) RNA isolated from PLB985 cells. A single sharp band that
indicated a 5
-untranslated region of 1.2 kb was detected using nested
primers in the 5
-region of the coding sequence, consistent with the
5
-untranslated region of the putative full-length clone (data not
shown).
The size of in vitro translated TF1phox was compared with the calculated molecular mass based upon the predicted amino acid sequence. In vitro translated protein was generated using the 1.5-kb clone as a template in rabbit reticulocyte lysate. SDS-polyacrylamide gel electrophoresis of the in vitro translation product demonstrated production of a protein of ~49 kDa, consistent with the predicted molecular mass. The in vitro translation product using the +1 to +1866 bp cDNA sequence as a template was ~20 kDa, consistent with the predicted molecular mass if the first methionine in the 1.5-kb clone was indeed the site of translation initiation (Fig. 4B). In vitro translation of the full-length 2.69-kb clone also resulted in production of a 49-kDa protein.
DNA-binding site specificity of TF1phox was investigated
with recombinant TF1phox expressed as a glutathione
S-transferase fusion protein and purified by affinity
chromatography (TF1phox/GST). EMSA with
TF1phox/GST and the 194 to
244 bp promoter sequence
probe demonstrated binding of a protein complex (Fig.
5). The complex was not demonstrated with purified GST
or fusion protein generated by antisense TF1phox/GST (Fig.
5A). Binding of TF1phox/GST to the BID2-binding
site probe was not competed for by unrelated DNA sequences, including
GASGBP (33) (Fig. 5B). A mutant
194 to
244 bp sequence
unable to bind the BID2 complex due to disruption of the ISRE-like
element (19) also was unable to compete for TF1phox
binding. The overlapping
214 to
262 bp sequence, originally used to
identify TF1phox by expression screening, competed
efficiently for TF1phox binding. A competitor
oligonucleotide containing the BID1-binding site (19) also competed
efficiently for TF1phox binding, consistent with the
cross-reactive binding specificities of BID1 and BID2 (19). Identical
results were found in EMSA using the overlapping
214 to
264 bp
promoter sequence as a probe (data not shown). In vitro
translated TF1phox was not used in these experiments
because it bound poorly in EMSA using CYBB promoter sequence
probes.
TF1phox Antiserum Recognizes the BID2 Protein Complex Binding to the CYBB Promoter
To investigate whether
TF1phox was a component of the BID2 protein complex, a
polyclonal rabbit antiserum was generated. EMSA were performed with the
194 to
244 bp probe and nuclear proteins from IFN-
(200 units/ml
for 48 h)-treated PLB985 cells in the presence of
anti-TF1phox serum or preimmune serum. Incubation of either
IFN-
- or PMA-treated PLB985 nuclear proteins with
anti-TF1phox serum disrupted binding of the BID2 complex to
the
194 to
244 bp probe (Fig. 6A). The
presence of preimmune serum in binding reactions had no effect upon the
BID2 complex under the same incubation conditions. Binding of IRF-2
increased in the presence of TF1phox antiserum, coincident
with disruption of TF1phox binding (Fig. 6A).
This result suggested that disruption of BID2 complex binding by
TF1phox antiserum permitted increased binding of a factor
usually excluded from the site by the presence of the
TF1phox protein (as part of the BID2 complex) in
differentiated myeloid cells. Identical results were obtained with
nuclear proteins treated with PMA (0.1 µM for 48 h)
(data not shown). A protein complex immunoreactive with
TF1phox antiserum bound to the probe in EMSA with the
nuclear proteins of a variety of cell lines (Fig. 6B). Since
the probe used in Fig. 6 (A and B) was generated
in the same labeling reaction, panels A and B
demonstrate increased intensity of the TF1phox complex (the
BID2 band) in PLB985 cells upon treatment of the cells with IFN-
for
48 h (compare lane 1 from A with lane 1 from B).
