(Received for publication, February 27, 1996, and in revised form, September 10, 1996)
From the Emory Skin Diseases Research Core Center, Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia 30322
In response to interferon (IFN
),
intercellular adhesion molecule-1 (ICAM-1) is expressed on human
keratinocytes, a cell type that is critically involved in cutaneous
inflammation. An ICAM-1 5
regulatory region palindromic response
element, pI
RE, has been shown to confer IFN
-dependent
transcription enhancement. By electrophoretic mobility shift assays
(EMSA), pI
RE forms a distinct complex with proteins from
IFN
-treated human keratinocytes, termed
response factor (GRF).
Binding of GRF is tyrosine phosphorylation-dependent, and
mutations of pI
RE that disrupt the palindromic sequence or alter its
spatial relationship abrogate GRF binding. Supershift EMSAs using
antibodies to characterized STAT proteins suggest that GRF contains a
Stat1
-like protein; however, non-ICAM-1 IFN
-responsive elements
(REs) known to bind Stat1
homodimers fail to compete for GRF binding
in EMSA, and pI
RE does not cross-compete with these REs that complex
with homodimeric stat1
. The pI
RE·GRF complex also displays a
distinctly different electrophoretic mobility compared to that of
IFN
REs complexed to homodimeric Stat1
. These findings indicate
that a distinct complex containing a Stat1
-like protein mediates
IFN
-induced ICAM-1 gene transcription and identifies a subset of
IFN
-responsive genes that appear to be regulated by this
complex.
The initiation and evolution of localized inflammation is a consequence of homing and extravasation of leukocytes at sites of tissue injury or threat (1). Intercellular adhesion molecule-1 (ICAM-1)1, a cell surface glycoprotein that belongs to the immunoglobulin gene superfamily, serves as a specific ligand for receptors expressed by leukocytes (2). ICAM-1 plays a pivotal role in the adhesion and transmigration of leukocytes at sites of inflammation, and the up-regulation of ICAM-1 cell surface expression during inflammatory responses is essential in facilitating leukocyte migration (3, 4).
IFN, a pleiotropic cytokine produced by activated T lymphocytes,
plays a critical role in host defenses and inflammation (5). In the
skin IFN
induces de novo expression of ICAM-1 on human
keratinocytes (HK), cells that are critically involved in cutaneous
inflammatory processes (6). Upon binding to its receptor, IFN
initiates a signal transduction cascade that involves rapid activation
of two members of the Janus tyrosine kinase family, JAK-1 and JAK-2
(7), and consequent tyrosine phosphorylation and activation of a latent
cytoplasmic protein, originally referred to as p91 and now known as
Stat1
(8, 9). Stat1
is the first described member of a family of
proteins known as STATs, or signal transducers and activators of
transcription. When activated by IFN
, activated Stat1
homodimerizes through Src homology domains (10, 11) to form
-activated factor (GAF). The Stat1
homodimers, after
translocation to the nucleus, bind to the
-activated site (GAS),
first identified in the human guanylate-binding protein (GBP) gene, and
initiate gene transcription (8, 9, 12). Stat1
(p84) is an
alternatively spliced product of the gene for Stat1 and is a truncated
protein that lacks 38 amino acids at the carboxyl end of Stat1.
Stat1
also homodimerizes and is capable of binding GAS but does not
activate transcription (13). It has recently been shown that Stat1
,
which obligatorily requires tyrosine phosphorylation to become active,
also requires phosphorylation of a serine residue for maximal
activation of gene transcription (14).
In addition to formation of the homodimeric GAF, Stat1 also
participates in forming the heteromultimeric transcription complex ISGF3, composed of Stat1, stat2, and a non-STAT protein, p48 (15, 16, 17, 18). ISGF3 binds to the IFN-stimulated response element (ISRE),
which is an IFN
/
- and IFN
-inducible element involved in the
regulation of a variety of genes (11, 13, 17). Recently, multiple
cytokines and growth factors have been shown to mediate their
transcriptional effects through these and additional STAT proteins, of
which there are now six characterized members (19). However, Stat1
is frequently activated in response to a wide variety of extracellular
signals, including those involved in transcriptional activation of a
number of genes involved in immune responses (19).
