(Received for publication, June 20, 1996, and in revised form, November 22, 1996)
From the Department of Pathology and Kaplan Cancer Center, New York University School of Medicine, New York, New York 10016
Transcriptional responses to interferon (IFN) are
mediated by tyrosine phosphorylation and nuclear translocation of
transcription factors of the signal transducer and activator of
transcription (Stat) family. The Stat1 protein is required for all
transcriptional responses to IFN (both type I and type II). Responses
to type I IFN ( and
) also require Stat2 and the IFN regulatory
factor family protein p48, which form a heterotrimeric transcription complex with Stat1 termed ISGF3. Stat1 homodimers formed in response to
IFN-
treatment can also interact with p48 and function as transcriptional activators. We now show that Stat2 is capable of
forming a stable homodimer that interacts with p48, can be recruited to
DNA, and can activate transcription, raising a question of why Stat1 is
required. Analysis of the transcriptional competence, affinity, and
specificity of Stat2-p48 complexes compared with other Stat
protein-containing transcription factor complexes suggests distinct
roles for each component. Although Stat2 is a potent transactivator, it
does not interact stably with DNA in complex with p48 alone. Adding
Stat1 increases the affinity and alters the sequence selectivity of
p48-DNA interactions by contacting a half-site of its palindromic
recognition motif adjacent to a p48 interaction sequence. Thus, ISGF3
assembly involves p48 functioning as an adaptor protein to recruit
Stat1 and Stat2 to an IFN-
-stimulated response element, Stat2
contributes a potent transactivation domain but is unable to directly
contact DNA, while Stat1 stabilizes the heteromeric complex by
contacting DNA directly.
Interferon- (IFN-
)1 binding to
its receptor leads to the activation of a multiprotein DNA-binding
complex called IFN-stimulated gene factor 3 (ISGF3). Purification of
its component proteins led to the identification (1, 2) and cloning of
the first two members of a novel family of signal transducers and
activators of transcription (Stats), Stat1 (3) and Stat2 (4), and the DNA-binding protein ISGF3
p48 (5). The Stat1 and Stat2 proteins are
present in the cytoplasm of untreated cells; upon stimulation with
IFN-
, they become rapidly activated by tyrosine phosphorylation at a
single site (6) catalyzed by receptor associated Jak (Janus) kinases
(7-10). This activation subsequently allows them to assemble into
stable homo- and heteromeric complexes through specific SH2 domain-phosphotyrosyl interactions (11) and translocate to the nucleus
where they bind specific enhancer elements in the promoters of
IFN-inducible genes. Interestingly, Stat1 (but not Stat2) is also
activated by tyrosine phosphorylation in response to a number of other
cytokines (12-14), including IFN-
(15; for review, see Refs. 16 and
17).
ISGF3 p48 (p48) is unrelated to Stat proteins but is a member of the
IFN regulatory factor family (18) of DNA-binding proteins that
recognize the positive regulatory domain I (PRDI) of the IFN-
gene
(19, 20) and the IFN-
-stimulated response element (ISRE) of
IFN-
-stimulated genes (5, 21). p48 binds the ISRE more efficiently
than it binds PRDI (22), and it is required for many transcriptional
responses of IFN-
target genes containing ISRE enhancer sequences
(23, 24). By itself p48 has a low affinity for the ISRE sequence and no
transcriptional activity, but complexed with the Stat1-Stat2
heterodimer that is formed upon IFN-
treatment, it forms a
significantly more stable protein-DNA complex (5, 21, 22) capable of
transactivating gene expression. Sequence specific recognition by ISGF3
has been shown to involve multiple protein-DNA contacts by both the
Stat proteins and p48 (22, 25).