Both gp91phox gene expression and binding of the BID2
complex to the promoter region of the gene encoding
gp91phox were increased by differentiation of PLB985 cells
with IFN-; however, treatment with IFN-
had been demonstrated to
decrease gp91phox expression in myeloid cell lines.
Therefore, the effect of these two agents upon TF1phox
protein abundance was investigated by Western blotting. Western blots
of total cellular lysate proteins from 1 × 107 PLB985
cells before and after 48 h of treatment with IFN demonstrated an
immunoreactive band of ~49 kDa that was not altered in abundance by
treatment with IFN-
(Fig. 6C). However, a decrease in the 49-kDa TF1phox immunoreactive protein was found in the
total cellular proteins of PLB985 cells treated for 48 h with
IFN-
.
To investigate the effect of the
TF1phox protein upon the promoter region of the
CYBB gene, the TF1phox coding sequence was
subcloned into the mammalian expression vector pSR (27)
(TF1phox/pSR
), and stable transfectant pools of PLB985
cells were selected. Stable transfectants of PLB985 cells with empty
pSR
vector were also selected to serve as a control pool. Expression
of TF1phox mRNA was verified by the presence of a
1.5-kb mRNA that hybridized with the 1.5-kb clone probe in Northern
blots of total cellular RNA from TF1phox/pSR
, but not
control PLB985 transfectants (data not shown). Western blots of total
cell lysates demonstrated increased TF1phox immunoreactive
protein in the PLB985 TF1phox/pSR
transfectants in
comparison with the control pSR
transfectants (Fig.
7A).
Nuclear proteins were isolated from TF1phox/pSR and
control pSR
transfectants for use in EMSA of the
194 to
244 bp
promoter sequence (Fig. 7B). Nuclear proteins were extracted
from PLB985 transfectants that had not been treated with IFN-
or
PMA. EMSA with nuclear proteins from PLB985 control pSR
transfectants demonstrated a dominant slow mobility complex identical
to the complex found with nuclear extract proteins from untreated
PLB985 cells. However, EMSA using nuclear extract proteins from PLB985
TF1phox/pSR
transfectants demonstrated no low mobility
complex, but showed an increase in the BID2 complex (an apparent
decrease in the intensity of the 2a complex in lane 4 relative to lane 3 is due to increased protein loading in
lane 4). Therefore, overexpression of TF1phox in
PLB985 cells had the same effect upon in vitro protein
binding as treatment of the PLB985 cells with IFN-
or PMA: decreased binding of the low mobility complex and increased binding of the BID2
complex. These results were obtained with nuclear extract proteins
prepared from at least three independent pools of PLB985 TF1phox/pSR
or pSR
stable transfectants.
To determine whether recombinant TF1phox protein was
present in the BID2 complex in EMSA using nuclear proteins from PLB985
TF1phox/pSR transfectants, HAI-tagged
TF1phox was overexpressed in PLB985 cells in the mammalian
expression vector pSR
. EMSA of the
194 to
244 bp promoter
sequence was performed using nuclear proteins from PLB985
TF1phox (HAI-tagged)/pSR
transfectants. The BID2 complex
generated in EMSA using nuclear proteins from epitope-tagged
TF1phox-overexpressing PLB985 cells was supershifted by
incubation with anti-HAI antibody, but not by an irrelevant monoclonal
antibody (Fig. 7C).
To determine the effect of overexpression of
TF1phox upon endogenous gp91phox mRNA
levels, Northern blotting of RNAs from TF1phox/pSR and
control pSR
of PLB985 cells was performed. Each experiment was
repeated three times for each of three independent pools of PLB985
cells stably transfected with TF1phox/pSR
or control
pSR
. Northern blots of total cellular RNA were sequentially probed
for hybridization with various DNA probes (Fig. 8).