IFN-dependent induction of ICAM-1 gene expression is
regulated at the transcriptional level (20, 21). The 5
flanking region
of ICAM-1 gene contains an 11-base pair (bp) element, which we refer to
as palindromic IFN
response element (RE), or pI
RE, located
upstream of the ICAM-1 transcription initiation site between nucleotides
76 and
66. pI
RE is composed of the sequence
5
-TTTCCGGGAAA-3
. Several laboratories have demonstrated
that pI
RE is both necessary and sufficient for
IFN
-dependent gene transcription (20,
22).2
The present studies were designed to characterize the molecular events
and trans-acting factors involved in the IFN-induced regulation of
ICAM-1 gene transcription. The data presented show that the protein
complex activated by IFN
, which trans-activates ICAM-1 gene
expression by binding to pI
RE, shares both similarities and distinct
differences with previously characterized IFN
-activated STAT
complexes. From these data we propose that the protein complex mediating IFN
-dependent ICAM-1 gene transcription, which
we refer to as the
response factor, or GRF, represents a distinct
form of IFN
-induced transcription trans-activator and likely
mediates trans-activation of a subset of IFN
-inducible early
response genes.
As described previously (22), HK were isolated from neonatal foreskins at the Emory Skin Diseases Research Center. HK were cultured in KGM supplemented with bovine pituitary extract (Clonetics Corp., San Diego, CA). Cultures were maintained at 37 °C in humidified 5% CO2 and passaged at 60-70% confluence using subculture reagents from Clonetics. Experiments with HK were conducted with cells in passage 3.
Cytoplasmic and Nuclear Extract PreparationCytoplasmic and
nuclear extracts were prepared as described previously (23) from cells
that were either left untreated or treated with 250 units/ml
recombinant human IFN (R&D Systems, Minneapolis, MN). Cells were
washed twice in ice-cold phosphate-buffered saline (Life Technologies,
Inc.), then quickly washed in buffer A (10 mM Hepes, pH
7.4, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 50 µM
phenylmethylsulfonyl fluoride (PMSF); all from Sigma). After centrifugation at 2,000 rpm (4 °C for 5 min), the cell pellet was resuspended in buffer A containing 0.1% Nonidet P-40 (U. S. Biochemical Corp.) and incubated on ice for 10 min. The nuclei were
collected by centrifugation at 4,000 rpm for 2 min at 4 °C. The
supernatant (cytosolic fraction) was removed and saved, and the pellet
was resuspended in buffer B (20 mM Hepes, pH 7.4, 1.5 mM MgCl2, 420 mM NaCl, 1 mM DTT, 50 µM PMSF, 0.2 mM EDTA,
25% glycerol). The nuclear pellet was incubated on ice for 30 min, followed by centrifugation at 14,000 rpm for 15 min. The protein concentrations of the cytosolic and nuclear fractions were determined using UV absorbance at 280 nm as described (24). Proteins were used
immediately in a binding reaction or aliquoted and stored at
70 °C.
The DNA binding reaction was performed for 30 min at room temperature in a volume of 20 µl, containing 5 µg of nuclear or cytoplasmic protein extract, 2.5 µg of bovine serum albumin (Life Technologies, Inc.), 2 µg of poly(dI-dC) (Sigma) 5 µl of 4 × binding buffer (1 × buffer: 12 mM Hepes, pH 7.8, 4 mM Tris, 60 mM KCl, 1 mM EDTA, 12% v/v glycerol, 1 mM DTT, 1 mM PMSF) with or without 10-100-fold molar excess of cold competitor DNA. Radiolabeled probe (1 × 105 cpm) was added for an additional incubation period of 20 min. In supershift EMSA, cytoplasmic or nuclear extracts were incubated with experimental or isotype control antibody, at supplier's recommended concentrations, prior to the addition of the 32P-labeled probe. Polyclonal antibodies to Stat1 and monoclonal antibodies to Stat1 through Stat6 and to p48 were obtained from Transduction Laboratories, Lexington, KY. Polyclonal antibodies to both the amino and carboxyl termini of Stat1 were kindly provided by J. E. Darnell, Rockefeller University, New York, NY. DNA binding reactions were separated on 4% native polyacrylamide gels. Gels were subsequently dried and autoradiography performed. The autoradiographs were scanned on a La Cie flat bed scanner (La Cie Ltd., Beaverton, OR) utilizing Adobe Photoshop software (Adobe Systems, Inc., Mountain View, CA). Subsequently the digitized image was labeled in Microsoft Power Point (Microsoft Corp., Redmond, WA) and printed on a high resolution laser printer. Each figure represents a computer-generated image of the autoradiograph, and each is typical of the autoradiograph in the context of relative band and background densities.