Phosphorylated Stat1 homodimers are formed following treatment with
either IFN- or IFN-
(26). As a dimer (known as gamma-activated factor, or GAF) Stat1 binds directly to an enhancer element called gamma-activated site, or GAS (27) and activates transcription of target
genes containing this site in their promoters. A major difference
between transcription factor binding at the GAS and the ISRE sequences
is the presence of a p48 recognition element in the ISRE which allows
p48 binding and thus Stat protein recruitment to this distinct site. As
might be predicted from the sequence similarity between Stat1 and
Stat2, it was found that Stat1 homodimers, in addition to Stat1-Stat2
heterodimers, also interact with p48 and bind the ISRE in response to
IFN-
treatment (28). This Stat1-p48 complex binds the ISRE with a
recognition specificity similar to ISGF3. Together the three distinct
multimeric complexes activated by IFN-
and IFN-
(ISGF3, GAF, and
Stat1-p48) contribute to the characteristic patterns of gene
transcription induced by these cytokines (18).
Mutagenesis of Stat2 suggested that this protein contributes the
transactivation function of ISGF3 (29), prompting us to question the
role for Stat1 in ISGF3. We found that Stat2, like other Stat proteins,
is capable of forming a stable homodimer when phosphorylated in
response to IFN-. In conjunction with p48, Stat2 homodimers are
capable of activating transcription from ISRE-containing genes in the
absence of Stat1. However, Stat2-p48-DNA complexes are very unstable,
only forming under conditions where these proteins are abundant. The
increased affinity of ISGF3 over Stat2-p48 appears to be caused by
additional sequence-specific DNA contacts contributed by Stat1 adjacent
to the primary p48 binding sequence. Stat2, in contrast, is incapable
of directly contacting the ISRE sequence.
Cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 5% calf serum.
Human fibrosarcoma 2fTGH cells and U3A mutant cells, kindly provided by
G. Stark and I. Kerr, have been described previously (30-32).
Transfection of U3A cells and selection of stable cell lines were
carried out by standard procedures. Transformed human embryonic kidney
293 cells (33) expressing simian virus 40 large tumor antigen (293T
cells) were a gift from R. Schneider (New York University). Cells were
treated with recombinant human IFN--2a (Hoffmann-La Roche) or
IFN-
(Boehringer Mannheim) at 500 units/ml.
Total RNA from U3A, U3A-Stat1, or
U3A-Stat2-p48 cells lysed in guanidinium thiocyanate solution was
isolated by standard methods (34). IFN-stimulated gene (ISG54) RNA was
quantified by RNase protection (35) following hybridization to a
250-base pair radiolabeled cRNA probe from exon 2 of the human gene and
digestion with RNase T2, as described previously (36). ISG54 induction
was quantified by PhosphorImager analysis (Molecular Dynamics),
normalizing RNA loading to the constitutively expressed -actin
mRNA (37). Stat2 and GAPDH RNA were quantified by reverse
transcription-polymerase chain reaction according to standard methods
(38). Primers were designed which distinguished between mRNA from
endogenous Stat2 or the transfected gene. Standard amplification
conditions involved 30 cycles of denaturation for 1 min at 94 °C,
annealing for 1 min at 55 °C, and extension for 2 min at 72 °C.
Specific primers used for reverse transcription-polymerase chain
reaction were: Stat2(5
) GCGGATCCCTGAGCCAATGGAAATCT; Stat2(3
)
TGGCTCAGCATCTGTTCT; GAPDH(5
) ACCACAGTCCATGCCATCAC; GAPDH(3
)
5
-TCCACCACCCTGTTGCTGTA-3
.
Full-length cDNA expression
constructs for ISGF3 p48 (5), Stat2 (4), Stat1
(3), and Jak1 (7)
were driven by the cytomegalovirus promoter (39). To identify Stat2
homodimer interactions, constructs described previously (29) and kindly provided by S. Qureshi and J. E. Darnell, Jr., expressing either full
length Stat2 incorporating HA influenza epitope 12CA5 (40), a
carboxyl-terminally truncated version missing amino acids
carboxyl-terminal to residue 752 (
Stat2), or epitope-tagged Stat2
containing a mutation of tyrosine 690 to phenylalanine (Stat2(F)) were
expressed and analyzed by immunoprecipitation. An IFN-responsive
reporter gene (pISG54-LUC) was constructed by incorporating a fragment of the hamster ISG54 promoter from
429 to +31 (41) fused to the
luciferase gene.