Increased gp91phox message was consistently detected in the
TF1phox/pSR
transfectants in comparison with control
pSR
transfectants. To investigate whether TF1phox
overexpression was specifically affecting gp91phox
expression or was inducing differentiation and therefore causing increased gp91phox message indirectly, the blots were
probed for p47phox (30), p67phox (31), and CD18
(32) messages. All of these messages increase during terminal myeloid
differentiation, similar to gp91phox. The abundance of
mRNAs for p67phox, p47phox, and CD18 was
not increased in PLB985 TF1phox/pSR
transfectants
relative to control pSR
transfectants. Blots were also probed for
-actin to control for RNA loading. Stable transfectant pools of HeLa
cells with TF1phox/pSR
or control pSR
also were
generated. No gp91phox message was detected in Northern
blots of RNA isolated from either of these lines (data not shown).
To investigate whether overexpressed TF1phox was acting
upon the promoter of the gene coding for gp91phox, the
concatenated duplex 194 to
244 bp promoter sequence was subcloned
into a vector with a linked minimal promoter and reporter gene
(p-TATA/CAT) (29). Plasmids were constructed with four copies of the
194 to
244 bp sequence in the sense orientation. PLB985 stable
TF1phox/pSR
and control pSR
pools were transiently
transfected with either control p-TATA/CAT vector or promoter sequence
containing vector (p-cybbTATA/CAT), and cell extracts were analyzed
after 72 h for CAT activity. CAT activity was normalized for
-galactosidase activity from a cotransfected plasmid
(pCMV/
-galactosidase) to control for transfection efficiency. Two
independent stable transfectant pools of both
TF1phox/pSR
and control pSR
were used in these
experiments.
Cell extracts from PLB985 pSR transfectants with p-TATA/CAT had
58.1 ± 29.5% (n = 7) more CAT activity than
transfectants with p-cybbTATA/CAT, consistent with the action of a
repressor upon the promoter element of the gene coding for
gp91phox in undifferentiated PLB985 cells (Fig.
9A). In contrast, cell extracts from PLB985
TF1phox/pSR
transfectants with p-cybbTATA/CAT had
120.3 ± 44.0% (n = 7) more CAT activity than
p-TATA/CAT transfectants, consistent with the action of a
transcriptional activator on the CYBB promoter element in
the TF1phox-overexpressing cells. The absolute values of
CAT activity for the p-TATA/CAT construct were not significantly
different between the TF1phox/pSR
and control pSR
stable transfectant pools. These results were unaffected by the
orientation of the promoter sequence relative to the minimal
promoter.
These constructs were also analyzed by cotransfection into the human
promyelocytic leukemia cell line U937. U937 cells are more committed
than PLB985 cells and are able to differentiate to monocyte/macrophages
with PMA or IFN- (39). U937 cells were cotransfected with either
p-TATA/CAT or p-cybbTATA/CAT and either control pRc mammalian
expression vector or TF1phox/pRc. Cell extracts were
analyzed 24 h after transfection, and CAT activity was normalized
for
-galactosidase activity as described above. Cell extracts from
transfectants with p-TATA/CAT had 12.6 ± 7.8% (n = 4) more CAT activity that transfectants with p-cybbTATA/CAT when
cotransfected with control pRc, consistent with the action of a
repressor upon the promoter element (Fig. 9B). Therefore, less repression was demonstrated in U937 cells than in PLB985 transfectants. However, transfectants with 30 µg of
TF1phox expression construct demonstrated 142.8 ± 23.8% (n = 4) more CAT activity with p-cybbTATA/CAT
than with p-TATA/CAT, and transfectants with 15 µg of
TF1phox expression construct (with 15 µg of pRc plasmid,
to control for total DNA content) demonstrated 55.6 ± 21.4% more
CAT activity with p-cybbTATA/CAT than with p-TATA/CAT, consistent with
the dose-dependent action of a transcriptional activator on
the CYBB promoter element.