To test the requirement of tyrosine phosphorylation for DNA-protein
complex formation, nuclear protein extracts were exposed to 10 units of
protein-tyrosine phosphatase (PTPase) 1-B from Yersinia
pestis (Upstate Biotechnology, Inc., Lake Placid, NY) in the
presence or absence of the PTPase inhibitor sodium orthovanadate (1 mM). These reactions were carried out for 30 min at
30 °C in a 15-µl reaction containing 20 mM Tris-HCl,
pH 7.4, 0.5 mM DTT, 0.1 mM EGTA. Treated
extracts were then incubated with 32P-labeled pIRE as
probe and analyzed by EMSA.
All oligonucleotides used for probes or as cold competitors were
synthesized at the Emory University Microchemical Facility. Double-stranded oligonucleotides were prepared by annealing
complementary strands as described (25). The pIRE oligonucleotide
was synthesized to include the IFN
-responsive site (in bold letters)
found in the ICAM-1 gene promoter
(5
-
TTTCCGGGAAA
-3
). The underlined sequences in the pI
RE oligonucleotide represent restriction sites that were used to create overhangs for labeling with
50 µCi of [
-32P]dCTP (DuPont NEN) by fill-in
reaction using Klenow (Stratagene, La Jolla, CA) as described (25).
Alternatively, a short primer, GGGATCCTTTCC, complementary to the 3
end of the above sequence was used to label pI
RE by a
primer-extension fill-in reaction using Klenow and 50 µCi of
[
-32P]dCTP (25). Unincorporated nucleotides were
removed by column chromatography over G25 Sephadex columns (Boehringer
Mannheim). The [
-32P]dCTP-labeled probe was used in
analysis of the phosphatase-treated or untreated proteins in EMSA. In
all other EMSAs 5
end-labeled probes were prepared with 100 µCi of
[
-32P]ATP (Amersham Corp.) using T4 polynucleotide
kinase (New England Biolabs, Beverly, MA) and were gel-purified as
described (25).
Oligonucleotides corresponding to IFNREs (written in bold letters) of
the genes indicated were used as radiolabeled probes or as cold
competitors. For all oligonucleotides used, non-wild type restriction
sites (underlined) were incorporated flanking the identified REs for
cloning and labeling purposes. Oligonucleotides used were as
follows: GAS of the GBP gene:
AGTTTCATATTACTCTAAATC
(26); ISRE of the GBP gene:
CGAAGTACTTTCAGTTTCTATTA
(9); ISRE of the 6-16 gene:
CCCTTTTACTTTGA
(27);
GRR of the Fc
R1 gene:
TTTCTGGGAAA
(28); pIRE
of the ICSBP gene:
TTTCCGAGAAA
(29);
IFNRE of the IRF-1 gene:
TTTCGGGGAAA
(30).
In experiments comparing complexes and mobility of pIRE
versus the GAS element, a short primer (GGGATCCGATTTAGA)
complementary to the 3
end of the GAS sequence of the GBP gene, was
used to label GAS by a primer-extension fill-in reaction using Klenow and 50 µCi of [
-32P]dCTP. Unincorporated nucleotides
were removed by column chromatography over G25 Sephadex columns.
