(Received for publication, October 13, 1995; and in revised form, December 22, 1995)
From the
An upstream inverted repeat (IR) element mediates
transcriptional activation of the interferon response factor-1 gene (IRF-1) by interferon (IFN)- and IFN-
. IFN-
and
IFN-
fail to induce IRF-1 in cells that lack signal
transducer and activator of transcription 1 (STAT1), and STAT1
homodimers bind to IR elements in extracts of IFN-
-treated cells.
We now report that STAT2 also plays an important role in the
IFN-
-mediated transcriptional activation of the IRF-1 gene. A new factor, most likely a STAT1-STAT2 heterodimer, was
detected with an IR probe in extracts of IFN-
-treated cells. STAT1
and STAT2 are already known to combine with p48, a DNA-binding protein,
to form IFN-stimulated gene factor 3 (ISGF3), which binds to
IFN-stimulated response elements (ISREs) distinct from the IR of the IRF-1 gene. In extracts of U2A cells, which lack p48,
STAT1-STAT2 heterodimers were still formed, indicating that they do not
contain p48. We manipulated the intracellular levels of STAT1-STAT2
heterodimers and STAT1 homodimers to examine their roles in the
induction of IRF-1 by IFN-
. Although both dimers can
induce IRF-1 transcription, the heterodimers are more potent
and thus may be the major activators in vivo. Deletion
analysis reveals that the C-terminal domain of STAT2 is important for
transcriptional activation mediated by both STAT1-STAT2 heterodimers
and ISGF3.
Many cytokine and growth factor signaling pathways utilize
proteins of the JAK ()and STAT families (1, 2, 3) . The IFN-
pathway involves
activation of TYK2, JAK1, STAT1, and STAT2. The two STAT proteins are
phosphorylated on conserved tyrosine residues by the JAK family kinases
when IFN-
binds to the receptor complex(3) . Activated
STAT1 and STAT2 associate with the DNA-binding protein p48 to form the
transcription factor ISGF3, which recognizes an interferon-stimulated
response element (ISRE, consensus: AGTTTCNNTTTCN(C/T)) (2) present in many promoters activated by IFN-
(for
examples, see (4, 5, 6) ). All three proteins
of ISGF3 make contact with DNA(7) .
IFN- triggers the
tyrosine phosphorylation of STAT1, but not STAT2 (8) .
Activated STAT1 forms homodimers, known as GAF(9) . In
IFN-
signaling, a complex containing STAT1, biochemically similar
to GAF, has been reported(10) . The GAS DNA sequences
(consensus: TTNCNNNAA) recognized by GAF serve as binding sites for
various cytokine- or growth factor-activated STAT proteins, including
STAT3 homodimers(11, 12) , STAT1-STAT3
heterodimers(11, 12) , STAT4(13) ,
STAT5(14, 15, 16) , and STAT6(17) .
Transcription of the IRF-1 gene is inducible by both
IFN- and IFN-
. A 16-kb 5`-flanking region that mediates a
response to either IFN-
or -
does not contain an ISRE, and
the induction of IRF-1 by IFN-
is independent of the p48
subunit of ISGF3 (18, 19, 20) .
Transcriptional activation of this IRF-1 promoter segment by
IFNs requires a palindromic GAS element, the IR element, which lies
about 110 bases upstream of the transcription start site. IFN-inducible
transcription factors containing STAT1 bind to the IR element in
vitro.
In this report, we demonstrate the formation of a novel
IFN--inducible DNA-binding factor consisting of STAT1 and STAT2.
Although this factor does not include p48, the level of p48 protein
does affect the balance between the novel factor and ISGF3. We propose
that transcriptional activation of the IRF-1 gene involves
interaction of the IR element with either a STAT1-STAT2 heterodimer or
a STAT1 homodimer.
Figure 1:
STAT1-STAT2 heterodimers in extracts of
IFN--treated cells. A, EMSA was performed using cell
extracts prepared from 2fTGH or U6A cells, untreated(-) or
treated with IFN-
(
) or IFN-
(
) for 15 min. An IR
probe was used. Complexes A, B, and C are indicated by arrows.
