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INTRODUCTION |
The interferon
(IFN)1 family of cytokines
regulates antiviral, antitumor, and immune defenses in the
vertebrates by inducing expression of a number of cellular
IFN-stimulated genes (ISGs) (1). ISG induction has been attributed
largely to the activation of the well established Janus tyrosine
kinase-signal transducing activators of transcription (STAT)
pathway, wherein tyrosine-phosphorylated STATs regulate the expression
of downstream genes directly (2-4). In addition to STATs, some members
of the IFN gene regulatory factor (IRF) family regulate IFN responses
(5-7). It is now well established that Janus tyrosine kinase 1 and
Tyk2, recruited to the ligand-engaged IFN-
/
receptor,
induce the phosphorylation of STAT1 and STAT2. These then associate
with ISGF3
(p48 or IRF-9), a member of the IRF family of
transcription factors (8). Although the published literature describes
the ISGF3 protein as p48, it will be referred to as IRF-9 in the
light of the unified nomenclature suggested by the International
Society for Interferon and Cytokine Research. This trimeric complex,
known as ISGF3, migrates to the nucleus and stimulates transcription
after binding to the IFN-stimulated response element (ISRE) (2, 3, 8).
In a number of tumors and viral oncogene-expressing cell lines,
down-regulation of the physical levels or inactivation of the
components of ISGF3 serves as a mechanism for evading the action of
IFNs (9, 10). ISGF3 is targeted by adenovirus (10), human papilloma
(11, 12), Epstein-Barr (13), and hepatitis C
viruses2 for evading the
antiviral action of IFNs.
IRF-9 is an IFN-regulated gene, like certain other members of the IRF
family (14). Pretreatment of cells with IFN- causes a robust
activation of IFN--induced gene expression and consequent biological
responses (15-17). This latter effect is achieved through an elevation
of cellular IRF-9 levels (14, 16, 17); however, its delayed expression
and sensitivity to protein synthesis inhibitors, unlike other IRFs,
suggest that the IRF-9 gene is regulated in an atypical manner (18).
Previous investigations from this laboratory have revealed the
molecular basis for such regulation. Analysis of the murine IRF-9
promoter led to the discovery that an unusual IFN- response element,
GATE, and its cognate transacting factors regulate the transcription
(18). These studies did not reveal the molecular nature of these
factors. In the present study using the GATE binding property as a
basis, we have identified a GATE-binding factor (GBF) from an IFN-
stimulated macrophage cDNA library. Sequence analysis revealed that
it codes for a novel protein, which we have named GBF-1. It regulates
GATE-dependent gene expression in an
IFN-dependent manner. GBF-1 homologs are present in other species. Although recombinant GBF-1 alone does not bind DNA on its own,
its expression in cultured cells permits DNA binding. Thus, GBF-1 is a
novel factor that mediates the pleiotropic effects of IFN-. This
study uncovers a novel mechanism of IFN action.
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MATERIALS AND METHODS |
Reagents--
Recombinant murine and human IFN-, mouse
IFN- (Pestka Biomedical Laboratories, Piscataway, NJ), IPTG,
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal;
Invitrogen) cDNA synthesis kits (Stratagene), mouse kidney cDNA
library (CLONTECH), restriction and modifying
enzymes (New England Biolabs), and nitrocellulose membranes (Schleicher
& Schuell) were used in these studies. Rabbit polyclonal antibodies
specific for c-Jun and Bcl2 were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Mouse monoclonal antibodies, FLAG
epitope tag, and actin were obtained from Sigma. Murine multiple tissue
Northern blots, pEGFP, and GFP-specific antibodies were purchased from CLONTECH Inc.
Cell Culture and Plasmids--
The murine macrophage cell line
RAW (RAW264.7) was grown in RPMI 1640 supplemented with 10% fetal
bovine serum (18). HeLa cells were grown in Eagle's minimum essential
medium with 5% fetal bovine serum. Human embryonic kidney 293 (HEK-293) cells were grown in Dulbecco's modified Eagle's medium with
5% fetal bovine serum. Primary mouse embryo fibroblasts were derived
from 9-day-old embryos of C57BL/6 mice. pIRE-tk-Luc and
ISRE-tk-Luc constructs were provided by Keiko Ozato, National
Institutes of Health, Bethesda, MD, and Nancy Reich, SUNY, Stony Brook,
NY, respectively. Wild type murine and monkey GBF-1 open reading frames
were also cloned into the mammalian expression vector pCMV-FLAG.
Initial experiments also used monkey GBF-1 cloned into the mammalian
expression vector pME18S under the control of the SR
promoter. The expression vector for C/EBP- was provided by Richard
W. Hanson, Case Western Reserve University, Cleveland, OH. Murine IRF-9
promoter and its mutants were described previously (18). This promoter
contains GATE and a Myc-Max-stimulated element (MSE). The A6 construct
was derived by inserting the native promoter elements of the murine
IRF-9 gene upstream of a luciferase reporter in the promoterless
pGL3-Basic vector (Promega). The GATE pm and MSE
pm constructs bear mutations in the respective elements in
the context of the native promoter. The P4 construct is generated by
inserting a 74-bp element of the IRF-9 gene, harboring GATE, upstream
of the SV40 early promoter in pGL3 promoter vector (Promega). The P3
mutant is similar to P4 but lacks the GATE sequence. The GW, GM-1,
GM-2, GM-3, and GM-13 constructs, bearing minimal wild type or mutant
GATE, were generated by cloning synthetic complementary double-stranded
oligonucleotides upstream of the SV40 early promoter in the pGL3
promoter vector. The terms GW and GM refer to wild type and mutant GATE
sequences, respectively. The sequences of a single-stranded
oligonucleotide are shown below. The GM-13 construct was generated
spontaneously during the cloning of the GW oligonucleotide. It lacked a
single T residue at position 13 of wild type GATE. Its IFN-
sensitivity is diminished significantly compared with the wild type.
All constructs had single-copy insertions of the elements as revealed
by DNA sequencing. The sequences of GATE oligonucleotides are as
follows:
GW, 5'-GATCTCCCGAGGAGAATTGAAACTTAGGGTGGGG- TAA3';
GM-1, 5'-GATCTCCCGAGGAGAAAACTTAGGGTGGGGTAA-3';
GM-2, 5'-GATCTCCCGAGGAGAATTGATAGGGTGGGGTAA-5';
GM-3, 5'-GATCTCCCGAGGAGAAGCGGCCGCTAGGGTGGGG- TAA-3'.
Gene Expression Analyses--
Southern, Northern, and Western
blot analyses, transfection, -galactosidase and luciferase assays,
electrophoretic mobility shift assays (EMSA), SDS-PAGE, and sequence
analysis were performed as described in our earlier publications (18).