In addition, four oligonucleotides incorporating distinct mutations,
insertions, or deletions in the wild type pIRE sequence were also
used as competitors or probes. These pI
RE mutants, with mutated
nucleotides shown in italics, were: MUT 1,
TCACCGGGAAA
; MUT 2,
TTTCCGGGAGA
;
MUT 3,
TTTCCATGCATGCATGGAAA
; MUT 4,
TTTCC(X)GGAAA
. Each mutant
incorporates a distinct class of change; MUT 1 has a two-nucleotide
mutation in the 5
side of the of pI
RE, MUT 2 has a one-nucleotide
mutation in the 3
side of pI
RE, MUT 3 has a 10-bp insertion
separating the 5
and 3
sides of the palindrome, and MUT 4 deletes the
central G-C hinge in the palindrome. In addition, two different 30-bp sequences from regions of ICAM-1 located upstream from the pI
RE were
used as irrelevant competitor DNA fragments.
We examined by EMSA
whether specific factors that can bind to pIRE are activated in HK
by IFN
treatment. As seen in Fig. 1A,
stimulation of cells with IFN
led to the induction of a distinct DNA-binding protein complex (GRF), resulting in retarded mobility of
the labeled pI
RE probe. However, no pI
RE binding activity was
observed in nuclear extracts isolated from untreated HK. Binding of GRF
to labeled pI
RE was not competed by excess unlabeled, irrelevant DNA
as competitor, while excess unlabeled pI
RE competed for GRF binding,
indicating the specificity of the retarded complex. In order to
determine the kinetics of induction of GRF, HK were treated with IFN
for various periods of time before preparing both cytoplasmic and
nuclear extracts for EMSA. As seen in Fig. 1B, the binding
activity of cytoplasmic extracts from HK peaked after 30 min of IFN
treatment, decreased after 4 h, and declined significantly by
8 h. By 24 h, binding activity was not present. Stimulation
of HK with IFN
led to the induction of GRF in both cytoplasmic as
well as nuclear fractions within seconds (data not shown), consistent
with the characteristic rapid activation of latent proteins by this
cytokine (13).
pI
We next addressed
whether targeted mutations, insertions, or deletions within the 11-bp
pIRE palindromic sequence had any effect upon GRF binding. As seen
in Fig. 2A, mutations that disrupted either
the 5
or the 3
end of the pI
RE palindrome failed to compete with
wild type pI
RE for binding to GRF. Moreover, substitution with a
10-bp spacer (allowing for one full helical turn) between the
palindromic halves or deletion of the G-C hinge of the palindrome abrogated the ability to compete with wild type pI
RE for binding to
GRF. When used as radiolabeled probes, these mutated oligonucleotides also failed to display any retarded complexes upon incubation with
lysates from IFN
-treated cells. Because the mutant pI
RE sequences
failed to display any retarded complexes and failed to compete with
pI
RE for GRF complex formation at 10-fold molar excess, we
investigated whether these mutants displayed a
concentration-dependent competition for GRF at higher molar
ratios. Unlabeled mutant oligonucleotides ranging from 10-fold to
1000-fold molar excess were used in a competition EMSA. As seen in Fig.
2C, the mutants failed to display any competition with
pI
RE for GRF complex formation even at a 1000-fold molar excess.
Furthermore, unlabeled double-stranded oligonucleotide corresponding to
the GAS sequence also failed to display competition for GRF complex
formation when used at a 1000-fold molar excess. Finally, while the
unlabeled double-stranded irrelevant oligonucleotide also failed to
display competition for GRF at a 1000-fold excess, a competition EMSA
utilizing unlabeled pI
RE displayed a detectable diminution of the
labeled GRF complex at a unlabeled to labeled ratio of 0.01 to 1 (Fig.
2B), and a significant competition at 0.1 and 1 to 1 molar
ratios (Fig. 2, B and C). These data demonstrate
a high and specific binding affinity of pI
RE for GRF binding, a
requirement for a specific sequence and spatial arrangement within this
element, and a lack of competition by an oligonucleotide containing the
classic GAS element previously demonstrated to bind homodimeric
Stat1
.
