(Received for publication, October 19, 1995)
From the
STAT (signal transducer and activator of transcription) proteins
combine with cytokine receptors and receptor-associated kinases in
distinct protein/protein interactions that are critical for
STAT-dependent signal transduction events, but the nature of any
subsequent STAT interactions with DNA-binding proteins in the nucleus
is less certain. Based on assays of DNA/protein binding and activity of
transfected reporter plasmids, we determined that occupation of
contiguous DNA-binding sites for Stat1 (the first member of the STAT
family) and the transcriptional activator Sp1 are both required for
full activation of the intercellular adhesion molecule-1 gene by
interferon-. Thus, Stat1 binding to DNA cannot by itself be
equated with biologic actions of Stat1. In co-immunoprecipitation
experiments, we also obtained evidence of direct and selective
Stat1/Sp1 interaction (in primary culture cells without
overexpression), further indicating that Stat1/Sp1 synergy confers an
element of specificity in the pathway leading to cytokine-activated
transcription and cytokine-dependent immunity and inflammation.
STAT ()proteins act as critical intermediates in
cytokine-dependent gene activation based on their dual capacities for
signal transduction (at the cell surface) and activation of
transcription (in the nucleus) (1) . Signal transduction
depends on programmed assembly of cytokine receptors,
receptor-associated JAK kinases, and in some cases serine kinases, that
recruit and activate specific STAT
proteins(2, 3, 4) . Phosphorylated/activated
STATs then dimerize, translocate to the nucleus, and direct
transcription of specific target genes. For example, the first member
of the STAT family (designated Stat1
) undergoes tyrosine 701 and
serine 727 phosphorylation in response to IFN-
(5) . This
activation step is triggered by IFN-
-dependent oligomerization of
the IFN-
receptor and consequent cross-phosphorylation of
receptor-associated Jak1 and Jak2 kinases and the receptor
-chain(6) . Receptor phosphorylation enables
-chain
recruitment of Stat1 via its SH2 domain. Stat1 then undergoes
phosphorylation and release from the receptor as a homodimer that can
translocate to the nucleus and bind to a specific DNA element (7, 8) . Thus, distinct protein/protein interactions
are critical for Stat1-dependent signal transduction events at the
IFN-
receptor, but the nature of Stat1 interactions with other
proteins (especially other transcription factors) in the nucleus is
less certain. In the present report, we take advantage of a primary
cell culture model with selective IFN-
responsiveness of the
intercellular adhesion molecule-1 (ICAM-1) gene (9, 10) in order to study the basis for
Stat1-dependent transcription. The results offer the first evidence
that Stat1-mediated transactivation depends on synergistic interaction
with another transcriptional activator (Sp1).
To investigate the basis for STAT-dependent transcription, we
used a primary culture epithelial cell (hTBEC) model that exhibits
IFN--dependent ICAM-1 expression under the control of an IRE at
nucleotides -130 to -94 of the ICAM-1 gene(10) .
This IRE contains an inverted repeat (at -116 to -106) that
is critical for forming an IRE-binding complex (IRE-BC) and for
IFN-
responsiveness of the ICAM-1 gene, and this inverted repeat
is similar to a motif found in several other IFN-
-responsive
genes(7, 20, 21) . In each case, the inverted
repeat motif is sufficient for binding phosphorylated
Stat1(11, 22, 23, 24, 25) ,
and this capacity is also exhibited by the inverted repeat in the
ICAM-1 gene in transformed cell lines (26, 27, 28) . DNA-protein cross-linking
experiments using the ICAM-1 gene inverted repeat and nuclear proteins
from IFN-
-stimulated hTBECs indicate that Stat1 directly contacts
the DNA at this site (Fig. 1).
Figure 1:
Activated Stat1
binds directly to the ICAM-1 gene inverted repeat. Nuclear proteins
from IFN--stimulated hTBECs (100 units of IFN-
/ml for 1 h)
were subjected directly to immunoblotting with anti-Stat1 Ab (lane
1) or were mixed with
P-labeled, BrdUrd-containing
ICAM-1 gene sequence (-120 to -98). The mixture underwent
nondenaturing 5% PAGE, and the
P-labeled band was excised,
treated with or without UV irradiation, and then subjected to 10%
SDS-PAGE and electrophoretic transfer to PVDF membranes for anti-Stat1
Ab immunoblotting (lanes 2 and 3) and
P
autoradiography (lanes 4 and 5). The slower migrating
Stat1 bands represent Stat1
and Stat1
covalently bound to the
ICAM-1 gene inverted repeat (designated Stat1
/IR and
Stat1
/IR), and the faster migrating bands represent unbound
Stat1
and Stat1
.