Luciferase activities were normalized to the activity of a
cotransfected CMV--galactosidase reporter. Triplicate
transfections/sample were performed to evaluate the statistical
significance of stimulation. In some experiments 4 µg of
pCMV-FLAG-GBF-1 and 1 µg of pEGFP vectors were electroporated into
cells, and the fluorescent cells (green) were sorted out using flow
cytometry. Three separate transfectants were sorted to generate a
sufficient number of cells. Sorted cell lysates were prepared for
monitoring the expression of endogenous IRF-9, GBF-1, and GFP using
specific antibodies after Western blotting. Experiments were repeated
at least three times to obtain consistent results. In vitro
transcription and translation were performed using the commercially
available RiboMax system (Promega).
cDNA Libraries--
A cDNA library was generated from
poly(A)+ mRNA isolated from RAW cells stimulated with
400 units/ml murine IFN- for 0, 4, 8, and 12 h. A commercially
available kit (Stratagene) was employed for cDNA synthesis with
pooled poly(A)+ RNA as template. The cDNA library was
cloned into the EcoRI and XhoI sites of the
-ZAPII vector (Stratagene). The resultant library was packed
in vitro and was used to infect Escherichia coli
to obtain the final library. This library consisting of more than 99%
recombinants was used for identifying the GATE-binding proteins. Induction of the protein encoded by the cloned cDNA was achieved by
IPTG treatment. Three million plaques were screened using a 32P-labeled, concatamerized GATE as described previously
(19). Positive clones identified in the first round were subjected to two more rounds of screening with wild type
(5'-CCCGAGGAGAATTGAAACTTAGGG-3') and mutant GATE probes
(5'-CCCGAGGACTGTTGCTCGGCGAGGG-3'). The mutated bases are italicized and underlined. Nucleotide
changes were introduced primarily on the basis of our earlier
observation that this sequence exhibited a partial homology (18) to
ISRE. In each case a corresponding complementary oligonucleotide was synthesized and annealed. These double-stranded oligonucleotides were
concatamerized, labeled with 32P using a nick translation
kit (Amersham Biosciences), and used in the experiments. At the end, 13 independent phage clones expressing GATE-binding proteins were
isolated. These were grouped into three based on Southern blot analyses
of the rescued inserts and partial sequence analysis. Among these, four
clones expressing various sizes of the same cDNA were grouped as
GBF-1. None of the clones contained the full-length sequence and lacked
the 5'-end. Inserts in the phage were rescued by in vivo
excision of the inserts, which allowed the transfer of cDNA into
pBluescript phagemid. Inserts were sequenced and used for further
analysis. A commercially available mouse kidney cDNA library
(CLONTECH) was screened further to obtain
full-length inserts.
Bacterial Expression--
The partial cDNA was used
for generating the recombinant GBF-1 protein in E. coli. An upstream primer (5'-CGGAATTCCTGGGGCTGTACCACACCGTG-3') and
a downstream primer (5'-CGCCTCGAGTAGCAACTAGTCTTTGCAGCTAGG-3') were used for amplifying the coding region, and the resultant PCR
product (850 bp) was cloned into the EcoRI and
XhoI sites of the bacterial expression vector pET32A(+).
After transformation into the E. coli BL21 codon plus
strain, the cells were induced with 1 mM IPTG in the growth
medium for 2 h. Sonicated cell extracts were prepared and passed
though a Ni-NTA-agarose column for purifying the recombinant protein.
The protein was eluted using 50 mM imidazole. The protein
was dialyzed and passed through the Ni-NTA-agarose column again to
remove the trace contaminants. The expression tag was removed by
thrombin digestion, and the recombinant GBF-1 was collected in the
unbound fraction, after passing the digest through a Ni-NTA-agarose column.
Immunization--
Electrophoretically pure recombinant murine
GBF-1was used for immunization. 20 µg of the antigen was prepared in
Freund's complete adjuvant and administered into the footpads of three BALB/c mice. Two weeks later two booster immunizations, at biweekly intervals, were performed with subcutaneous injection of 10 µg of
antigen prepared in Freund's incomplete adjuvant. Serum was collected
after tail vein bleeding, and the presence of high titer antibody was
determined using ELISA with recombinant GBF-1 as antigen. A negative
control ELISA with similarly produced unrelated recombinant GRIM-19
protein was also performed. The antisera did not react significantly
with the negative control. Blood collected after cardiac puncture of
animals and serum was saved. Nonimmune serum was also collected prior
to immunization.
Immunofluorescence--
Cells were grown on sterilized cover
glasses in six-well dishes (Nalgene) and then fixed with 2%
paraformaldehyde in phosphate-buffered saline for 10 min and then with
methanol for 20 min at room temperature. The cells were then washed
three times with phosphate-buffered saline containing 0.15% glycine
and 0.5% bovine serum albumin. The cells were then incubated with
antibodies specific for GBF-1 (1:250 dilution) in the same buffer for
1 h at 37 °C. Cells were washed with phosphate-buffered saline
and then incubated with Texas Red-labeled goat anti-mouse IgG (1:1,000
dilution) for 1 h. They were then washed with phosphate-buffered
saline and observed under a Nikon fluorescent microscope. These cells
were also counterstained with 0.1 µg/ml DAPI to visualize the nuclei.
In some experiments, FLAG-GBF-1 was introduced into cells by
transfection, and cells were fixed and stained as described above
16 h after transfection.
Expression in Mammalian Cells--
The mouse GBF-1 was PCR
amplified using a forward primer
(5'-CGGAATTCCatggcccaggcggcgcgcctttc-3') and a reverse primer
(5'-CGCCTCGAGTAGCAACTAGTCTTTGCAGCTAGG-3'), digested with
EcoRI and XhoI, and cloned into the pCMV-FLAG
vector. A full-length GBF-1 cDNA was isolated by the Japanese
consortium on the monkey (Macaca fasicularis) genome. The
open reading frame of monkey GBF-1 (here forth Mk-GBF-1) was PCR
amplified by using an upstream primer
(5'-CGGAATTCCGGCAGAGCGAACATGGCCCCG-3') and a downstream primer
(5'-CGCGGATCCCTAGACATTCAGTGCGCTG-3'). The resultant PCR product (1.1 kb) was cloned into the pCMV-FLAG vector between EcoRI and
BamHI for expressing as an N-terminal FLAG-tagged protein.
Subcellular Fractionation--
HeLa cells were transfected with
the FLAG-tagged GBF-1 construct. 36 h later cells were
lysed in an ice-cold buffer consisting of 20 mM HEPES, pH
7.4, 0.5% Nonidet P-40, 10 mM KCl, 1.5 mM
MgCl2, 1 mM EDTA, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and a
commercially available protease inhibitor mixture (Sigma) as described
previously (20). The lysates were overlaid on a 50% sucrose cushion.
After centrifugation at 2,000 × g for 10 min, the
overlaid fraction was collected and centrifuged at 10,000 × g for 25 min. The supernatant was collected and used as the cytosolic fraction. The pellet formed after sucrose cushion
centrifugation was collected, washed, and examined under a
phase-contrast microscope to ensure the presence of nuclei. It
contained greater than 99% pure nuclei. This fraction was extracted
further with high salt and dialyzed. The dialyzed fraction was
centrifuged at 10,000 × g for 30 min, and the
supernatant was collected as the nuclear fraction. An aliquot from the
initial lysate was saved for determining the total GBF-1 content.
Typically, 70-90 µg of total protein extract from each sample was
analyzed by Western blot analysis.
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RESULTS |
Isolation of GBFs--
The GBFs were identified using a
Southwestern technique based on the DNA binding property of a protein
(19). An expression library was prepared using the pooled
poly(A)+ RNA isolated from the IFN--stimulated murine
macrophage cell line RAW and cloned into -ZAPII vector (see
"Materials and Methods"). Protein encoded by the cloned cDNA in
the phage plaques was induced by IPTG treatment. The plates were
overlaid with nitrocellulose membranes, and the membranes were probed
with a 32P-labeled, concatamerized GATE. Clones identified
in the first round, using wild type GATE probe, were subjected to two
more rounds of screening. Replica membranes from the same plate were probed with mutant or wild type GATE, separately. Because GATE exhibits
a partial homology (18) to the ISRE, a mutant GATE was designed by
mutating the residues that were homologous to ISRE. In particular, the
GAA and AAACTT nucleotides in the middle of the sequence were changed
to CTG and CTCGGC, respectively. A phage clone from the first round,
named GBF-1, scored positive with wild type GATE and did not bind to
mutant GATE in the second and third round analyses (Fig.