The changes in the pIRE palindromic sequence that abrogated GRF
binding and complex formation were consistent with additional transcriptional activation studies using reporter gene constructs. None
of the described mutations were capable of driving reporter gene
expression, while wild type pI
RE was sufficient to confer IFN
inducibility of heterologous reporter gene constructs (data not shown).
These results indicate that GRF requires an intact pI
RE palindromic
sequence to bind to pI
RE in vitro and pI
RE is
necessary to function in vivo.
The
importance of protein-tyrosine phosphorylation in the activation of
trans-acting proteins involved in IFN-induced transcription of other
genes (19, 31) led us to investigate whether pI
RE binding activity
induced upon IFN
treatment of HK was dependent upon tyrosine
phosphorylation. Purified recombinant PTPase 1-B, isolated from
Y. pestis, is a close homologue of the human
tyrosine-specific phosphotase PTPase 1 and has been found to
specifically catalyze the removal of phosphate from tyrosine residues
(32). We assessed the effect of PTPase 1-B on the pI
RE·GRF complex
formation. When the pI
RE probe was incubated with IFN
-treated HK
cell lysates in the presence of PTPase 1-B, formation of the
pI
RE·GRF complex was abrogated (Fig. 3). However,
addition of the PTPase inhibitor sodium orthovanadate prior to
incubation with the enzyme prevented disruption of complex formation,
while the inhibitor itself had no effect upon the complex formation.
These results indicate that tyrosine phosphorylation is needed for
formation of the pI
RE·GRF complex, a property consistent with GRF
containing a STAT-like protein (13).
Anti-Stat1
Since IFN
activation of Stat1
is known to involve tyrosine phosphorylation of
quiescent cytoplasmic proteins, we investigated the possibility of
Stat1
involvement in GRF using anti-Stat1
antibodies in
supershift EMSAs. As shown in Fig. 4, addition of polyclonal anti-Stat1
antibodies directed against either the amino-terminal or the carboxyl-terminal portions of Stat1
to IFN
-treated cell lysates supershifted the pI
RE·GRF complex, and
these supershifted complexes were competed away by excess unlabeled
pI
RE. Irrelevant antibodies had no effect upon complex formation or
mobility (data not shown). Since polyclonal antibodies raised against
both the amino- and the carboxyl-terminal portions of Stat1
supershift the pI
RE·GRF complex, and since Stat1
lacks the 38 carboxyl-terminal amino acids that are included in Stat1
, it is
extremely unlikely that the Stat1-like protein identified through these
studies is Stat1
. Monoclonal anti-Stat1
antibodies used in
subsequent studies, while specific for Stat1
, were raised against
peptide regions common to Stat1
and Stat1
, and thus do not
provide evidence to include or exclude Stat1
as a possible component
of the pI
RE·GRF complex. However, polyclonal antibody supershift
data presented in Fig. 4 provide strong evidence that the GRF complex
does not contain Stat1
.
Antibodies to Other STAT Proteins Do Not Supershift the pI
Since recent reports have shown that
numerous STATs and non-STAT proteins interact with Stat1 in nuclear
transcription complex formation (19), we investigated whether GRF
contained any other known STAT proteins by supershift EMSA. As shown in
Fig. 4, both polyclonal and monoclonal antibodies to Stat1 were able
to supershift the pI
RE·GRF complex. However, antibodies to Stat2,
Stat3, Stat4, Stat5, and Stat6 did not interact with this complex (Fig.
5). Antibodies to p48, a component of the ISGF3 complex,
also did not react with the GRF (data not shown).
GRF Contains a Distinctly Different DNA-Protein Complex
In
order to investigate further whether the anti-Stat1 antibody
supershifted GRF complex contained a form of Stat1 that could be
competed by GBP·GAS or GBP·ISRE, we performed competition
supershift EMSA. As seen in Fig. 6, the supershifted GRF
when bound to pI
RE was not competed by excess unlabeled
double-stranded oligonucleotide corresponding to either the GAS or ISRE
elements of the GBP gene. However, this complex was competed by excess
of unlabeled pI
RE and was also competed by double-stranded DNA
corresponding to the IFNRE of the IRF-1 gene, an IFN
RE that varies
in sequence from pI
RE by only a single nucleotide (see Table
I).