Although a series of
IFN--responsive genes share a capacity to bind Stat1, it is not
clear that Stat1 binding alone is sufficient for full responsiveness to
IFN-
. For example, deleting DNA elements surrounding the inverted
repeat leads to a marked decrease in the maximal level of
responsiveness to IFN-
that is often linked to a concomitant
decrease in (cytokine-independent) basal promoter activity (10, 23, 25, 27) . Basal activity
and IFN-
responsiveness are restored when the Stat1-binding site
is placed in front of a heterologous promoter(10) , but these
systems may include other DNA elements that could synergize with the
Stat1 site (as noted below). We were therefore interested in further
analyzing the types of DNA/protein and protein/protein interactions in
the ICAM-1 gene promoter region that serve to fully activate
IFN-
-driven transcription.
A distinct feature of the ICAM-1
gene IRE is the presence of a putative binding site (a GC box at
-99 to -94) for the Sp1 transcription factor (29) at the 3`-end of the element (Fig. 2A).
Accordingly, we performed gel mobility-shift assays using the ICAM-1
gene inverted repeat and the adjacent GC box. When the GC box and
adjoining sequence were included in the oligonucleotide probe, a second
more slowly migrating complex (designated IRE-BC) was
observed (Fig. 2B). This behavior fits with
observations that: (i) in general, the GC box alone is necessary but
not sufficient for Sp1-binding so that some less specific adjoining
sequence is also required(30) ; and (ii) for the ICAM-1 gene,
the footprint for Sp1 extends to nt -81 by DNase I protection
mapping. (
)Formation of IRE-BC
was decreased by
mutation of the GC box but not by mutation of the inverted repeat,
whereas formation of the original, faster migrating complex (designated
IRE-BC
) was decreased by inverted repeat but not by GC
mutation (Fig. 2C). In addition, IRE-BC
was
present in unstimulated and IFN-
-stimulated cells and was
supershifted with anti-Sp1 Ab, whereas IRE-BC
was present
only in IFN-
-stimulated cells and was reactive only with
anti-Stat1 Abs (Fig. 2, C and D). Thus, two
distinct sites in the ICAM-1 gene IRE (an inverted repeat at -116
to -106 and a GC box at -99 to -94) are responsible
for formation of two DNA-protein complexes: IRE-BC
, which
contains Stat1; and IRE-BC
, which contains Sp1.
Figure 2:
Sp1 binds 3`-adjacent to Stat1 in the
ICAM-1 gene IRE. A, design for wild-type (WT) and
mutant (MU) ICAM-1 gene sequences that were tested for their
capacity to bind nuclear proteins from unstimulated and
IFN--stimulated hTBECs in gel mobility-shift assays. B,
wild-type ICAM-1 sequences prepared as
P-labeled,
double-stranded oligonucleotides of varying lengths were used to define
binding sites mediating formation of two IRE-binding complexes
(IRE-BC
and IRE-BC
). Note that the GC box alone
is necessary but not sufficient for Sp1-binding (lanes 1 and 2 versus lanes 3-6) so that some less specific adjoining
sequence is also required. C, ICAM-1 sequence (-130 to
-81) without or with inverted repeat or GC box mutations were
used to define binding sites for Stat1 and Sp1. D, nuclear
protein extracts from unstimulated hTBEC monolayers were treated with
no antiserum (lane 1) or with Abs against Sp1 (lane
2), AP-2 (lane 3), Stat1 (lane 4), or nonimmune
IgG (lane 5) and then mixed with
P-labeled
oligonucleotides containing ICAM-1 gene sequence (-130 to
-81). Arrows indicate the positions of binding and
supershifted (*) complexes. E, nuclear protein extracts were
prepared from IFN-
-stimulated hTBEC monolayers and assayed for DNA
binding as in D.
The
functional significance of Sp1- and Stat1-binding sites for
IFN--responsiveness of the ICAM-1 gene was determined using a
series of ICAM-1/luciferase-reporter gene constructs in transient
transfection assays of unstimulated versus IFN-
-stimulated epithelial cells. Reporter constructs
contained either wild-type ICAM-1 gene 5`-flanking sequence or the same
sequence with mutation of the Stat1-binding inverted repeat (-116
to -106) or the Sp1-binding GC box (-99 to -94).
Mutations that blocked protein binding to either site also blocked
IFN-
responsiveness in transfection experiments using constructs
containing ICAM-1 gene sequence from -130 to -35 (i.e. using the ICAM-1 gene endogenous promoter, Fig. 3A) or ICAM-1 sequence from -130 to
-94 fused to a minimal promoter construct containing the Herpes simplex thymidine kinase gene promoter (i.e. using a heterologous promoter, Fig. 3B). Thus, a
functional Sp1 site (located close to the transcription initiation
site) was required for full IFN-
responsiveness.