1). Clones positive in the second round
are indicated with arrows. These differences were not the result of differential labeling of the two probes because both sequences were labeled to the same efficiency (~109
cpm/µg). Positive phage clones were purified. Three independent plaques coding the same insert were identified. Sequence analysis revealed that they coded for a novel protein and carried only the
partial cDNAs. All of them lacked the 5'-end including the open
reading frame area, and the longest one contained a 1.04-kb insert.
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Fig. 1.
Isolation of GBF-1. A phage preparation
that scored positive in the first round of screening with wild type
GATE was infected into the E. coli XL-1 blue MRF' host and
plated. Protein expression was induced upon overlaying 10 mM IPTG-impregnated nitrocellulose membranes. Duplicate
membranes from the same plate were incubated with a concatamerized
double-stranded DNA corresponding to mutant or wild type GATE (see
"Materials and Methods") in a binding buffer (50 mM
Tris-HCl, pH 7.5, 50 mM KCl, 2 mM EDTA, 1 mM dithiothreitol, and 5% nonfat dry milk powder) at
4 °C for 12 h. The probes were labeled with 32P
using a nick translation kit to a comparable specific activity
(~109 cpm/µg). The filters were washed three times at
room temperature with the binding buffer containing 0.25% nonfat dry
milk powder; they were then dried and exposed to x-ray films to detect
the clones expressing GATE-binding protein. Phage was plated at ~150
and 20 plaque-forming units/plate in the second and third rounds,
respectively. Positive clones after the second round screen are shown
with arrows. The third round screen yielded a pure
clone.
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Identification of Full-length GBF-1, a Novel Protein--
A
1.43-kb long incomplete cDNA was obtained after screening a mouse
kidney cDNA library. An extremely GC-rich nature of the 5'-end
prevented the isolation of a full-length cDNA. Therefore, two
rounds of 5'-RACE were performed to obtain the 5'-region. The 5'-RACE
products were fused to the cDNA to generate the final cDNA
~1.6 kb. BLAST analyses identified highly homologous expressed sequence tag cDNAs from mouse (GenBank accession no. BC004846), human (AK024100 and BC011613), monkey (M. fasicularis,
AB046026), and Drosophila melanogaster (AJ250307). The
sources of these cDNAs were quite disparate. The mouse cDNA was
isolated from kidney, mammary gland, and blastocyst libraries. The
monkey cDNA was identified from a brain cDNA library. The human
cDNAs with variable sizes (1.6-1.8 kb) were isolated from uterine
and neuroblastoma cell lines. The longest human cDNA (BC011613) had
190- and 450-nucleotide 5'- and 3'-untranslated regions, whereas the
shortest human cDNA (FLJ14038) had 19- and 450-nucleotide 5'- and
3'-untranslated regions, respectively. The mouse cDNA in the
data bases had 17- and 389-nucleotide 5'- and 3'-untranslated regions,
respectively. The monkey cDNA (2.041 kb) had 451- and
456-nucleotide 5'- and 3'-untranslated regions, respectively. The mouse
and human cDNAs map to chromosomes 2 and 9 in their respective
species. The precise location of the human GBF-1 is 9q34.13.
The mouse, human, and monkey GBF-1 cDNAs encode 384, 377, and 377 amino acids, respectively. An assignment of the name GBF-1 was based
primarily on amino acid homologies. The monkey and mouse proteins
functions as GBF-1s (see below). The mouse and human GBF-1 polypeptides
are 73% identical and 77% similar. The mouse and monkey GBF-1
proteins are 73% identical and 77% similar. Most of these cDNAs were
capable of encoding ~38-40-kDa polypeptides. The mouse and
Drosophila homologs exhibit a 38% identity and 58% similarity. Most of the homology resides between amino acids 96 and
371. The high sequence homology between monkey and human (97%) predicts that the human cDNA codes for a GBF-1-like function, although this was not tested experimentally.
ClustalW alignment of the mouse, monkey, human, and
Drosophila sequences showed extensive homology throughout
their polypeptide backbones (Fig.
2A). The mouse and
Drosophila sequences have a longer C terminus than those of
human and monkey. Drosophila has a 14-amino acid internal
peptide between positions 105 and 120, which is absent in the mammalian
GBF-1. A glutaredoxin-like motif is present between amino acids 103 and
119 (Fig. 2B). BEAUTY analysis (21) indicates that GBF-1 has
a homologous domain located between amino acids 146 and 220 (32%
identity and 49% similarity) to a domain present in mouse, human
protein-tyrosine phosphatases 
and µ. In addition, regions between
amino acids 182 and 315, 167 and 242, and 183 and 246 have similarities
to the regions conserved in eukaryotic RNA polymerases, eukaryotic and
viral ribonucleoside diphosphate reductases, respectively. These
features are consistent with its DNA binding property. Phylogenetic
analysis revealed a close relationship among the protein sequences
(Fig. 2C). As expected, the vertebrate homologs cluster
together, whereas the invertebrate homolog formed a distal branch.
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Fig. 2.
GBF-1 codes for a novel protein.
A, sequence analysis of GBF-1. A number of complete DNA
sequences from expressed sequence tag data bases were identified using
Blast analysis. A conceptual translation of the cDNA sequences was
performed, and the resultant open reading frames were compared in a
ClustalW analysis. The mouse and monkey cDNAs were characterized in
this study, and their GBF properties are confirmed. An assignment of
GBF-1 to human and Drosophila cDNAs is primarily on the
basis of sequence homology but not function. B, a domain map
of mouse GBF-1. Various predicted domains and their locations are
indicated on the polypeptide backbone. PTP, protein-tyrosine
phosphatase; RDR, ribonucleotide diphosphate reductase.
Numbers flanking the lines show amino acid
coordinates. C, phylogenetic relationships of GBF-1
homologs. Using the GCG software the sequence divergence among various
GBF-1 homologs was compared. All GBF homologs seem to have a common
ancestry. D, in vitro translation of full-length
GBF-1 and the cloning vector pGEM7zf. 3 µg of plasmid DNA linearized
with XhoI was used as a template to generate RNA using SP6
RNA polymerase. The resultant RNA was programmed into nuclease-treated
rabbit reticulocyte lysates (Promega), and translation was carried out
for 2 h at 30 °C in the presence of 100 µCi of
[35S]methionine (Amersham Biosciences). 20-µl reaction
components were separated on a 10% SDS-polyacrylamide gel and
visualized after fluorography. Positions of the molecular mass
standards (kDa) are indicated on the right.
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Sequence analysis revealed that mouse GBF-1 codes for a novel protein
with an apparent molecular mass of ~38 kDa. To test whether
the cDNAs code for predicted proteins, the cDNAs were cloned
into the pGEM7Zf(+) vector, under the control of the SP6 promoter.
Using SP6 RNA polymerase, an mRNA was generated in vitro from the cloned inserts and programmed into rabbit reticulocyte lysates
in the presence of [35S]methionine. The resultant
products were separated on a 10% SDS-PAGE and fluorographed. The
mouse and monkey cDNAs yielded proteins of ~39 and 42 kDa,
respectively (Fig. 2D). The difference in sizes could be
caused by post-translational modifications.