|
Our data from
the supershift EMSA using anti-Stat1 antibodies suggests that
Stat1
, or a Stat1
-like protein, is part of the GRF that binds to
the ICAM-1 pI
RE, but both the GAS and ISRE elements failed to
compete with pI
RE for complex formation. Therefore, we investigated
whether pI
RE could cross-compete with GAS. In addition, because
Stat1
had previously been shown to bind to the GAS element of
several genes as a homodimer referred to as GAF (8), we also
investigated whether the complexes formed with pI
RE and GAS
displayed any differences in EMSA. Using pI
RE or GAS as probes with
IFN
-treated HK cell lysates, we observed striking differences in the
mobility of complexes formed with pI
RE and GAS when run in the same
gel (Fig. 7). GRF displayed a distinctly slower mobility
compared to the GAF complex. Furthermore, unlabeled pI
RE at a
1000-fold molar excess failed to compete with GAS for the binding of
GAF complex. However, a 1 to 1 molar ratio of unlabeled ISRE displayed
significant competition with labeled GAS for binding GAF, while a
10-fold molar excess of unlabeled ISRE completely competed for
GAF·GAS complex formation. More importantly, unlabeled GAS displayed
a concentration-dependent competition for binding of GAF with a
significant diminution of the labeled GAS·GAF complex when unlabeled
GAS was added at only a 0.1 to 1 molar ratio. However, neither
unlabeled GAS or ISRE, when added to labeled pI
RE reactions,
displayed any ability to compete for GRF complex formation even when
added at a 1000-fold molar excess (Fig. 7). Interestingly, excess
unlabeled IFNRE of the IRF-1 gene displayed a similar
concentration-dependent competition for GRF complex
formation to that displayed by unlabeled pI
RE, suggesting similar
binding affinities of GRF to these two IFN
-responsive elements, as
was the case using the Fc
R1·GRR element as competitor for GRF
binding in earlier experiments (Fig. 2C). These results indicate that GRF, which complexes with pI
RE of ICAM-1, IFNRE of
IRF-1, and GRR of Fc
R1, is distinct from the classic GAF complex that binds to GAS.
GRF Displays Similarity with the DNA-Protein Complex Formed with the IFN
In addition to the ability of
IFNRE of the IRF-1 gene to compete for GRF binding to pIRE (Fig. 6
and 7), studies in our laboratory revealed that the IFN
response
element pIRE of the ICSBP gene (29) also functioned well as a
competitor for GRF binding (data not shown). We therefore investigated
whether the complexes formed with pI
RE and those formed with a
representative element of those genes that contain IFN
REs with
sequences very similar to pI
RE (see Table I and below) displayed
similar or different mobilities in EMSA. Using labeled oligonucleotides
of equal size (25 bp) containing either pI
RE or the GRR of the
Fc
R1 gene (28) as probes, we compared EMSA mobilities of the
DNA-protein complexes formed when these probes were incubated with
IFN
-treated HK cell lysates. As seen in Fig. 8, the
complexes that formed with pI
RE and GRR displayed similar mobility.
GRR also competed for GRF binding to pI
RE, as seen in Fig.
2C, and unlabeled pI
RE displayed a similar competition
for GRF binding to GRR (data not shown). In addition, both complexes
were supershifted in a similar manner with monoclonal antibodies to
Stat1
.
Taken together, the data presented in these experiments indicate that
pIRE of the ICAM-1 gene, GRR of the Fc
R1 gene, IFNRE of the IRF-1
gene, and pIRE of the ICSBP gene form a subset of IFN
response
elements that bind to a trans-activating complex,
response factor,
that contains a Stat1
-like protein. This GRF complex clearly appears
to be distinct from the homodimeric Stat1
complex that forms GAF and
binds to the GAS element of other IFN
-responsive genes, such as that
characterized in the guanylate-binding protein gene. Comparison of the
identified critical sequences of these GRF-binding and non-GRF-binding
elements and their distinct differences, as well as the mutations used
in the above studies, are shown in Table I. These data and comparisons
indicate that a potential consensus sequence, TTTCNGNGAAA, is required
for binding of GRF. They also indicate that elements such as GAS and
ISRE, which do not contain the characteristic 11-bp palindrome sequence
displayed by pI
RE, bind to Stat1
containing complexes but not to
the complex typified by the
response factor, which binds to pI
RE
and similar elements identified in a subset of IFN
-responsive
genes.