U3A, U3A-Stat2,
U3A-p48, or U3A-Stat2-p48 cells were seeded at 5 × 105 cells/6-cm dish. The next day, cells were transfected
with the ISG54-LUC reporter gene construct (1 µg) alone or together
with expression plasmids directing the synthesis of Stat1, Stat2, or p48, using the calcium phosphate precipitation method (42). After
overnight incubation with the precipitate, incubation was continued in
the absence or presence of human IFN--2a (500 units/ml) for an
additional 6 h. Luciferase activity in cell extracts was determined according to the Luciferase Assay System (Promega).
Immunoprecipitation and immunoblotting were performed as described previously (7) using the influenza-specific monoclonal antibody 12CA5 (40), Stat2-specific rabbit serum (3) (kindly provided by Chris Schindler), and rabbit anti-human p48 (5).
Electrophoretic Mobility Shift Assays (EMSAs)Cytoplasmic
and nuclear protein extracts were prepared from transiently transfected
293T cells, or transiently or stably transfected U3A cells, and assayed
for ISRE binding activity using the high affinity ISG15 ISRE sequence,
as described previously (22). Cells in 6- or 10-cm dishes were
transfected with expression plasmids directing the production of p48,
Stat1, Stat2, Stat2-HA, Stat2, or Stat2(F) (2.5 µg).
Ligand-independent activation of the different Stat2 proteins was
achieved by cotransfection with 0.4 µg of an expression plasmid for
the Jak1 tyrosine kinase. Antibodies against p48, Stat1, Stat2, Stat3,
and HA (5, 6, 40, 43) were used at a dilution of 1:200. ISG15 ISRE
(44), a high affinity binding site for ISGF3 complexes in
vitro (22), PRDI from the IFN-
promoter (45) which fails to
bind ISGF3 (21), or a synthetic high affinity GAS sequence that binds
Stat1 (12) were used as competitors at 20-fold molar excess. To
determine the sequence recognition specificity of the Stat2-Stat2-p48,
Stat2-Stat1-p48, and Stat1-Stat1-p48 complexes, binding was tested to
wild type (GGCTTCAGTTTCGGTTTCCCTTTCCCGAGGATC) and point-mutated
(T89: GGCTTCAGTTTCGGTTT
CCTTTCCCGAGGATC and
T101: GGCTT
AGTTTCGGTTTCCCTTTCCCGAGGATC)
ISRE probes (22) labeled to equal specific activities.
Dissociation rates of protein-DNA complexes were determined essentially as described (1), using cytoplasmic extracts containing DNA-binding complexes composed of p48, Stat2-p48, or Stat2-Stat1-p48 (ISGF3). The approximate half-life of each complex was determined by quantitative PhosphorImager analysis.
In response to
IFN-, tyrosine phosphorylated Stat1 and Stat2 multimerize to form
either Stat1 homodimers that bind GAS sequences through an intrinsic
DNA binding domain (46) or Stat1-Stat2 heterodimers that bind ISRE
sequences through the DNA binding domain of p48 (22). To explore the
possibility that Stat2 may homodimerize in a manner analogous to
phosphorylated Stat1, we first examined the ability of Stat2 to bind
ISRE sequences in the presence of p48 (Fig.
1A). Tyrosine phosphorylated Stat2 and p48 in
extracts from transfected 293T cells were assayed for protein-DNA interaction by gel mobility shift using an ISRE probe. Cell extracts containing activated Stat2 (lane 1) or p48 (lane
2) displayed no ISGF3-like ISRE binding activity. However, mixing
protein extracts containing activated Stat2 with extracts containing
p48 produced a slowly migrating complex (lane 3). This
complex bound antibodies specific for p48 (lane 4) or Stat2
(lane 5), whereas antibodies against Stat1 (lane
6) or Stat3 (lane 7) had no effect, showing that
endogenous Stat1 protein did not participate in formation of this
complex. The sequence recognition specificity of the complex was tested
by competition assay. A 20-fold molar excess of unlabeled ISRE
abrogated all binding to the probe (lane 8). In contrast, unlabeled PRDI (an ISRE-related sequence oligonucleotide; lane 9) or GAS (lane 10) competed poorly or failed to
compete. These results indicated that Stat2 binds DNA in the presence
of p48 with a specificity similar to ISGF3.