GBF-1 Induces GATE-dependent Gene
Expression--
Because the GBF-1 cDNA has been isolated by virtue
of its interaction with GATE, it is necessary to determine whether it
is a transcriptional activator or repressor. Therefore, we have
subcloned the GBF-1 open reading frame under the control of a
constitutive enhancer in the mammalian expression vector pCMV-FLAG and
tested whether it induces the luciferase reporter. Initial studies used a construct P4, driven by a 74-bp enhancer element consisting of GATE
region of p48. GBF-1 expression vector but not the control vector
induced the luciferase expression, in a dose-dependent manner. Although at a higher molar ratio GBF-1 slightly inhibited the
gene expression, luciferase activity was significantly more than the
vector alone at that dose (Fig.
3A). This inhibitory effect
may be the result of a competition for limited amounts of general
transcriptional coactivators such as histone acetylases available in
the cell. A similar construct, P3, lacking GATE did not respond to
GBF-1 (Fig. 3C; see below).
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Fig. 3.
Induction of gene expression by GBF-1.
Transfection was carried out using LipofectAMINE reagent (Invitrogen)
and normalized to an internal control, the -actin--galactosidase
reporter gene (18). A, expression vector (pCMV-FLAG) or its
derivative expressing GBF-1 was transfected along with the 0.4-µg
P4-Luc construct into RAW cells, and the expression of luciferase was
measured using 30 µg of total cell protein at 30 h
post-transfection. Numbers on the x axis refer to
molar-fold of the expression vector or GBF-1 relative to the P4
reporter. B, a comparison of the gene-inductive effects of
mouse (Mu-GBF-1) and monkey (Mk-GBF-1). RAW cells were transfected with
the indicated GBF-1 expression plasmid or the corresponding empty
vector. Cells were treated with 200 units/ml IFN- for 16 h.
Each bar represents the mean ± S.E. of triplicate
measurements. C, GATE-dependent induction of
gene expression by GBF-1. Cells were transfected with 0.4 µg of the
indicated reporter genes and 0.1 µg of GBF-1 expression vector. The
-fold induction of luciferase gene was calculated, relative to that of
vector (pCMV-FLAG) transfected in each case. Data represent the mean of
triplicate measurements. Construction of luciferase reporters was
described earlier (18). A6, wild type p48 promoter;
MSE pm, mutant of Myc-stimulated element; GATE
pm, mutant of GATE. The P4 and P3 constructs contain the p48
promoter sequences (thick black bar), placed upstream of a
heterologous promoter (SV40). Cells were treated with 200 units/ml
IFN-, where indicated. D, effect of GBF-1 on endogenous
IRF-9 expression. Primary MEFs were electroporated with the indicated
expression vectors along with a pEGFP expression vector. GFP-expressing
cells were sorted by flow cytometry, and the cell lysates were
monitored for IRF-9, FLAG, and GFP expression using specific antibodies
in Western blot analysis. E, a quantification of IRF-9
expression. Empty and filled bars correspond to
the vector and GBF-1 transfectants, respectively. Each bar
represents mean densitometric intensity ± S.E. of triplicates.
Cells were stimulated with 200 units/ml IFN- for 16 h.
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Because GATE was an IFN--inducible element and GBF-1 induced
GATE-dependent gene expression, we next determined the
influence of IFN- on GATE-dependent gene expression
(Fig. 3B). We also compared the gene-inductive effects of
monkey and mouse GBF-1 in these experiments. RAW cells were transfected
with the P4 reporter, along with an empty expression vector or the
corresponding vector with mouse or monkey GBF-1 (Fig. 3B).
Cells were exposed to IFN- for 16 h, and luciferase activity
was measured. Both mouse and monkey GBF-1 caused an elevation of basal
expression, and IFN- treatment led to further induction. These data
suggest that either an IFN--dependent post-translational
modification such as phosphorylation or an increase in GBF-1 levels
(see Fig. 8) or both contribute to the transcriptional up-regulation.
Monkey GBF-1 was marginally better at inducing gene expression in
several independent experiments.
The effect of Mu-GBF-1 was also determined in the context of wild type
promoter. The A6 construct carried a 1,056-bp upstream sequence derived
from the mouse IRF-9 promoter. This promoter carries GATE and a MSE.
Two other mutants, each bearing the targeted mutations in the GATE
(GATE pm) and MSE (MSE pm), were also included in
this experiment. Luciferase gene expression from the wild type and MSE
pm was induced by GBF-1 (Fig. 3B). In contrast,
the GATE mutant (GATE pm) did not respond to GBF-1 under
these conditions. GBF-1 also induced transcription from the P4
construct. In contrast the P3 construct, which lacked GATE, did not
respond to GBF-1. Only those reporters that possessed a wild type GATE
responded further to IFN-, significantly (Fig. 3C). These
results, together with the binding data (Fig. 1), indicate that GBF-1
mediates both basal and inducible expression from GATE-driven promoters.
We next examined the effect of GBF-1 on the endogenous IRF-9. Our
attempts to overexpress this protein stably did not yield productive
transfectants. This may be caused by the toxic effects of overexpressed
GBF-1. Therefore, we used transient transfection to examine the
influence on the expression of endogenous IRF-9 protein. Mouse embryo
fibroblasts were transfected with an empty expression vector
(pCMV-FLAG) or the same vector expressing FLAG-GBF-1 along with the
pEFGP plasmid. After treating the cells with IFN- for 16 h,
transfected cells (on the basis of green fluorescence) were sorted out
using flow cytometry. Cell lysates were prepared from the sorted
transfectants and monitored for IRF-9, GBF-1, and GFP using specific
antibodies. Although IFN- induced the expression of endogenous IRF-9
in the vector-transfected cells, overexpression of GBF-1 further
augmented the IFN- induced expression of IRF-9, significantly (Fig.
3D). IFN- had no effect on the expression of GBF-1 and
GFP. There was marginal induction of the basal IRF-9 level. A
quantification of the IRF-9 expression is shown in Fig. 3E.
A modest but significant stimulation of endogenous IRF-9 protein
occurred in the presence of GBF-1. The difference in the extent of the
stimulatory effects of GBF-1 on the luciferase reporters (Fig. 3,
B and C) and the endogenous IRF-9 (Fig. 3, D and E) could be the result of different
conformations and locations of these promoters: the former is located
on a plasmid, and the latter is part of the genome.
GBF-1 Does Not Stimulate GAS- or ISRE-dependent
Transcription--
Because GATE possesses a partial homology to ISRE,
and it promotes IFN--responsive gene expression, we next tested
whether GBF-1 could stimulate transcription from other known
IFN-stimulated elements. Two luciferase reporters, one driven by ISRE
and the other by pIRE (GAS) were used for this study. RAW cells were
transfected with the reporters along with pCMV-FLAG or pCMV-Mu GBF1.
pIRE-Luc-transfected cells were exposed to 200 units/ml IFN- for
16 h. ISRE-Luc-transfected cells were treated with 200 units/ml
IFN- for 16 h. Cell extracts prepared after 16 h of IFN
treatment were measured for luciferase activity. As shown in Fig.
4, GBF-1 had no significant effect compared with the vector control on ISRE- or pIRE-driven reporters. These data indicate the specific effect of GBF-1 on
GATE-dependent gene expression.
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Fig. 4.