We have characterized a specific DNA-binding complex, which we
have termed GRF, that binds to the minimal IFN-responsive element of
the ICAM-1 gene, pI
RE. Formation and binding of GRF is dependent on
IFN
-induced activation of pre-existing proteins, as demonstrated by
the rapid activation and binding of GRF and by the activation of this
complex in the presence of cycloheximide.3
In addition, we have demonstrated that the pI
RE sequence is necessary for binding GRF and that mutant pI
RE sequences neither bind GRF nor compete with pI
RE for the pI
RE·GRF complex even when used at high molar ratios. Moreover, pI
RE displays a high and
specific binding affinity for GRF.
IFN signaling involves activation of Stat1 by phosphorylation of a
tyrosine residue in order to assemble active transcription-stimulating complexes (33, 34). Treatment with PTPase 1-B, which specifically dephosphorylates tyrosine residues, has been shown to abrogate binding
of these transcription complexes as shown by EMSA (34, 35). Our data
indicate that IFN
-induced activation of GRF is tyrosine
phosphorylation-dependent as well.
It has been shown that Stat1 is activated by a number of other
agonists, in addition to IFN
and IFN
, based largely on reactivity of binding complexes with anti-Stat1
antibodies (19, 36, 37, 38). Our
supershift data using antibodies to the known STAT proteins suggest
that Stat1
, or a protein with antigenic similarities to Stat1
, is
a component of GRF. In addition, studies using polyclonal antibodies to
either the amino-terminal or the carboxyl-terminal portions of Stat1
appear to exclude Stat1
as a possible component of the GRF complex.
These data are in agreement with the recently reported GAF-like factor
binding to the IFN
RE of ICAM-1 gene (39) and the recent finding of
Stat1
involvement in the complex by immunoblotting using anti-Stat1 antibodies (40). Previous reports indicate that Stat1
is a common
component of the IFN-activated trans-acting complexes GAF and ISGF3.
Stat1
binds to GAS as the homodimer GAF, and the multimeric ISGF3
complex, which binds to ISRE, contains Stat1
in addition to Stat2
and p48 (19). However, in the present studies, neither a
double-stranded oligonucleotide corresponding to GBP·GAS nor GBP·ISRE used as unlabeled competitors competed with pI
RE for the
binding of GRF. Further, we have observed that double-stranded oligonucleotides corresponding to the IFN
REs of the ISRE from the
6-16 gene (27) and the GAS sequence
(GCGGATCCTTTCCTGTAAAAGCTTGC) from the Ly6A/E gene (41) also
fail to compete with pI
RE for GRF complex
formation.4
Our studies demonstrate a lack of cross-competition between GAS and
pIRE for formation of their respective DNA-protein complexes. However, ISRE from the GBP gene, which binds Stat1
as part of the
heterotrimer in the ISGF3 complex and does not compete with pI
RE for
complex formation, clearly competes with GAS for the GAS·GAF complex.
We have observed that both GAS and ISRE do not compete with pI
RE
even at a 1000-fold excess in our experiments using HK cell lysates.
Our results are in agreement with the report that shows GBP·GAS does
not compete with the ICAM-1 IFN
RE in airway epithelial cells (20)
and are in contrast to those obtained by others in MeL JuSo cells
(42).