Phosphorylated Stat2 Can Form Homodimers
Formation of an
ISGF3-like complex composed of Stat2 and p48 suggested that Stat2 might
homodimerize, analogous to Stat1. This possibility was tested by
coexpressing full-length and truncated, recombinant Stat2 (Fig.
1B). Full-length Stat2 tagged with an HA epitope (Stat2-HA)
and a shorter, untagged form (Stat2) that lacks the
carboxyl-terminal 100 amino acids were expressed in 293T cells.
Extracts containing Stat2-HA (lane 1),
Stat2 (lane 2), or Stat2-HA and
Stat2 together (lane 3) showed
equal expression levels for the different proteins. A smaller protein,
apparently a proteolytic product of recombinant Stat2-HA lacking the
carboxyl terminus and epitope tag, was also detected (lanes
1 and 3). Endogenous Stat2, which is expressed in 293T
cells at much lower levels than recombinant protein, was not detected
at this exposure. When these extracts were precipitated with a
monoclonal antibody against the HA epitope followed by Western blotting
using the Stat2 antibody (lanes 4-6), untagged
Stat2 was
recovered from extracts of cells where it was coexpressed with Stat2-HA
(lane 6), but not from extracts containing Stat2-HA (lane 4)
or
Stat2 (lane 5) alone. Efficient interaction between
Stat2-HA and
Stat2 required tyrosine phosphorylation, as
indicated by a need for Jak1 kinase and by a lack of interaction with a
Stat2 mutant in which tyrosine 690 was replaced by phenylalanine (data
not shown).
The same cell extracts were also assayed by gel shift (Fig.
1C). Extracts containing phosphorylated Stat2-HA, mixed with
extracts from p48-transfected cells, displayed a slowly migrating
complex (lane 1), which was supershifted by antibodies
against HA (lane 2) or Stat2 (lane 3). Under the
same conditions, extracts containing Stat2 displayed a faster
migrating complex (lane 4) which was only disrupted by
Stat2-specific antibody (lane 6), but not by antibody
against HA (lane 5). Both complexes, together with a new
complex of intermediate mobility, were observed when extracts from
cells coexpressing Stat2-HA and
Stat2 were mixed with extracts containing p48 (lane 7). Antibodies against Stat2 disrupted
all three complexes (lane 9), whereas the antibody specific
for HA supershifted only the slower two complexes (lane 8).
These results confirmed that the slower mobility complex contained
Stat2-HA homodimers, the faster complex contained
Stat2 homodimers,
and the intermediate complex consisted of Stat2-HA-
Stat2
heterodimers. Overall, these data demonstrated that activated Stat2
formed homodimers that bind the ISRE when complexed with p48.
Formation of the Stat2-Stat2 gel shift complexes, like the
interactions detected by immunoprecipitation (Fig. 1B),
depended on tyrosine phosphorylation of Stat2. Extracts containing a
mutant Stat2 in which tyrosine 690 was changed to phenylalanine
(lanes 10 and 11) no longer displayed ISRE
binding activity when mixed with p48-containing extracts. Similarly,
extracts from cells coexpressing Stat2(F) and
Stat2 only displayed
the faster migrating complex (lane 11) consisting of
Stat2 homodimers. Formation of all of these complexes required the
presence of an active tyrosine kinase (not shown).
IFN-unresponsive U3A
cells lack Stat1 protein (32), and extracts of U3A cells displayed no
IFN-inducible ISRE binding activity when assayed by gel shift (Fig.
2A, lanes 1 and 2). The
same was true for U3A cells transiently transfected with an expression plasmid for Stat2 (lanes 3 and 4, respectively),
whereas cells transfected with a p48 expression plasmid displayed a
rapidly migrating complex characteristic of p48, present in untreated (lane 5) as well as IFN-treated cells (lane 6).