These results were sustained when the data were analyzed in terms of absolute CAT activity. If the absolute CAT activity of the U937 cells transfected with p-TATA/CAT and pRc (30 µg) was normalized to 1.0, the CAT activity of U937 cells transfected with p-TATA/CAT and TF1phox/pRc (30 µg) was 0.995 ± 0.034. Therefore, no significant effect of TF1phox was demonstrated on the expression vector. Using the same approach, the CAT activity of U937 transfectants with p-cybbTATA/CAT and pRc (30 µg) was 0.935 ± 0.0.059, that with p-cybbTATA/CAT and TF1phox/pRc (30 µg) was 2.843 ± 0.183, and that with p-cybbTATA/CAT and TF1phox/pRc (15 µg) was 1.422 ± 0.115. These results were consistent with those obtained for PLB985 transfectants.
During terminal myelopoiesis, a number of phagocyte-specific genes
become transcriptionally active. Treatment of leukemia cell lines with
IFN- or PMA resulted in monocytoid differentiation over a 48-h time
period, accompanied by increased expression of gp91phox,
which was maximal at 48 h (8). BID2 complex binding to the CYBB promoter was temporally associated with transcription
of the gene encoding gp91phox (19), suggesting that BID2
component proteins might be of interest in understanding myeloid gene
regulation. We cloned a cDNA by affinity of the expressed protein
for the BID2-binding site sequence (24). The predicted amino acid
sequence of the protein, referred to as TF1phox, included
an RNA-binding domain and a nuclear localization domain, suggesting
that it was a nuclear protein.
The presence of an RNA-binding site in a DNA-binding protein was not without precedent. Y box-binding proteins, a family of highly conserved RNA-binding proteins (55, 56), bind to the MHC class II gene promoter (57) and repress transcription (58). Myef-2, identified by screening a cDNA library for proteins binding the myelin basic protein gene promoter, had homology to the RNA recognition motif of heterogeneous ribonucleoprotein M4 and repressed transcription of the myelin basic protein gene (59). Characterization of TF1phox was of potential interest in further clarification of the role of dual function DNA/RNA-binding proteins in gene regulation.
Two independent binding assays demonstrated that TF1phox
specifically recognized the BID2-binding site sequence.
TF1phox, expressed in gt11 and immobilized on
nitrocellulose filters, bound to the
214 to
262 bp promoter
sequence of the gene encoding gp91phox. TF1phox
binding was competed for by excess homologous (but not heterologous) oligonucleotide or homologous oligonucleotide with a specific mutation
in the BID2-binding site.2
TF1phox expressed as a GST fusion protein also had
sequence-specific DNA binding in EMSA. Therefore, TF1phox
might be relevant to gp91phox expression since
TF1phox/CYBB promoter binding was not the result
of nonspecific DNA-protein interaction.
Although TF1phox expressed as a GST fusion protein bound DNA probes, in vitro translated TF1phox bound the DNA probes poorly. This difference may be due to a characteristic of the reticulocyte lysate, such as the presence of an inhibitor of TF1phox binding or inappropriate post-translational modification of TF1phox. Or, the difference may be due to a characteristic of the GST fusion protein, such as improved DNA binding by the TF1phox/GST protein due to dimerization of the GST regions. The latter explanation suggests a possible partner protein to facilitate TF1phox DNA binding.
Western blots demonstrated TF1phox immunoreactive protein
in human cells, and anti-TF1phox serum disrupted BID2
complex binding to the CYBB promoter. However, confirmation
of identity of the endogenous BID2 nuclear protein to cloned
TF1phox will require purification of BID2 to homogeneity
and peptide sequencing. Although IFN- or PMA treatment of PLB985
cells increased in vitro DNA binding of TF1phox,
neither of these treatments increased TF1phox mRNA or
total cellular TF1phox immunoreactive protein. Therefore,
other mechanisms, such as post-translational modifications or
protein-protein interactions, may be involved in regulation of
TF1phox binding.