Consistent with our results, GRF that is supershifted by anti-Stat1
antibodies is not competed away by excess unlabeled GAS or ISRE from
the GBP gene. On the other hand, excess unlabeled IFNRE from the IRF-1
gene displayed competition for the supershifted GRF. In fact,
semi-palindromic IFN
REs from other IFN
-responsive genes, such as
the IFNRE of the IRF-1, pIRE of the ICSBP gene and GRR of Fc
R1 gene,
show somewhat closer sequence homology with that of pI
RE than do the
sequences of various GAS and ISRE elements as shown in Table I. It is
precisely these elements that share greater sequence homology with
pI
RE that can function as competitors in EMSAs with pI
RE for
complex formation in IFN
-induced HK cell lysates. Interestingly,
these REs show differences in their sequences in nucleotides
immediately flanking the G-C hinge of the pI
RE. The mutations that
rendered pI
RE completely nonfunctional in vitro and
in vivo in our study are either in the 5
or 3
end of the
palindrome or the G-C hinge of the palindrome, and suggest binding
specificity of GRF for these specific sequences. Comparison of the
sequences of IFN
REs of the IRF-1, ICSBP, and Fc
R1 genes with the
pI
RE sequence of ICAM-1 and mutations of pI
RE sequence reveals a
potential consensus sequence, TTTCNGNGAAA, that is required for binding
of GRF. Among the GAS and ISRE sequences of genes such as GBP, 6-16,
and Ly6A/E, the GAS sequence of Ly6A/E displays closest homology to
this GRF-binding consensus sequence. However, the sequence of Ly6A/E
diverges from the GRF-binding consensus sequence at two sites, the
central G-C hinge and in the 3
half of the palindrome. We have been
unable to demonstrate any competition for GRF binding with the
LY6A/E·GAS. Whether this inability to bind GRF results from
divergence of the nucleotide in the hinge or the 3
half of the
palindrome has not yet been determined.
Finally, the significantly slower mobility of the pIRE·GRF complex
compared to that of the GAS·GAF complex, using identical IFN
-treated HK cell lysates with GAS and pI
RE as separate probes in the same EMSA, cannot be accounted for by differences in the sizes
of the radiolabeled oligonucleotides. In fact, the smaller pI
RE
probe (25 bp) formed a more slowly migrating DNA-protein complex when
compared to the GAS·GAF complex bound by the larger (36 bp) GAS
probe. In contrast, the IFN
-activated DNA-protein complexes formed
with either the Fc
R1·GRR or the ICAM-1·pI
RE clearly display
similar mobility on EMSA, and both complexes supershift in a similar
fashion with anti-Stat1
antibodies. The IFNRE of the IRF-1 gene and
the pIRE of the ICSBP gene form a DNA-protein complex with
IFN
-treated HK cell lysates, which also displays a mobility similar
to that of the pI
RE·GRF complex in EMSA (data not shown).
Other investigators have recently shown that FcR1·GRR does not
compete with GBP·GAS in competition EMSA (43). Further, Fc
R1·GRR
was also shown to bind a Stat1
-like protein that interacted with an
additional 43-kDa protein in response to IFN
-stimulation (35, 44).
The semi-palindromic IFN
RE of the MIG gene, which displays
significant sequence homology to ICAM-1·pI
RE, has been reported to
bind an IFN
-activated trans-activating factor (
RF-1) that is
composed of at least two proteins of 95 and 130 kDa (45). Furthermore,
RF-1 was shown to exhibit differences in electrophoretic mobility
distinct from GAF and to contain one or more subunits antigenically
related to Stat1
(45).
These data thus indicate that pIRE represents a distinct subset of
IFN
REs found in a number of early response genes that mediate
trans-activation in response to IFN
signaling through a DNA-binding
protein complex (GRF) that is distinctly different from the previously
characterized IFN
-activated complex, GAF. While GRF certainly
appears to contain a Stat1
-like protein, identification and
characterization of all of the components of this distinct
trans-activating complex, their relationship to other STAT and non-STAT
proteins, and the specific biochemical pathways involved in their
activation will further elucidate the molecular mechanisms by which
IFN
initiates differential responses at sites of localized
inflammation.
We thank Dr. J. E. Darnell for the kind gift of polyclonal anti-Stat1 antibodies and Dr. Josiah N. Wilcox for invaluable assistance in preparation of the figures for this manuscript. We gratefully acknowledge the advice, technical help, and critical review of this manuscript by Dr. Johannes Bauer, Georgetta Cannon, Dr. Jennifer Duff, Dr. Tri Nguyen, and Virginia Secor.