(Received for publication, October 10, 1995; and in revised form, January 30, 1996)
From the
Interferon- (IFN-
)-induced signal transduction is
mediated by the phosphorylation-activation of the signal transducer and
activator of transcription (STAT) proteins Stat1, Stat2, and Stat3.
Previous studies have shown that these activated STATs dimerize to form
four distinct STAT complexes which translocate to the nucleus and
activates transcription by binding to specific promoter elements. The
interferon-stimulated gene factor-3 (ISGF3) consists of Stat2 and Stat1
heterodimers in association with a DNA-binding protein, p48, that binds
to the interferon stimulated response element. Homo- and heterodimers
of Stat1 and Stat3 bind to the palindromic interferon response element
(pIRE). In this report we demonstrate the utility of a biochemical
procedure that we have developed, based on genomic DNA affinity
chromatography, for the identification of IFN-
-induced STAT
complexes. Using this approach, we identified ISGF3-independent
Stat2-containing STAT complexes. Results from the analysis of Stat2
complexes in the electrophoretic mobility shift assay were consistent
with genomic DNA affinity chromatography results and identified a
Stat2:1 complex that binds with low affinity to the pIRE of the
interferon regulatory factor-1 gene. Immunoprecipitation studies of
Stat2 revealed an IFN-
dependent co-precipitation of both Stat1
and Stat3. Taken together, our results suggest that IFN-
activates, in addition to ISGF3, other Stat2-containing STAT complexes,
one of which binds to an element related to the interferon regulatory
factor-1 pIRE.
Interferons (IFNs) ()are secreted proteins engaged in
many diverse biological activities, including regulation of cell growth
and differentiation, inhibition of viral replication, and modulation of
the immune system(1) . IFNs target responsive cells by
interacting with distinct cell surface receptors. The high affinity
interaction between IFN-
/
and its specific cell surface
receptor leads to the activation of the receptor-associated cytoplasmic
tyrosine kinases of the Jak family, Jak1 and
Tyk2(2, 3) . This membrane proximal event correlates
with the activation of effector molecules leading to gene
activation(4) . These effectors are members of a family of
latent cytoplasmic transcription factors of the signal transducer and
activator of transcription (STAT) family(5, 6) ,
implicated in cytokine signal transduction(7) . STAT proteins
possess both Src homology-2 (SH2) and Src homology-3 (SH3)
domains(6) . STATs associate via their SH2 domains with
specific phosphotyrosine residues in the cytoplasmic domains of the
activated receptor components, leading to a Jak-dependent
phosphorylation/activation of receptor associated
STATs(8, 9, 10) . The STAT proteins then
dimerize via SH2-phosphotyrosyl interactions (11) and
translocate to the nucleus were they bind specific promoter sequences,
thereby regulating gene expression.
IFN- induces the
phosphorylation of the STAT proteins Stat1, Stat2, and
Stat3(12, 13) . To date, these activated STATs have
been shown to form four distinct STAT complexes. A STAT complex
designated the interferon-stimulated gene factor 3 (ISGF3) is formed
between Stat2 and Stat1 (Stat2:1) in association with a DNA-binding
adapter protein, p48, of the interferon regulatory factor (IRF) family
of
proteins(5, 14, 15, 16, 17, 18) .
ISGF3 transcriptionally activates a subset of interferon-stimulated
genes (ISGs) that contain an interferon-stimulated response element
(ISRE), with a consensus sequence AGTTTCNNTTTCNC/T(19) .
Additionally, homo- and heterodimers of Stat1 and Stat3 (Stat1:1,
Stat1:3, and Stat3:3) recognize an element designated the palindromic
interferon response element (pIRE), with a consensus sequence
TTC/ANNNG/TAA, regulating expression of a distinct subset of
ISGs(20, 21) . Studies with mutant cell lines that are
deficient in response to IFN-
indicate that alternative pathways
to regulate gene expression, via elements distinct from the previously
described ISRE or pIRE, may exist(22) .
To date, the
procedure to identify STAT complexes induced by IFN- was dependent
on the prior identification of the target DNA elements
involved(23, 24) . However, other IFN-
-induced
STAT complexes may form that target yet unidentified DNA elements. In
this report we describe a novel biochemical approach utilizing genomic
DNA affinity chromatography (GDAC) for the identification of
IFN-
-inducible STAT complexes. This technique permits the
identification of STAT complexes irrespective of the DNA recognition
elements involved. We show that this technique can be used to identify
the previously characterized IFN-
-inducible STAT complexes that
recognize both the ISRE of the 2`-5` oligoadenylate synthetase (OAS)
gene and the pIRE of the interferon regulatory factor-1 (IRF-1) gene.