Effect of GBF-1 on ISRE- and
GAS-dependent gene induction. RAW cells were
transfected with expression vector (pCMV-FLAG) or the same vectors
carrying GBF-1 (pCMV-GBF-1) (0.25× molar concentration with respect to
the reporter) along with 0.4 µg of luciferase reporters driven by
ISRE or pIRE (GAS). 0.2 µg of a CMV-galactosidase reporter was also
included for normalizing luciferase activity. An empty vector
(pCMV-FLAG) was cotransfected for a control. Where indicated with a + sign pIRE-Luc- or ISRE-Luc-transfected cells were stimulated with 200 units/ml mouse IFN- or 200 units/ml mouse IFN- for 16 h,
respectively. Luciferase activity was measured as described under
"Materials and Methods."
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Mutant GATE Does Not Support GBF-1-stimulated
Transcription--
In the next part of this study we aimed at defining
the nucleotides required for the gene stimulatory activity of GBF-1 via GATE. Four mutants were used for this purpose. Oligonucleotides bearing
wild type GATE or specific base changes were synthesized and cloned
upstream of the SV40 early promoter into the pGL3-promoter vector. The
sequences of wild type GATE (GW) and mutants GM-1, GM-2, and GM-3 are
shown under "Materials and Methods." One of these mutants, GM-13,
was generated spontaneously during the cloning of wild type GATE. It is
identical to wild type GATE except for a deletion of the T residue at
the 13th position. The reporter constructs bearing GATE mutations were
introduced into RAW cells along with murine and monkey GBF-1 via
transfection and tested for induction of the luciferase reporter (Fig.
5A). GM-1, GM-2, and GM-3 were
unresponsive, whereas GM-13 had a severely reduced sensitivity (~20%
of the wild type) to IFN- compared with the GW construct. The GW
construct responded normally to GBF-1 (Fig. 5A). In the
presence of mouse or monkey GBF-1, a 2-3-fold enhancement of basal
transcription occurred, and it was augmented synergistically by IFN-
treatment.
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Fig. 5.
GATE sequences necessary for stimulating gene
expression. A, RAW cells were transfected with
luciferase vectors driven by various mutant GATE sequences and
CMV--galactosidase. Along with these reporter plasmids the pCMV-FLAG
or the same vector carrying Mu-GBF-1 or Mk-GBF-1 was cotransfected, and
luciferase activity was measured. Empty bars, no treatment;
filled bars, 200 units/ml IFN- for 16 h. Luciferase
activity was normalized to -galactosidase activity. Because the
scale is too large for distinguishing the effects of GBF-1 on GM-13,
these data were replotted on a different scale and are presented in the
inset. B, a comparison of wild type and mutant
GATE sequences to the CBS of other cellular genes. The core CBS is
indicated with a line over the sequences. The conserved T
residue has also been underlined in CBS and wild type GATE.
Mutated bases in GATE have been italicized and
underlined. The  sign in GM-13 indicates the deleted T
residue. C, effect of C/EBP- and Mu-GBF-1 on the GM-13
reporter. RAW cells were transfected with the indicated GBF-1 or
C/EBP- expression vectors (0.25× relative to the reporter) along
with the GM-13 reporter. Cells were stimulated with 200 units/ml
IFN- for 16 h as described in A. Luciferase activity
was determined as described under "Materials and Methods."
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Although GM-13 had a strongly reduced response to IFN-, monkey and
mouse GBF-1 still induced basal and IFN-stimulated transcription (Fig.
5A, inset). Previous studies from this laboratory
and that of others have shown that the GATE responds to transcription
factor C/EBP- (22, 23). A consensus C/EBP- binding site (CBS), homologous to the one present in several cellular genes, is present in
the center of GATE (Fig. 5B). Because GM-1, GM-2, and GM-3 did not respond to IFN, and the mutations were within the CBS, the
location of a GBF-1 response element was not revealed. These mutants
also did not respond to C/EBP- (data not shown). GM-13 has a highly
reduced response to IFN- (Fig. 5A). A well conserved T
residue, present in most C/EBP binding sites, is absent in GM-13 (Fig.
5B). This mutation is within the CBS of GATE. Therefore, we
tested whether this mutant responded to C/EBP- or GBF-1. RAW cells
were transfected with the GM-13 reporter along with an empty expression
vector, C/EBP- expression vector, or GBF-1 expression vector (Fig.
5C). They were then treated with IFN- for 16 h, and
luciferase activity was measured. In vector- and
C/EBP--cotransfected cells a similar extent of IFN-stimulated
expression of luciferase gene was noted. In contrast, GBF-1 enhanced
both basal and IFN-stimulated response significantly. By comparing the
sequences of the GATE mutants, we have inferred that GBF-1 requires
both part of the CBS and the adjacent sequences for stimulating gene
expression. Thus, the residues required for GBF-1 response appear to be
GAACTTAG in the context of full-length GATE.
Binding of GBF-1 to GATE--
Initial studies showed that
recombinant GBF-1 alone did not bind to 32P-labeled GATE
(data not shown). Therefore, for conducting these experiments we have
expressed the mouse and monkey GBF-1s as FLAG epitope-tagged proteins
using the pCMV-FLAG vector. The plasmid was transfected into HEK-293
cells, and the nuclear lysate was employed for EMSA. To ensure the
production of the FLAG-tagged protein in the cells, the lysates were
subjected to a Western blot analysis with FLAG epitope-specific
antibodies (Fig. 6A). The FLAG
antibody detected an appropriate size band in the extracts from mouse
GBF-1 protein-expressing cells. The control vector expressed only the
FLAG epitope tag (1.5 kDa), which was too short to be retained on the
gel. Therefore, this band was not detected in lanes loaded with
vector-transfected cell lysates.
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Fig. 6.
DNA binding property of GBF-1.
A, expression of FLAG-tagged monkey and mouse GBF-1. HEK-293
cells were transfected with pCMV-FLAG or the same vector carrying
monkey or mouse GBF-1. After transfection of 2 µg of each plasmid DNA
for 24 h, equal quantities of cell lysates (50 µg) were prepared
and Western blotted. The blot was probed with a monoclonal antibody
that recognizes the FLAG epitope tag. Numbers on the
right indicate the migrating positions of size markers in
kDa. B, 50 ng of a wild type GATE, GW oligonucleotide was
labeled with 32P and used (70,000 cpm/reaction) in an EMSA
(for the sequence, see "Materials and Methods"). About 30 µg of
protein from transfected HEK-293 cells was employed for EMSA.
None, probe alone in the binding buffer; NSC,
nonspecific complex; a and b, endogenous
complexes. The GBF-1 complex is indicated. Where indicated, an excess
(50×) of GW or GM-1 oligonucleotides was incubated with cell extracts
prior to the addition of 32P-labeled GW to the reaction.
C, IFN--enhanced binding of GBF-1 to GATE.
Plasmid-transfected (indicated above the panel) HEK-293
cells were stimulated with 200 units/ml human IFN- for 1 h and
then used in EMSA, where indicated. D, Western blot analysis
of GBF-1 expression in the transfected cell lysates before and after
treatment with 200 units/ml IFN- for 1 h. HEK-293 cells were
transfected with 2 µg of pCMV-FLAG-GBF-1. 30 h later one plate
was stimulated with IFN-, and the other was left untreated. Cells
were lysed, and nuclear extract was prepared. 30 µg of nuclear
extracts were separated on a 10% SDS-PAGE, Western blotted, and probed
with anti-FLAG antibodies. E, specificity of mouse GBF-1
binding to GATE. HEK-293 nuclear lysates after transfection with 2 µg
of Mu-GBF-1 were employed in EMSA. In lanes 2 and
3 no competitor was used. In lane 2 nuclear
extracts were prepared after a 1-h IFN- treatment of cells. In
lanes 3, 4, and 5 a 50-fold molar
excess of unlabeled GW, GM-1, or GM-3 oligonucleotide was preincubated
with the extracts and then with labeled GW probe. Lane 7 is
an empty vector-transfected control, and the GATE complex formation
with this extract is not different from that of mock lysates. In
lanes 8 and 9 GBF-1-transfected, IFN-treated
lysates were incubated with FLAG epitope- or Myc epitope- (nonspecific
antibody control) tagged antibodies for 1 h before use in EMSA.