Interestingly, cells cotransfected with Stat2 and p48 plasmids
(lanes 7-14) displayed a novel, slowly migrating complex
inducible by IFN- (lane 8). Antibody reactivity
identified the composition of this complex. The slow mobility complex
as well as the p48 complex were supershifted by antibodies specific for
p48 (lane 11). Likewise, the slowly migrating complex, but
not the p48 complex, was disrupted by antibodies against Stat2
(lane 13). In contrast, antibodies against Stat1 (lane
12) or Stat3 (lane 14) had no effect on either complex, indicating
that a Stat2-p48-DNA complex formed in response to IFN-
.
The transcriptional potency of the Stat2-p48 complex was tested on an
ISRE-reporter construct. U3A cells were transfected with the
IFN--responsive reporter construct ISG54-LUC alone or together with
expression plasmids directing the synthesis of Stat2 or p48 (Fig.
2B). U3A cells failed to express ISG54-LUC in response to
IFN-
, nor did U3A cells transiently transfected with plasmids for
Stat2 or p48 alone show a significant response to IFN-
. However, cotransfection of all three plasmids (ISG54-LUC, Stat2, and p48) resulted in a 5-fold induction of luciferase activity in response to
IFN-
, a response that depended on the presence of the
carboxyl-terminal transactivation domain of Stat2. We conclude that the
Stat2-p48 complex can activate transcription in response to IFN-
provided that sufficient quantities of Stat2 and p48 proteins are
present. The level of transactivation was similar to that observed
following transfection of U3A cells with Stat1 cDNA (28).
The ability of Stat2-p48 complexes to
activate endogenous gene expression was examined. U3A cell lines were
generated which stably expressed Stat2 and p48 (indicated as U3A-Stat2,
U3A-p48, and U3A-Stat2-p48). As control, U3A cells were complemented by stable expression of Stat1 (U3A-Stat1). Similar to transiently transfected U3A cells (Fig. 2A), extracts from U3A-Stat2-p48
cells displayed an IFN-dependent, ISGF3-like protein-DNA
complex (Fig. 3A, lane 8). In
contrast, U3A cells transfected with either construct alone failed to
form this complex (lanes 3-6). Whereas U3A cells expressed
only minute amounts of endogenous Stat2 and p48 and these proteins
failed to accumulate in response to IFN- treatment, U3A-Stat2-p48
cells expressed levels of these proteins approximately equal to those
observed in U3A-Stat1 cells treated with IFN-
for 24 h (Fig.
3B). Expression of the endogenous ISG54 gene was induced in
response to IFN-
treatment in U3A-Stat2-p48 cells, but the level of
expression was approximately 30-fold lower, as judged by PhosphorImager
analysis, than in IFN-
-treated U3A-Stat1 cells (Fig. 3C,
compare lanes 2 and 4).
Similarly low levels of induction were observed for the endogenous
Stat2 gene (Fig. 3D). Polymerase chain reaction primers were
designed which allowed expression of endogenous Stat2 mRNA to be
distinguished from expression of the transfected gene. Expression of
endogenous Stat2 was 6-fold inducible in U3A-Stat1 cells in response to
IFN-, whereas it was induced only approximately 2-fold in
U3A-Stat2-p48 cells (compare lanes 2 and 4).
Thus, although Stat2 is a potent transactivator when overexpressed by
transient transfection where small numbers of transfected cells
probably express high levels of Stat2 and p48, the Stat2-p48 complex is a poor transactivator when expressed at physiological levels.
To explore possible
reasons for the poor transcriptional activity observed for Stat2-p48
complexes, the stability of the Stat2-p48 and ISGF3 protein-DNA
complexes was determined from dissociation rate measurements (22).
Stat2-p48, ISGF3, and p48 complexes bound to DNA were challenged with a
500-fold molar excess of unlabeled ISRE oligonucleotide. At serial time
points after addition of competitor, aliquots were removed and loaded
directly onto a running polyacrylamide gel (Fig.