Our studies also identified ISGF3-independent Stat2-containing STAT
complexes. Since we are able to demonstrate that an IFN-
-inducible
Stat2:1 complex binds with low affinity to the IRF-1 pIRE, in the
absence of p48, and Stat3 will co-precipitate with Stat2 following
IFN-
treatment of cells, we infer that multiple Stat2-containing
complexes are induced upon IFN-
treatment of responsive cells.
Figure 1:
Identification of IFN--induced
STAT complexes by GDAC. Actively growing U266 cells were incubated with
or without IFN-
in the presence of 10 mM NaF. Cytoplasmic
extracts were prepared and analyzed for DNA-binding STAT complexes
using GDAC in the presence of pIRE, ISRE, and mutant ISRE
oligodeoxynucleotides. Eluates from genomic DNA were resolved by
SDS-PAGE (7%) and analyzed by Western blotting. Blots were probed first
with anti-phosphotyrosine (Ptyr) antibody and then with
antibody to Stat2, Stat3, and finally Stat1. MWM, molecular
weight markers. IFN-
-induced phosphotyrosine containing proteins
and STAT proteins are indicated by lines and arrows,
respectively.
Our initial results suggested that GDAC can be used to
identify IFN--induced STATs, irrespective of the DNA recognition
elements involved. To date, IFN-
-induced STAT complexes have been
shown to recognize either the ISRE or the pIRE. Hence, addition of
excess DNA recognition element to the cytoplasmic extracts prior to
GDAC can be used to identify specific IFN-
-induced STAT complexes.
The addition of competing IRF-1 pIRE oligodeoxynucleotides prevented
Stat1, Stat3, and, to a lesser extent, Stat2 (see below) binding to
genomic DNA (Fig. 1). Stat1 and Stat3 have been shown to form
homo- and heterodimers that specifically recognize the
pIRE(12, 31) . Competition with 2`-5` OAS ISRE
oligodeoxynucleotides reduced Stat2 and Stat1, but not Stat3 binding to
genomic DNA. This is consistent, in part, with published results
indicating that the ISRE binding STAT complex, ISGF3, contains Stat1
and Stat2 but not Stat3(6) . The absence of complete
competition of Stat2 by the ISRE, however, suggests that Stat2 may form
a STAT complex(es) that binds DNA at a site(s) distinct from the ISRE.
As a control, we included mutant ISRE oligodeoxynucleotides as
competitor. This element did not compete for any of the STATs.
Figure 2:
An ISGF3-independent Stat2 complex(es) is
induced by IFN-. A, cytoplasmic extracts were prepared
from U266 cells incubated with or without IFN-
in the presence of
NaF. NaF treatment was omitted for the preparation of nuclear extracts.
These extracts were then analyzed by GDAC in the presence of ISRE
oligodeoxynucleotides. Eluates from genomic DNA were resolved by
SDS-PAGE (4%-12%) and after Western blotting probed first with
anti-Stat2 antibody and then with anti-p48 antisera. B,
cytoplasmic extracts of IFN-
-induced U266 cells were analyzed by
GDAC in the presence of ISRE and increasing amounts of pIRE
oligodeoxynucleotides. GDAC eluates were resolved by SDS-PAGE (7%) and
after Western blotting probed first with anti-Stat2 antibody and then
with antibody to Stat3 and Stat1. C, ISGF3-independent Stat2
DNA binding was examined for sensitivity to phosphotyrosine (PY), phosphothreonine (PT), and phosphoserine (PS). Phosphoamino acid-treated (30 mM) cytoplasmic
extracts of IFN-
-induced U266 cells were analyzed by GDAC in the
presence or absence of ISRE oligodeoxynucleotides. GDAC eluates were
analyzed as described in the legend for A.
Earlier studies suggested that Stat2
does not bind the pIRE(32) . However, we found that the
presence of excess IRF-1 pIRE oligodeoxynucleotides affected the
genomic DNA binding activity of Stat2 (Fig. 1). Therefore, we
performed a titration with the pIRE to determine the DNA binding
characteristics of the ISGF3-independent Stat2 complex(es). Cytoplasmic
extracts of IFN--treated U266 cells were analyzed using GDAC in
the presence of excess 2`-5` OAS ISRE and increasing amounts of IRF-1
pIRE oligodeoxynucleotides. The high salt eluates were examined for the
presence of the IFN-
-induced STAT proteins Stat1, Stat2, and
Stat3. Whereas genomic DNA binding of both Stat1 and Stat3 was largely
eliminated, Stat2 was only mildly affected by the addition of 150 ng of
pIRE and, indeed, remained bound to genomic DNA in the presence of 600
ng of pIRE oligodeoxynucleotides (Fig. 2B). These
results indicate that the ISGF3-independent Stat2 complex(es), unlike
homo- and heterodimers of Stat1 and Stat3, exhibits only a low affinity
for the pIRE.