Overexpression of recombinant TF1phox in PLB985 cells resulted in increased abundance of gp91phox mRNA, but did not increase the abundance of several other messages indicative of differentiation in leukemia cells. This result suggested that overexpression of TF1phox did not stimulate differentiation, but acted more specifically upon gp91phox mRNA levels.
To demonstrate the effect of overexpression of TF1phox, artificial promoter constructs were employed. Inclusion of the BID2-binding site sequence in the reporter constructs resulted in repression of minimal promoter function in myeloid cell lines. This suggested that a repressor was acting on this promoter sequence in undifferentiated PLB985 or U937 cells, consistent with the level of expression of gp91phox. The lesser degree of repression in U937 cells may reflect greater commitment to differentiation in this line. Overexpression of TF1phox relieved the repression conferred by the CYBB promoter sequence. This suggested that a transcriptional activator was acting on the promoter sequence in TF1phox-overexpressing myeloid cells. However, it was possible that TF1phox was unrelated to gp91phox expression in native myeloid cells, but artificially blocked repressor binding and therefore permitted binding of genuine transcriptional activation factor(s) in the TF1phox transfectants.
Our investigations suggested the possible action of two repressor
proteins on the 194 to
244 bp promoter sequence in undifferentiated myeloid cells. Other investigators had demonstrated that CDP repressed gp91phox expression (10, 11). Although a slow mobility
protein complex that bound the
76 to
137 bp and
194 to
244 bp
promoter sequences was immunoreactive with CDP antiserum in EMSA using
nuclear proteins from HeLa cells, the complex generated using PLB985
nuclear proteins was not. These results suggested that the dominant DNA
binding activity in HeLa and PLB985 nuclear proteins might represent
the products of different genes. Alternatively, the two DNA binding activities might be non-cross-immunoreactive products of the same gene
that had undergone lineage-specific post-transcriptional or
post-translational modifications, or there might be masking of relevant
epitopes by a myeloid specific CDP protein partner. Further
investigation of these possibilities will be of interest in
understanding lineage-specific gene regulation.
The 194 to
244 bp region of the CYBB promoter also bound
the transcriptional repressor and oncogene IRF-2. IRF-2 and a
TF1phox immunoreactive protein competed for binding to this
promoter region. Although IRF-2 is usually described as a repressor of IFN-stimulated gene expression (60), it has been demonstrated to
function as a transcriptional activator under some conditions (61-63).
Determination of the effect of IRF-2 upon the CYBB promoter in cells capable of expressing gp91phox will be of
interest. For some IFN-responsive genes, IRF-2 repression is
antagonized by IRF-1 transcriptional activation (42). It is possible
that TF1phox functions as an alternative antagonist to
IRF-2 in the regulation of gene transcription.
Our data suggest that regulation of gp91phox expression is
complex (Fig. 10). Overexpression of recombinant
TF1phox resulted in decreased binding of a CDP-like protein
to the promoter, suggesting that TF1phox actively displaced
CDP. Blocking of TF1phox binding resulted in increased
binding of the potential repressor IRF-2, suggesting that
TF1phox also displaced IRF-2. Overexpression of
TF1phox not only lifted functional repression by the 194
to
244 bp region of the promoter, but resulted in transcriptional
activation by this element. Further investigation into the interaction
of TF1phox and other factors with the CYBB
promoter region will be important in understanding how the balance of
transcription factor activities regulates lineage-specific gene
transcription.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M66390[GenBank].
We thank Drs. T. Gabig, D. Miller, and A. Kraft for helpful discussions and suggestions during the progress of this study. We thank Dr. R. Pine for kindly providing the anti-IRF-1 antiserum, Dr. E. Neufeld for anti-CDP serum, Dr. K. Ozato for anti-ICSBP serum, Dr. K. Robertson for the human CD18 cDNA, and Dr. T. Leto for the p67phox and p47phox cDNAs. Additional thanks are due to R. Dodson and G. R. Pottler for technical assistance.