B, an extract from IFN-
-treated 2fTGH cells was assayed with
an IR probe. Antibodies against STAT1 (S1), STAT2 (S2), STAT3 (S3), or Waf-1 (W) were included
in the binding reactions as indicated. C, same as B,
except that extracts of U2A cells were
used.
Figure 2: Alteration of the relative amounts of STAT1-STAT2 heterodimers and STAT1 homodimers in U6A cells. The details are the same as in Fig. 1A, except that extracts from transfected U6A cells overexpressing STAT2 or a STAT2-STAT1 chimera (N2; see ``Materials and Methods'') were used.
Figure 3:
STAT1-STAT2 heterodimers and STAT1
homodimers both activate transcription of the IRF-1 gene in
response to IFN-. A, total RNAs, prepared from 2fTGH,
U3A, U6A, U6A/STAT2, and U6A/N2 cells, were analyzed by RNase
protection, using probes for IRF-1 and
-actin. The cells
were untreated or treated with IFN-
for 1 or 4 h. The protected
fragments are indicated by arrows. A shorter exposure is also
shown for the
-actin fragment. B, quantitation of the
experiment. The intensities of the bands were measured by use of a
PhosphoImager (Molecular Dynamics). The signals were normalized to
-actin.
It has been
proposed that the STAT1 homodimer also functions as a transcriptional
activator of this gene in IFN--treated cells(20) . To
evaluate the role of the homodimer in the absence of the heterodimer,
we expressed in U6A cells a chimeric protein, designated N2, in which
the N-terminal 305 amino acids of STAT1 are replaced by the N-terminal
315 amino acids of STAT2. We observed only STAT1 homodimers in
IFN-
-treated U6A/N2 extracts (Fig. 2, lane 5). In
response to IFN-
, IRF-1 induction was restored in U6A/N2
cells (Fig. 3), suggesting that STAT1 homodimers are also
transcriptional activators of the IRF-1 gene. The N2 protein
and endogenous STAT1 were both phosphorylated in response to IFN-
in U6A/N2 cells.
However, we did not detect
co-precipitation of N2 with STAT1 in these cells,
suggesting that N2 dimerizes poorly with STAT1. Furthermore,
complex C in U6A/N2 extracts is not likely to contain N2/STAT1
heterodimers or N2/STAT1 heterodimers because an amount of an antibody
against N-terminal STAT2 that could supershift all of complex A in
2fTGH cells (Fig. 1B, lane 3) showed only
minimal effect on complex C in U6A/N2 cells.
Although
both STAT1-STAT2 heterodimers and STAT1 homodimers can activate IRF-1, it is likely that the heterodimer is more potent. Upon
IFN- treatment, the level of homodimers in U6A/N2 cells was about
10-fold higher than the level of heterodimers in U6A/STAT2 cells (Fig. 2; compare complex C in lane 5 to complex A in lane 2). However, induction of IRF-1 gene expression
was stronger in U6A/STAT2 cells than in U6A/N2 cells 4 h after
IFN-
treatment (Fig. 3, A and B; compare
U6A/STAT2 to U6A/N2). Although there are more STAT1 homodimers than
STAT1-STAT2 heterodimers in IFN-
-treated 2fTGH cells (Fig. 1A, lane 2), the heterodimers may still
contribute to the activation of IRF-1 gene expression, since
they are more potent.
Figure 4: STAT2 proteins with C-terminal truncations form heterodimers with STAT1. Extracts were prepared from U6A cells transfected with wild-type STAT2 or STAT2 C-terminal deletion constructs, using clones with similar levels of expression. The end points of the deletions are indicated. The sizes of the STAT1-STAT2 heterodimers (arrows) decreased as the length of the deletions increased.
Figure 5:
Complementation of IFN--induced IRF-1 expression in U6A cells by full-length STAT2 or STAT2
proteins with C-terminal truncations. The amino acid positions at the
C-terminal ends of the deletions are indicated. Total RNAs were
analyzed by RNase protection, using probes for IRF-1 and
-actin.