DNA-binding STAT complexes contain dimers of STAT
proteins formed through SH2-phosphotyrosyl peptide
interactions(11, 17, 33) . These complexes
are sensitive to competition by soluble phosphotyrosine(31) .
To confirm that STAT dimers are required for ISGF3-independent Stat2
complex formation, we analyzed extracts of IFN--treated U266
cells, using GDAC in the presence of ISRE oligodeoxynucleotides, for
sensitivity to phosphotyrosine. The results shown in Fig. 2C demonstrate that the presence of phosphotyrosine abrogates the DNA
binding activity of the ISGF3-independent Stat2 complex(es), whereas
phosphothreonine or phosphoserine had no effect.
ISGF3-independent
Stat2 complex(es), as assayed in GDAC, exhibit a relatively low
affinity for the IRF-1 pIRE (see Fig. 2B). These
results suggest that IFN--induced Stat2 complexes may bind to the
pIRE under specific conditions that would allow detection in the EMSA.
In order to optimize detection of Stat2 in complex with the pIRE, we
incubated IFN-
-treated cytoplasmic extracts with a large amount
(up to 50 ng) of IRF-1 pIRE. Following EMSA, complexes were detected by
immunoblotting with antibodies to Stat2. A pIRE-dependent mobility
shift complex, that migrated with a higher mobility than the ISGF3-ISRE
complex, was detected in extracts of IFN-
-treated cells only when
>5 ng of pIRE probe was used (Fig. 3A, upper panel).
Apparently, the Stat2-pIRE complex forms with relatively low affinity,
since we were able to detect the ISGF3-ISRE complex using as little as
0.5 ng of ISRE probe. Immunoblotting with antibodies to p48 revealed
the association of p48 with the ISRE-Stat2 complex and not the
pIRE-Stat2 complex (Fig. 3B, lower panel). This
ISGF3-independent Stat2 complex was not found in extracts of untreated
cells, nor did it form when IFN-
-treated cell extracts were
incubated with a mutant 2`-5` OAS ISRE as probe (Fig. 3B). Addition of antibodies against Stat1 and
Stat2 to the protein-DNA reaction mixture affected the mobility of the
pIRE-Stat2 complex in the native gel. Antibodies to Stat3, however, had
no effect (Fig. 3B). These results indicate that a
Stat2:1 complex can bind, albeit with low affinity, to the IRF-1 pIRE
and confirm our results with GDAC.
Figure 3:
The ISGF3-independent Stat2:1 complex
exhibits pIRE binding activity in EMSA. A, cytoplasmic
extracts of IFN--treated U266 cells were incubated with increasing
amounts of IRF-1 pIRE or 2`-5` OAS ISRE oligodeoxynucleotides. pIRE-
and ISRE-bound Stat2-containing STAT complexes were identified in the
EMSA by immunoblotting with antibodies to Stat2 (upper panel).
In an identical experiment p48-ISRE complexes were detected with
antibodies to p48 (lower panel). Protein-DNA complexes are
indicated by arrows. B, cytoplasmic extracts of
untreated or IFN-
-treated U266 cells were incubated with or
without polyclonal antisera to Stat1, Stat2, Stat3, or preimmune sera,
followed by the addition of pIRE or mutant ISRE oligodeoxynucleotides
(50 ng). pIRE bound Stat2 containing STAT complexes were identified as
described in the legend for A. Nonsupershifted protein-DNA
complexes are indicated by the arrow.
Figure 4:
IFN- induces a Stat2:3 complex. A, lysates from U266 cells, either left untreated or treated
with IFN-
, were immunoprecipitated with polyclonal antisera to
Stat2 or preimmune antisera. Immunoprecipitates were resolved by
SDS-PAGE (7%) and after Western blotting probed first with anti-Stat3
antibody, then with antibody to Stat1, and finally antibody to Stat2. B, extracts were prepared from U266 cells treated with
IFN-
and immediately reacted with phosphotyrosine (PY),
phosphothreonine (PT), or phosphoserine (PS). The
anti-Stat2 immunoprecipitates were resolved by SDS-PAGE (7%) and after
Western blotting probed first with anti-Stat3 antibody and then with
antibody to Stat2.