SS, supershift complex. Note the slow moving complex with
FLAG tag-specific antibodies. F, expression of Mu-GBF-1 in
the transfected cell lysates. Western blot analysis of cell lysates was
performed with FLAG antibody. 35 µg of the cell lysate from each
sample was used for Western blot.
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After confirming the expression, the cell extracts were incubated with
32P-labeled GATE, and EMSA was performed to detect DNA
binding (Fig. 6B). Protein extracts from vector-transfected
cells formed two complexes with GATE in EMSA, denoted as a
and b (lane 1). Of these two, complex b is less
prominent, and its formation is variable. In contrast, lysates from the
FLAG-GBF-1-expressing cells formed an additional complex that migrated
above complex a (lanes 3 and 4). This complex can
be seen only after a long exposure of the blots (48 h). This
observation suggests that GBF-1 interacts weakly with GATE. This
complex corresponds to FLAG-GBF-1 because a similarly transfected pCMV
vector expressing cell lysates did not form the same complex
(lane 2). A fast migrating complex, denoted as
NSC (nonspecific complex), is detected in all
lanes. Formation of the band is variable in different
experiments. Preincubation of the nuclear extracts with an excess of
unlabeled wild type GATE probe resulted in the disappearance of
complexes GBF-1, a and b (lane 5). Unlabeled GM-1
oligonucleotide failed to inhibit the formation of this complex. In
addition, GBF-1-transfected cell lysates failed to form the same unique
complex when incubated with 32P-labeled GM-1, GM-2. or GM-3
(data not shown). The GATE-binding complexes formed with
IFN--treated HEK-293 cells is different from those observed in RAW
cells. In the human cell lines we have observed the formation of weaker
complexes (data not shown), presumably because of species differences.
Only one of them was slightly inducible. Furthermore, HEK-293 and RAW
cells represent human kidney and mouse macrophages, respectively. Thus,
these differences could account for variations in the EMSA banding patterns.
The effect of IFN- on the formation of the GBF-1 complex was
analyzed in the next experiment. Cells transiently transfected with
FLAG-GBF-1 expression vector were treated with IFN- for 1 h,
and the extract was used for EMSA (Fig. 6C). A short
treatment time was chosen to permit the post-translational
modifications. This also precludes the induction of endogenous GBF-1.
Cell extracts from empty expression vector formed complexes a and b
(lanes 2 and 3). Lysates from GBF-1-transfected
cells formed a specific slow migrating complex (lane 4)
whose binding was intensified after IFN- treatment (lane
5). In addition, the binding of complex a was also intensified.
The enhancement of DNA binding was not caused by differential amounts
of expressed protein in the treated and untreated lysates. Western blot
analysis of the extracts used for EMSA with FLAG epitope tag-specific
antibodies revealed that both untreated and IFN--treated cells had a
comparable expression of GBF-1 (Fig. 6D, compare lanes
3 and 4). As expected no such band could be detected in
the vector-transfected cell lysates (lanes 1 and
2).
Similarly, we determined the DNA binding specificity of muGBF-1, using
transfected HEK-293 cells (Fig. 6E). As expected, the GBF-1
complex was formed only with the lysates from muGBF-1 expression vector-transfected cells but not those transfected with empty vector
(compare lanes 2 and 3 with lane 7).
In lane 2, cells were treated with IFN- for 1 h
before preparing nuclear lysates. Therefore, its binding was enhanced
compared with the untreated cells. Formation of the GBF-1 complex was
inhibited by an excess of GW but not GM-1 and GM-3 oligonucleotides
(lanes 5 and 6). This complex was "supershifted" by the monoclonal FLAG epitope but not Myc
epitope-specific antibodies. Vector transfectants did not form any
complex different from that of mock transfected cells (compare
lanes 1 and 7). Expression of the transfected
gene product was confirmed by Western blot analysis of the lysates with
FLAG antibodies. No change in GBF-1 protein expression was noted after
IFN- treatment.
Expression of the GBF-1 Gene--
We next determined whether GBF-1
was expressed in normal tissues. A commercially available mouse
multiple tissue Northern blot was probed with a 32P-labeled
GBF-1 cDNA (Fig. 7). These blots
contained 2 µg/lane poly(A)+ RNA from each indicated
tissue. GBF-1 probing revealed that the gene was expressed at a higher
steady-state level in heart, brain, liver, and kidney. Its expression
was low in spleen, lung, skeletal muscle, and testis. Prolonged
exposure revealed two other weakly cross-hybridizing mRNAs in
testis and kidney (indicated with arrows). The identity of
these cross-hybridizing bands remains to be determined. This blot was
stripped and probed with a 32P-labeled -actin to confirm
equal loading. All lanes had a comparable amount of RNA. Because the
cardiac and skeletal muscles express an additional form of actin, an
extra band can also be seen in those lanes.
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Fig. 7.
Expression of GBF-1 gene in normal mouse
tissues. A commercially available multiple tissue Northern blot
bearing 2 µg/lane poly(A)+ RNA from the indicated tissues
was probed with a 32P-labeled GBF-1. Skel
Muscle, skeletal muscle. The position of GBF-1 is indicated with a
thick arrow. The upper and lower
arrows indicate the positions of weakly cross-hybridizing RNAs
from testis and kidney. These bands can only be seen after prolonged
exposure (3 days). The bottom panel shows the same blot
stripped and probed with -actin cDNA. The cardiac and skeletal
muscles express two forms of actin, and both can be seen in the
specific lanes of these blots. All other tissues express a
single form of actin.
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GBF-1 Is an IFN-stimulated Gene--
Given the observation that
GBFs are synthesized in response to IFN- (18), we next determined
whether IFN- induced the expression of GBF-1. RAW cells were treated
with IFN-, poly(A)+ RNA was isolated, and the expression
of GBF-1 mRNA was examined by Northern blotting. As shown in Fig.
8A, GBF-1 mRNA was induced in a time-dependent manner with maximal induction occurring
at 8 h poststimulus (5-fold) and a decline thereafter. A minor
species of mRNA (1.3 kb) is also in these cells, and it is not
significantly stimulated. This band was seen under low stringency
washing conditions or after a long exposure of the blots. Under these
conditions the expression of a housekeeping gene glyceraldehyde
3-phosphate dehydrogenase (GAPDH) mRNA was not altered with IFN-
treatment (Fig. 8A, bottom panel).
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Fig. 8.
IFN--induced
expression of GBF-1. A, Northern blot analysis of 2.5 µg
of poly(A)+ RNAs from 200 units/ml IFN--stimulated RAW
cells using a 32P-labeled GBF-1 probe. This blot was
stripped and probed with 32P-labeled GAPDH to ensure the
presence of equal loading of RNA. B, Western blot of RAW
cells probed with polyclonal antibodies raised against mouse GBF-1.
This blot was stripped and reprobed with an actin-specific monoclonal
antibody to confirm equal loading of protein in all lanes.
C, densitometric quantification of GBF-1 protein expression.
Each bar represents the mean band intensity ± S.E. of
triplicates. The bars in this graph correspond to the
lanes of B.