The cells were untreated(-) or treated with IFN-
for 4 h
(
). The positions of the protected fragments and undigested probes
are indicated by arrows. A shorter exposure is also shown for
the
-actin fragment.
Figure 6:
Alteration of the ratio of ISGF3 to
STAT1-STAT2 heterodimers by p48. A, EMSA was performed using
extracts of either U2A or p48-complemented U2A cells,
untreated(-) or treated with IFN- for 15 min. An ISRE probe
from the 9-27 gene was used (GGAAATAGAAACT)(5) . The
position of ISGF3 is indicated by arrows. We see two bands,
corresponding to the 91- and 84-kDa forms of STAT1. B, the
same extracts were assayed with an IR probe (see ``Materials and
Methods''). To show critically that complex A was not present in
the IFN-
-treated U2A/p48 cells, the amount of extract was doubled
in that lane. Complexes A and C are indicated by arrows.
We performed EMSA with various GAS elements (1) and
an extract of U6A/STAT2 cells; only the IR and Fc GAS elements
were found to bind STAT1-STAT2 heterodimers, suggesting that the
heterodimers probably prefer GAS elements with the core sequence
TTCCC(A/C)GAA. We did not identify a GAS element specific for
heterodimers since STAT1 homodimers were detected with the same probes.
It will be interesting to determine the optimal GAS sequence for
binding heterodimers by use of the polymerase chain
reaction(32) . However, the optimal sequence for STAT1-STAT2
heterodimers may be no more specific than the optimal sequence for
STAT1-STAT3 heterodimers, which also binds to STAT1 homodimers with
high affinity(32) .
STAT1-STAT2 heterodimers form in U2A
cells in the absence of p48. It is likely that p48 binds to preformed
heterodimers to form ISGF3, so that the level of p48 can influence the
steady-state amount of heterodimer. The expression of p48 is usually
low in most cell types but can be induced by IFN-. Thus, the
heterodimer may be directed either to form ISGF3 or to bind to a
selected set of GAS elements depending on the availability of p48,
which thus may modulate the response to IFN-
in an
IFN-
-dependent manner.
We manipulated the amounts of
STAT1-STAT2 heterodimers and STAT1 homodimers in transfected U6A cells
to reveal that both can function to stimulate transcription of the IRF-1 gene in response to IFN-. However, a small amount
of heterodimer is sufficient to promote a high level of IRF-1 induction, revealing that this novel factor is a potent
transcriptional activator. The STAT1 homodimers induced by IFN-
activate IRF-1 transcription less strongly. Our results
suggest that, depending on the level of p48, STAT1-STAT2 heterodimers
can play a major role in activating IRF-1 transcription in
response to IFN-
.
We showed by deletion analysis that the acidic domain of STAT2 is important for the transcriptional activation of the IRF-1 gene. The same region is also important for transcriptional activation of ISRE-containing genes(29) . It is possible that the acidic domain of STAT2 may interact with the same basic transcription factor(s) at the start sites of these genes. We detected truncated heterodimers and STAT1 homodimers in U6A cells transfected with a series of STAT2 proteins carrying C-terminal deletions and found that decreased IRF-1 induction correlated with shortening of the STAT2 acidic domain. The STAT1 homodimers formed in these cells are insufficient to induce IRF-1 transcription, probably because they fail to compete effectively with the defective heterodimers.
Although a STAT1-STAT3 heterodimer (complex B) was
detected using the IR element probe, this species is unlikely to be
important for IFN--mediated IRF-1 gene expression because
the amount of complex B is very low. The role of STAT3 in the IFN-
signaling pathway remains unclear.
In summary, we have demonstrated
an alternative transcriptional activation pathway mediated by a novel
transcription factor that is likely to be a STAT1-STAT2 heterodimer.
The heterodimer binds to the IR element of the IRF-1 gene and
the GAS element of the Fc gene and is a potent transcriptional
activator of the IRF-1 gene. Since the IRF-1 protein has been
found to be a tumor suppressor (33) and a mediator of
apoptosis(34) , the STAT1-STAT2 heterodimer may play an
important role in the antiproliferative response mediated by type I
IFNs.