Using GDAC we performed competition
experiments with oligodeoxynucleotides corresponding to the pIRE of the
IRF-1 gene and the ISRE of the 2`-5` OAS gene to identify
IFN--induced STAT complexes (Fig. 1). Our studies revealed
a Stat2-containing STAT complex(es) distinct from those identified
previously. This complex(es) is capable of binding genomic DNA in the
presence of excess ISRE (Fig. 2A). Competition
experiments with the ISRE indicated that the ratio of ISGF3 to total
DNA-binding Stat2 is lower in the nucleus than the cytoplasm (Fig. 2A). As p48 is a component of ISGF3 and is
required for DNA binding activity, this result likely reflects a lower
ratio of p48 to total DNA-binding Stat2 in the nucleus compared with
the cytoplasm. An excess of IRF-1 pIRE (600 ng) partially competed the
DNA binding activity of the ISGF3-independent Stat2 complex(es) (Fig. 2B), suggesting that this complex(es) can bind to
this element. However, as the DNA binding activity of both Stat1 and
Stat3 was largely eliminated by excess pIRE, these Stat2- containing
complex(es) likely bind with relatively low affinity. Nevertheless,
following prolonged autoradiographic exposure, residual low levels of
DNA binding Stat1 and Stat3 were apparent in the IFN-
-inducible
cell extracts from which ISRE- and pIRE-binding complexes had been
depleted (data not shown). The implications are that the
ISGF3-independent Stat2 complex(es) may require Stat1 and/or Stat3 for
DNA binding. The results in Fig. 2C reveal that these
complex(es) bind DNA as a dimer. Consistent with the observed low
affinity binding of Stat2 complex(es) to the pIRE in GDAC (Fig. 2B), low affinity, pIRE-binding Stat2:1
heterodimers were detected in EMSA (Fig. 3, A and B). Immunoprecipitation experiments identified an
IFN-
-inducible Stat2:1 dimer and a novel Stat2:3 dimer (Fig. 4). Moreover, in vitro studies involving
tyrosine-phosphorylated Stat2 purified from baculovirus infected insect
cells, identified Stat2 homodimerization. (
)Taken together,
these data suggest that the ISGF3-independent Stat2 complexes
identified using GDAC likely contain the STAT dimers Stat2:1, Stat2:2,
and Stat2:3. However, in the mobility shift assay we detected only a
Stat2:1 heterodimer in complex with the pIRE. The absence of both
Stat2:2 and Stat2:3 complexes may be a consequence of a lower affinity
and/or abundance of these complexes compared with the Stat2:1 complex.
Alternatively, Stat2:2 and Stat2:3 complexes may bind to DNA elements
distinct from the consensus ISRE and pIREs. We propose that one or more
high affinity DNA recognition elements for the ISGF3-independent Stat2
complexes exist that are related to the IRF-1 pIRE.
Recent evidence
suggests that Stat2, within the complex of ISGF3, does not contact DNA
directly. Instead it participates in the DNA binding activity of ISGF3
by interacting with the DNA-binding protein p48(17) .
Originally we hypothesized that the ISGF3-independent Stat2 complex(es)
may also require a p48-like adapter protein for DNA binding. The DNA
binding activity of ISGF3 is sensitive to alkylation by N-ethylmaleimide (NEM)(30) . The addition of extracts
from untreated cells to NEM treated extracts of IFN--induced
cells, restores ISGF3, suggesting that only p48 is sensitive to NEM. We
found that, although the ISGF3-independent Stat2 complex(es) was
sensitive to NEM treatment, the DNA binding activity could not be
restored by the addition of extracts from untreated cells (data not
shown). Given the relatively high frequency of cysteine residues within
STAT proteins, these results are not surprising; the conditions we
employed for NEM treatment promote alkylation of cysteine residues,
thus inactivation of STAT dimer binding to either an adapter protein,
or directly to DNA, could be expected. This NEM sensitivity of STAT
dimer(s) precludes the use of this assay to predict the presence of a
p48-like protein(s) within STAT complex(es).
In this report, we
demonstrate for the first time Stat2 DNA binding activity independent
of ISGF3. We propose that these Stat2-containing STAT dimers regulate
transcription by interacting with an element(s) related to the IRF-1
pIRE that is present in the promoter region of specific ISGs.
Identification of this promoter element(s) will shed light on the role
of these complexes in IFN- signal transduction.