4A). Quantitation of the decay of each complex over time by PhosphorImager analysis indicated that
dissociation of the ISGF3-ISRE complex was slow, displaying a half-life
between 10 and 30 min. In contrast, the Stat2-p48-ISRE interaction was much less stable, with a half-life of <1 min, similar to the short half-life observed for p48 bound to the ISRE alone (22). These results
suggest that the presence of Stat1 greatly increases the low intrinsic
affinity of p48 for DNA, whereas Stat2 homodimers are unable to
stabilize this interaction. This difference in affinity of >20-fold is
consistent with the approximately 30-fold lower levels of induction of
endogenous ISG54 gene expression in Stat2-p48 cells (Fig.
3C).
The increased affinity of ISGF3 for DNA over that of p48 alone
correlates with an altered sequence specificity (22). p48 binds a
9-nucleotide core element, whereas ISGF3 makes additional base-specific
contacts extending beyond the core sequence. The contribution of Stat1
and Stat2 to this altered binding specificity was determined by gel
shift using oligonucleotide probes with altered ISRE sequences (Fig.
4B). Complex binding to wild type sequences was compared
with binding to oligonucleotides containing either a C89T or C101T
mutation. These mutations were chosen to disrupt two potential GAS
half-sites (TTC) flanking the core ISRE element that could be contact
points for Stat proteins (47). As shown previously (22), ISGF3 failed
to bind the T89 oligonucleotide (lanes 1-3), whereas
Stat2-p48 was capable of binding all three sequence elements (compare
lanes 4-6). This binding specificity is similar to that
defined for p48 alone (22), indicating that the higher affinity and
altered recognition specificity of ISGF3 result from additional
protein/nucleotide contacts contributed by Stat1 but not by Stat2.
Additional evidence for this idea came from examining ISRE binding
characteristics of Stat1-p48 complexes that form in response to IFN-
(28). Both the C89T and the C101T mutations prevented Stat1-p48
complexes from binding DNA (lanes 7-9), indicating that
Stat1 either directly or indirectly contacts DNA to alter the stability
and specificity of p48 binding.
Both IFN- and IFN-
activate transcription by inducing the
tyrosine phosphorylation of Stats. Once phosphorylated, these proteins
can form a number of distinct transcription factor complexes. These
include Stat1 homodimers that bind GAS sequences in response to either
IFN type, Stat1 homodimers that bind ISRE sites in conjunction with
p48, predominantly in response to IFN-
, and Stat1-Stat2 heterodimers
that bind the ISRE in conjunction with p48 (ISGF3). Since Stat2 is only
phosphorylated in response to IFN-
, this complex only forms in
IFN-
-treated cells. We now show that another p48-containing complex
can form in IFN-
-treated cells, composed of Stat2 homodimers without
Stat1. However, this complex displayed only limited affinity for
DNA.
Analysis of the different Stat complexes capable of interacting with p48 suggests a model for transcription factor assembly. The heteromeric ISGF3 complex composed of Stat1, Stat2 and p48 binds a composite DNA sequence defined by a p48 recognition element plus a flanking sequence not contacted by p48 directly. This flanking sequence resembles a half-site of the GAS element recognized by Stat1 homodimers, which can be considered a spaced palindrome of two TTC half-sites (47). The capability to bind spaced palindromic half-sites is inherent in Stat1 dimers, presumably due to a DNA binding domain created by juxtaposition of protein regions between amino acids 400 and 500 (46, 48). Therefore, it would seem likely that DNA binding by ISGF3 relies on the same Stat1 domain, but in this case dimerized with an analogous region of Stat2. Although Stat dimers are incapable of binding to a single half-site, a low affinity interaction at a half-site would be stabilized through protein-protein contacts with DNA-bound p48. Additional evidence for Stat1-Stat2 dimers being capable of forming a functional DNA binding domain has been provided recently by Li et al. (49), who showed that such dimers could bind a palindromic GAS sequence. These Stat1-Stat2 complexes bind a DNA sequence also recognized by Stat1 homodimers, indicating that Stat2 does not affect the specificity of Stat1, probably because it fails to contribute directly to DNA binding. Likewise, analysis of ISGF3 formation by protein-DNA cross-linking failed to detect stable interaction between Stat2 and DNA (25).