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We next determined whether induction of GBF-1 mRNA also resulted in
a corresponding rise in protein levels. IFN--stimulated RAW cell
extracts were monitored for GBF-1 levels by Western blot analyses with
mouse polyclonal antibodies raised against recombinant mouse GBF-1
protein. Indeed, IFN- caused a time-dependent increase in the levels of GBF-1 protein (Fig. 8B). A small,
inducible, faint band can also be seen in this blot. It may be a
proteolytic product or a cross-reacting protein. This blot was reprobed
with an antibody raised against actin to confirm equal loading (Fig. 8B, lower panel). A quantification of Western
blot is shown in Fig. 8C. Although a significant stimulation
of GBF-1 protein occurred at 2 h after IFN- treatment, an
~6-fold induction was observed at 16 h. Similar induction of
GBF-1 protein was observed in primary mouse embryo fibroblasts and HeLa
cells (data not shown).
To provide further evidence that GBF-1 is an IFN--inducible gene, we
have determined its induction in vivo. BALB/c mice were injected with 50,000 units of IFN- via the tail vein, and total RNA
from various tissues was extracted after 12 h. These RNAs were
used for Northern blotting, and the blots were probed with 32P-labeled GBF-1 cDNA. As shown in Fig.
9A GBF-1 mRNA was readily induced by IFN- treatment in all tissues examined, whereas a basal
level of its expression was seen in most tissues. Probing of this blot
with 32P-labeled GAPDH showed the presence of a comparable
amount of RNA in all lanes (Fig. 9B). The band intensities
were quantified using a PhosphorImager. GBF-1 expression data were
normalized to that of GAPDH and are shown in Fig. 9C. A
3-4-fold induction of the mRNA was noted in most tissues, although
the overall magnitude of expression varied among the tissues.
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Fig. 9.
Induction of GBF-1 mRNA by
IFN- in vivo. Where indicated
with a plus sign, BALB/c mice were treated with 50,000 units/ml of IFN- for 12 h by tail vein injection. A minus
sign indicates saline treatment. 50 µg of total RNA from each
sample was loaded in the lanes. The blot has been probed
with a 32P-labeled GAPDH to quantify RNA loading in the
tracks (B). C, quantification of GBF-1 mRNA.
The expression of GBF-1 was normalized to that of GAPDH.
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Intracellular Localization of GBF-1--
To determine the
intracellular location of GBF-1 we took two different approaches. In
one, primary mouse embryo fibroblasts (MEF) were transfected with a
FLAG-tagged GBF-1 or the control vector lacking GBF-1. The cells were
incubated with FLAG tag-specific antibody and then with a second
antibody labeled with Texas Red. The cells were counterstained with
DAPI to visualize the nuclei. As shown in Fig.
10A, the FLAG epitope
tag-specific antibodies recognized a protein both in the cytosol and
nucleus. The vector-transfected cells were not stained significantly
with Texas Red. This result was further confirmed using fractionated
nuclear and cytosolic extracts (Fig. 10B). HeLa cells were
transfected with FLAG vector or FLAG-tagged GBF-1. Cell lysates were
prepared and fractionated into nuclear and cytosolic extracts. The
fractionated extracts were analyzed by Western blot with FLAG
tag-specific antibodies. GBF-1 was present in both nuclear and
cytosolic extracts. These extracts were also probed with antibodies
that specifically recognize c-Jun (nuclear protein) and Bcl2
(cytosolic). As shown at the bottom portion of the
panel, these antibodies recognized proteins in the
appropriate fractions.
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Fig. 10.
Intracellular location of GBF-1.
A, primary MEFs were transfected with pCMV-FLAG
(Vector) or the same vector-expressing mouse GBF-1
(GBF-1). Cells were fixed, permeabilized, and incubated with
a monoclonal antibody against the FLAG epitope tag (1:250 dilution).
They were then washed and incubated with a goat anti-mouse IgG tagged
with Texas Red (1:2,000 dilution). The cells were also counterstained
with 0.1 µg/ml DAPI to detect the nuclei. A representative field is
shown. Magnification is ×40. B, fractionation of cell
extracts to define the intracellular locus of GBF-1. GBF-1- or
vector-transfected MEFs were fractionated into nuclear
(Nucl) and cytoplasmic (Cyto) compartments as
described under "Materials and Methods." A fraction of the total
extract was saved and also used in Western blots. These blots were
first probed with FLAG tag-specific antibodies to detect GBF-1
(B1). They were then stripped and probed with c-Jun-
(B2) or Bcl2- (B3) specific antibodies.
C and D, localization of endogenous GBF-1 in HeLa
and MEFs. Cells were grown on cover glasses, fixed, permeabilized, and
incubated with a polyclonal antibody against GBF-1. They were then
stained with a second antibody tagged with Texas Red (as in
A) and DAPI. Cells were photographed at ×100 (C)
and ×60 (D) magnification.
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In the second approach we stained cells with mouse antibodies specific
for GBF-1. Permeabilized MEF and HeLa cells were first incubated with
nonimmune or anti-GBF-1-specific antibodies and then with a secondary
antibody tagged with Texas Red. Similar results were obtained with both
cell types. The nonimmune antibody did not detect any signals. In
contrast, the GBF-1-specific antibodies readily stained the nuclear and
cytosolic compartments. The nuclear compartment was more intensely
stained. IFN- treatment caused a high intensity nuclear staining
compared with the untreated cells. This observation is consistent with
the rise in GBF-1 protein levels. These data indicate that GBF-1 is
primarily a nuclear protein, although a significant portion of it can
be found in the cytosol. Most importantly, a punctate staining is
observed in both nuclear and cytosolic compartments, suggesting that
GBF-1 is part of a subcellular domain/organelle. The exact nature of these complexes is not clear at this stage. There was no significant shuttling of protein from the cytosol to nucleus after IFN-
treatment. However, a perinuclear rearrangement of cytosolic protein
was observed in some fields (data not shown).
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DISCUSSION |
IFN- is central to a wide spectrum of biological
responses in vertebrates, including immune cell regulation and
antiviral, antimicrobial, and antitumor activities (24-26). A number
of ISGs that mediate IFN action have been identified using a variety of techniques (27, 28). All IFN responses are attributed to the activation
of Janus tyrosine kinase-STAT signaling pathways, a transiently
activated process (2, 3). Although the activation of STATs and
subsequent expression of certain ISGs are independent of de
novo RNA or protein synthesis, induction of several
IFN--regulated genes requires the synthesis of other protein factors
(1, 28). Furthermore, unlike IFN-/, the IFN--stimulated genes
are induced with variable kinetics (1) and often require new protein
synthesis. Additionally, induction of certain ISGs does not correspond
to the kinetics of STAT-activation and down-regulation (1, 28). Thus,
factors other than STATs regulate the expression of these "late
induced genes." Indeed, recent analyses have shown that IFNs can
induce some genes in cells lacking STAT1 (29, 30). Although
most IFN--induced genes are not stimulated in the absence of Janus
kinase Tyk2, a fraction of the IFN- response is retained in the U1A
cells that lack Tyk2 (31). Recent studies with
TYK2
/ mice also showed only a partial
impairment IFN-/ signaling in the absence of Tyk2 (32, 33). These
observations indicate the presence of other potential IFN-regulated
pathways and genes, although these could be a minor population relative
to the number of total ISGs. Indeed a number of such factors, such as
hXBP1, RF-X, IRF-1, class II transactivator (CIITA), and IFN-consensus sequence-binding protein (ICSBP) represent the secondary mediators of
signals (7, 34-40). However, these factors can only partially account
for the pleiotropic nature of IFN response, and additional undefined
IFN-induced regulators may exist.