Figure 3:
Stat1 and Sp1 sites are both required for
maximal IFN--responsiveness of the ICAM-1 gene. A, hTBECs
were transfected with a luciferase-reporter plasmid containing ICAM-1
gene sequence (-130 to -35) or the same sequence with
mutations in the inverted repeat (Stat1-binding site) or GC box
(Sp1-binding site). After transfection, cells were incubated without (open bars) or with (dark bars) IFN-
and then
assayed for luciferase activity. B, the same transfection
protocol was followed using constructs with ICAM-1 gene IRE sequence
(-130 to -94) with or without mutation of the inverted
repeat or GC box driving a heterologous minimal promoter (TK,
thymidine kinase). C, cells were transfected with reporter
plasmids containing ICAM-1 gene sequence (-130 to -35) or
the same sequence with the GC box replaced by Gal4-binding sites. Cells
were cotransfected with expression plasmids for Gal4 DNA-binding domain
alone (Gal4) or with this domain fused to the transactivating domain(s)
for Sp1 (Gal4/Sp1) or p65 (Gal4/p65). Results for Gal4/Fos and Gal4/E1a
chimeras were similar to those for Gal4/p65, and expression level of
each chimeric protein was verified by immunoblot against anti-Gal4 Ab
(data not shown). In A-C, each value is the average of
duplicate samples and is representative of three experiments; similar
values were obtained in experiments with pRSV-CAT cotransfection to
control for transfection efficiency (data not
shown).
To determine
whether the effect of Sp1 was specific or could be conferred by any
transcriptional activator, Sp1 and a series of activators were brought
to the promoter as fusion proteins containing a Gal4 DNA-binding
domain. Direct evidence that Sp1 is needed for full IFN-
responsiveness was obtained when co-transfection of a Gal4/Sp1 fusion
protein restored full responsiveness to a relatively inactive construct
containing ICAM-1 5`-flanking sequence with a Gal4-binding site in
place of the Sp1-binding site (Fig. 3C). Specificity
for Sp1 was indicated when its capacity to restore full responsiveness
was not shared with the transactivating domains of several other
transcription factors (including p65/RelA, Fos, or E1a), even though
each of these factors was capable of increasing basal promoter activity
to a similar level (Fig. 3C). Taken together, the
results indicate that both Stat1 and Sp1 binding are required for
maximal IFN-
activation of the ICAM-1 gene. The proximity of the
two binding sites and the selective effect of Sp1 also suggested a
specific interaction between the two transcription factors.
Accordingly, we next examined the capacity of Sp1 and Stat1 for
direct protein/protein interaction. Co-immunoprecipitation experiments
using either nuclear or whole cell extracts from unstimulated or
IFN--stimulated cells indicated direct Sp1/Stat1 interaction (Fig. 4, A and B). This interaction was
therefore not dependent on Stat1 phosphorylation state. Additional
immunoblotting indicated that hTBECs also contain Stat2, -3, -5, and
-6; however, only Stat3 appeared to directly interact with Sp1 (data
not shown). This finding correlates with a higher degree of homology
between Stat1 and Stat3 than among other STAT family
members(31, 32, 33, 34, 35, 36) .
Immunofluorescence microscopy demonstrated that Stat1 was localized to
the cytosol in unstimulated cells and to the nucleus in
IFN-
-stimulated cells, whereas Sp1 was found in the nucleus under
both conditions (data not shown). Predominant Sp1/Stat1 interaction
must therefore occur in the nucleus and is limited by nuclear
translocation of Stat1.
Figure 4:
Stat1 and Sp1 interactions in the control
of the ICAM-1 gene. A and B, whole cell protein
extracts from unstimulated and IFN--stimulated hTBEC monolayers
were immunoprecipitated with anti-Sp1 antibody and Protein A-Sepharose
and then subjected to SDS-PAGE and electrophoretic transfer to PVDF
membrane for immunoblotting against Abs to Stat1 (A) or Sp1 (B). Control Ab gave no detectable signal. C and D, model for DNA/protein and protein/protein interactions that
control transcription rate of the ICAM-1 gene under basal (C)
and IFN-
-stimulated conditions (D). Under basal
conditions, Sp1 binds to the GC box (at -99 to -94), but
under IFN-
-stimulated conditions, Stat1 (as a phosphorylated
homodimer) binds to the inverted repeat (at -116 to 106) and
interacts with Sp1 to cause maximal IFN-
-dependent gene
activation.
In summary, we find that maximal
IFN--driven activation of the ICAM-1 gene depends on two critical
DNA/protein interactions: 1) an inverted repeat motif that mediates
direct binding of activated/phosphorylated Stat1, and 2) a GC box motif
that recruits constitutively active Sp1. We also present evidence for
Stat1/Sp1 interaction that may serve to confer an additional element of
specificity for transcriptional activation (Fig. 4, C and D). In this model, Sp1 may help recruit Stat1 to the
promoter and/or may serve to better link Stat1 action to the basal
transcription complex. Interestingly, IFN-
-dependent activation of
a second immune response gene in airway epithelial cells, the
interferon regulatory factor-1 gene, is also Stat1-dependent
and also contains a GC box with a corresponding DNA footprint
downstream of the inverted repeat motif(20, 37) .
Thus, IFN-
-driven activation of an immune response subset may be
linked by a signal transduction pathway that includes transcriptional
synergy between Stat1 and Sp1. Single-cytokine activation of this
subset in the epithelial barrier provides an efficient molecular basis
for mediating mucosal immunity and inflammation.