Each of the three polypeptides comprising ISGF3 appears to provide a
distinct function. p48 contributes most of the DNA binding specificity
by recognizing the core sequence of the ISRE (22). Stat2 contains a
transactivation domain that is essential for transcriptional activity
of ISGF3 (29). Stat1, which also contains a transactivation domain that
functions in Stat1 homodimer complexes (32), does not appear to
contribute significantly to the transcriptional potency of ISGF3 (29).
Rather, Stat1 contributes necessary contacts with DNA which raise the
affinity of ISGF3 for DNA above a minimal threshold provided by p48
alone. Indeed, the Stat1 isoform, which lacks the transactivation
domain used by Stat1 homodimers, is capable of stabilizing ISGF3
formation (32).
Further evidence that the Stat1 DNA binding domain is involved in
interactions at the ISRE was obtained from analysis of the recognition
specificity of the ISGF3-like complex composed of Stat1 homodimers and
p48. Again, GAS-like half-sites appear involved in stabilizing the
protein-DNA interaction. Interestingly, the Stat1-p48 complex contacted
two TTC half-sites on either side of the p48 recognition sequence,
unlike ISGF3 where Stat1 interacted with only the 3 TTC. It is
possible that the presence of Stat1 homodimers in this complex forms a
symmetric complex, whereas Stat1-Stat2 heterodimers form an asymmetric
complex that restricts Stat1 binding to a single side of p48. Similar
asymmetric interactions of heterodimeric transcription factors have
been reported for NF-AT·AP-1 complexes on the interleukin-2 receptor
enhancer (50).
All Stat proteins, with the exception of Stat2, appear to bind palindromic DNA sequences as homodimers. It is possible that Stat2 homodimers also could bind DNA on their own. Indeed, Stat2-containing complexes have been detected which bind genomic DNA, but neither the nature of the complex nor the sequence of the DNA has been characterized (51). Our data would argue that, even if Stat2 is capable of binding DNA, it is specific for a distinct sequence other than the ISRE. The presence of Stat2 in an ISGF3 complex did not appear to add nucleotide contacts to those provided by Stat1. Also, Stat2 homodimers bound to DNA in conjunction with p48 appeared to have a DNA binding specificity identical to that of p48 alone. The presence of Stat2 did not increase the affinity for DNA, again suggesting that Stat2 was not in contact with DNA. On the other hand, it is entirely possible that Stat2 homodimers are capable of binding to a DNA sequence distinct from either the ISRE or the GAS elements.
The biological significance of Stat2-p48 complexes is difficult to
assess. Although this transcription factor is capable of being formed
in response to IFN-, its low DNA binding affinity for an ISRE
sequence precludes transcriptional responses at modest protein
concentrations. Thus, U3A cells that lack Stat1 but express Stat2 do
not detectably express ISRE-containing genes (32). Similarly, we and
others have produced mouse strains lacking Stat1, and these animals are
also impaired for transcription of IFN-
-stimulated genes (52, 53).
However, in both U3A cells and in Stat1
/
mice, constitutive levels
of p48 are reduced relative to wild type and neither p48 nor Stat2
levels can be induced in response to IFN treatment. Therefore, active
Stat2-p48 complexes would not be expected to be detected. It remains
possible that natural situations of abundant Stat2 and p48 exist where
Stat2-p48 complexes could mediate a weak transcriptional response to
IFN-
. It also remains possible that alternative DNA sequences or
alternative adaptor proteins exist which allow Stat2 homodimers to be
recruited to DNA and activate transcription without the direct
participation of Stat1.
We thank Chris Schindler for antisera against
Stat1 and Stat2; S. Qureshi and J. E. Darnell, Jr., for gifts of Stat2
expression constructs; G. Stark and I. Kerr for U3A cells; R. Schneider
for expression vectors and cell lines; and Hoffmann-La Roche for
IFN-.