We have shown earlier that the murine IRF-9 gene is regulated by a
novel IFN- response element GATE (18). Others have shown IFN-induced
expression of the chemokine RANTES (regulated on activation normal T
cell expressed and secreted) (41) and major histocompatibility complex
class IB (42) are mediated by novel elements that appear to be
dependent on transcription factors other than ISGF3. To define the
transacting factors that interact with GATE we have screened a cDNA
expression library derived from an IFN--stimulated mouse macrophage
cell line, using a method based on the Southwestern approach (Fig. 1),
and we identified the candidate cDNAs. One of these is a known
transcription factor, C/EBP- (22). An independent study also
confirmed that C/EBP- stimulates GATE-regulated transcription (23).
In this study we have characterized a novel factor, GBF-1. GBF-1
exhibits the characteristics of a GATE-binding protein: it binds to
GATE, it is an IFN--inducible protein, and it activates transcription. It also activates only GATE-driven reporters, not other
IFN-responsive promoters. The last observation indicates the
specificity of GBF-1 in mediating IFN--induced transcriptional responses.
GBF-1 exhibits a weak DNA binding property. Bacterially expressed GBF-1
does not bind to GATE. There could be several reasons for this
inability: the recombinant protein lacks appropriate post-translational
modifications; it requires other cellular factors for binding to GATE;
it may have lower affinity for GATE under the conditions of EMSA
experiments. Because we have isolated GBF-1 on the basis of its GATE
property (Fig. 1), it is surprising to note that it does not bind to
DNA in an EMSA. The DNA binding screen differs from EMSA in the
following respects. In the former method a multimerized GATE is used to
detect DNA binding. In contrast, a monomeric GATE is employed in the
latter. Multimerized GATE may support a low affinity binding of GBF-1,
whereas a monomeric GATE might not. Furthermore, the Southwestern
detection of DNA-binding proteins (Fig. 1) is an in situ
method, whereas EMSA is an in vitro method. However, after
expression in the mammalian cells, GBF-1 was able to bind to DNA. Our
previous studies identified two GATE-binding complexes, GIF-1 and
GIF-2, in the murine macrophage RAW cells (18). We were unable to
assign which of these two complexes contains GBF-1 because our
antibodies are incapable of neutralizing or supershifting. A better
quality antibody is required for examining this aspect. A number of
other IFN-regulated transcription factors such as ICSBP (IRF-8) and
CIITA do not bind to DNA on their own but can activate transcription
very well. They form complexes with other factors to activate
transcription (43-45). CIITA additionally possesses histone
acetyltransferase and GTP binding activities required for its
transactivating function (46, 47). Like CIITA, GBF-1 activates both
basal and inducible responses (48). Interestingly, ICSBP was also
discovered (49) using a method similar to that described here. Although
defined as a repressor of ISRE-dependent transcription
(50), subsequent studies showed that it activates transcription in a
context-specific manner (51-53). Previously, we have shown that
transcription factor C/EBP- binds to GATE and stimulates
transcription. A C/EBP- consensus sequence (CBS) is present in GATE
(Fig. 5B) and is necessary for stimulating transcription in
a variety of cells (22, 23). The fact that GM-1, GM-2, and GM-3 do not
respond to IFN- and have a disrupted CBS indicates that C/EBP-
binding site plays a large role in regulating this promoter. The fact
that GM-1, GM-2, and GM-3 do not respond to GBF-1 indicates that it
acts as part of the complex formed on CBS with C/EBP- for promoting transcription (Figs. 1 and 5). The weak but significant transactivation of GM-13 construct by GBF-1 suggests that it mediates a minor fraction
of the IFN- response in association with a weak complex formed with
another protein at GATE. Thus, GBF-1 requires part of the CBS and the
adjacent sites for driving gene expression. Consistent with this, GBF-1
synergistically induces GATE-dependent reporter in the
presence of C/EBP-.3
However, it is also possible that GBF-1 may interact with other proteins to generate unknown biological responses, which need to be
investigated further. This may include both transcriptional and
nontranscriptional functions, given its location in cytoplasmic and
nuclear compartments. The observation that IFN- stimulates GBF-1-dependent gene expression significantly further
suggests that an inducible post-translational modification regulates
its activity. This observation is consistent with our earlier studies, which showed that inhibitors of protein kinases block IFN--induced expression from GATE (18). Our recent studies show that inhibitors of
extracellular signal-regulated kinase 1/2 activation suppress IFN-induced gene expression (54). Preliminary studies with
mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase 1/2-specific and p38 mitogen-activated protein kinase-specific
pharmacologic inhibitors do not significantly inhibit
GBF-1-dependent IFN--stimulated induction of the GM-13
reporter (data not shown). This observation is also consistent with the
absence of any potential mitogen-activated protein kinase-specific
phosphorylation sites in GBF-1 protein. It is likely that other factors
may control its phosphorylation status. This aspect is currently being studied.
The immunocytochemical analyses in mouse and human cells revealed that
GBF-1 was present both in the nucleus and cytoplasm. This observation
suggests that GBF-1 might mediate other unknown biological responses in
the cells. We have described one of its functions in this study,
namely, the GATE-dependent transcriptional activation.
GBF-1 protein has a well conserved redox center. This domain may play a
role in regulating the transcription. Interestingly, other
transcription factors also possess redox domains or are modulated by
redox factors. For example, the upstream stimulating factor (55), D0, a
RIPE3b1-like transacting factor (56), Hox B5 (57), and thyroid
transcription factor (58) possess redox-sensitive domains. Furthermore,
a number of transcription factors of the Fos-Jun/AP-1 family (59, 60),
p53 (61, 62), hypoxia-inducible factor-1 (63), and PEB-2 (64) are
regulated via a modulation of their redox status. Ref-1, a redox
protein, maintains the redox status of some of transcription factors
described above (58, 62, 64-66). Future studies are required for an
understanding of the contribution of the gluatredoxin-like
domain to the transactivating function of GBF-1.
GBF-1 is also an IFN-inducible gene (Fig. 8). The induction of the
GBF-1 gene is not a cell line-specific effect because it is also
induced in various tissues by IFN-, in vivo (Fig. 9). Preliminary studies show that GBF-1 protein is also induced by other
proinflammatory cytokines, interleukin-1 and -6 (data not shown).
Therefore, its expression may be uniquely regulated by IFN- and
other cytokines. A computer-aided search of published interleukin-1- or
-6-inducible gene promoters for GATE-like elements did not reveal any
strong homologies. The only partial homology we could find was to the
C/EBP- binding sites, which was consistent with the data shown in
Fig. 5. On a similar note, we did not find GATE-like elements in the
promoters of the published ISG promoters. It is likely that there could
be a separate set of genes controlled by GBF-1. A separate
investigation is required to resolve these issues. Finally, the other
physiological roles of GBF-1 can be realized only after generating a
knockout mouse. Our study assigned one function to this novel protein,
i.e. an enhancement of IFN-stimulated gene expression
through GATE. While this paper was in review a new report showed that
monkey GBF-1 has membrane-bound prostaglandin E synthase activity (67).
The functional relevance between this enzyme activity and
IFN--activated transcription needs to be investigated further.
Finally, other transcriptional targets and binding partners of GBF-1
need to be defined.
-dependent Gene Expression*
§,
TOP
ABSTRACT
INTRODUCTION
