From the Sidney Kimmel Comprehensive Cancer Center
and ¶ Department of Molecular Biology and Genetics, and The Johns
Hopkins University School of Medicine, Baltimore, Maryland 21231
Received for publication, December 11, 2002, and in revised form, January 17, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transcription factors of the interferon
regulatory factor (IRF) family have been identified as critical
mediators of early inflammatory gene transcription in infected cells.
We have shown previously that IRF-5, like IRF-3 and IRF-7, is a
direct transducer of virus-mediated signaling and plays a role in the
expression of multiple cytokines/chemokines. The present study is
focused on the molecular mechanisms underlying the formation and
function of IRF-5/IRF-7 heterodimers in infected cells. The interaction between IRF-5 and IRF-7 is not cooperative and results in a repression rather than enhancement of IFNA gene transcription. The
formation of the IRF-5/IRF-7 heterodimer is dependent on IRF-7
phosphorylation, as shown by the glutathione S-transferase
pull-down and immunoprecipitation assays. Mapping of the interaction
domain revealed that formation of IRF-5/IRF-7 heterodimers occurs
through the amino terminus resulting in a masking of the DNA binding
domain, the consequent alteration of the composition of the
enhanceosome complex binding to IFNA promoters in
vivo, and modulation of the expression profile of
IFNA subtypes. Thus, these results indicate that
IRF-5 can act as both an activator and a repressor of
IFN gene induction dependent on the IRF-interacting
partner, and IRF-5 may be a part of the regulatory network
that ensures timely expression of the immediate early inflammatory genes.
Type I interferons
(IFN)1 play an essential role
in the innate immune response against virus infection (1). In
uninfected cells, the expression of IFN genes is tightly
regulated. Virus infection activates transcription of type I
IFN genes, and the cis-acting virus-responsive elements
(VRE), located within the 110 nucleotides 5' of the transcription
initiation site, are sufficient for virus-mediated activation (2, 3).
The VRE of IFNA genes contain purine-rich GAAANN motifs that
constitute the specific binding sites (IRF-E and PRDI/III) for the
proteins of the interferon regulatory factor (IRF) family, whereas the
VRE in the promoter of the IFNB gene contain not only
PRDI/PRDIII elements but also an NF- Nine cellular IRF and three viral homologues (vIRF) have been
identified (4-9). All of the cellular IRF share a region of homology
in the amino terminus encompassing a highly conserved DNA binding
domain (DBD) that is characterized by five tryptophan repeats (4).
Three of the repeats contact DNA recognizing the GAAA or AANNGAAA
sequences (10-12). KSHV-encoded IRF that contain an imperfect DNA
binding domain are not able to bind DNA with the same specificity as
cellular IRF.
Several of the cellular IRF were implicated in the regulation of type I
IFN gene expression in virus-infected cells.
IRF-1 was first identified as an activator of the
IFNB gene, whereas IRF-2 antagonized the
IRF-1-mediated activation and acted as a suppressor
(13-15). Whereas the infected embryonic fibroblasts from
IRF-1 In the murine system, induction of type I IFN genes in
virus-infected primary embryonic fibroblasts was proposed to proceed by
two sequential phases. During the initial phase, which does not require
protein synthesis, transcription of IFNB and
IFNA4 genes was activated. The second phase, during which
the rest of the IFNA subtypes were induced, depended on the
IFN-mediated induction of IRF-7 expression (23, 29).
However, in recently generated mice with a homozygous deletion of the
IRF-7 gene, production of IFN In uninfected cells, these three IRF proteins are generally present in
the cytoplasm. Viral infection induces phosphorylation of IRF-3, IRF-5,
and IRF-7 by yet unidentified kinase(s), and these IRF are then
retained in the nucleus where they interact with the histone
transacetylases p300/CBP or PCAF
(32-35).2 Of note, IRF-5
contains two nuclear localization signals and translocates to the
nucleus in uninfected cells, although less efficiently than in
virus-infected cells (5). Interestingly, although a large number of RNA
and DNA viruses, as well as double-stranded RNA, induce phosphorylation
of IRF-3 and IRF-7, phosphorylation of IRF-5 seems to be
virus-specific, and this factor is not induced or phosphorylated by
double-stranded RNA (5). Structure-function analysis of IRF-3, IRF-5,
and IRF-7 proteins revealed that all of these IRF contain an
auto-inhibitory domain, which in transient transfection assays
suppresses the transcriptional activity of these factors (5, 22, 36,
37). Furthermore, results from a study with the IRF-7
dominant-negative mutant suggested that IRF-3 and IRF-7 form homo- and
heterodimers and that these interactions are critical for the
stimulation of the transcriptional activity of endogenous
IFNA genes (22, 38).
Although the promoters of all IFNA genes are highly
homologous, differential expression of these genes, as a function of
cell type and inducing agent, has been observed (39, 40).
IFNA1 was identified as the major subtype expressed in human
fibroblasts expressing IRF-3 and ectopic IRF-7 (25) but not IRF-5. In
contrast, in cells that express ectopic IRF-5, but not IRF-7, NDV
predominantly induced the IFNA8 subtype (21). Analysis of
the transcriptional complex/enhanceosome assembled on the promoter of
the IFNA1 and IFNB genes revealed that both the
IFNA and IFNB enhanceosomes contain IRF-3 and
IRF-7, as well as IRF-1 (19, 41). In cells that express IRF-5, but not
IRF-7, both IRF-3 and IRF-5 were found to be bound to IFNA
VRE and the endogenous IFNA promoters (5, 21). However, we
have observed recently3 that
in monocytes, and particularly in precursor dendritic cells (pDC2) that
are high producers of IFN The goal of this study was therefore to gain more insight into the
molecular mechanisms underlying the expression of human IFNA
genes in cells expressing both IRF-7 and IRF-5,
and to determine whether the expression of these two IRF in the cell is
associated with high levels of IFNA production, as seen in
pDC2 cells (42). To this effect, we have generated 2fTGH cell lines
expressing ectopic IRF-7 or IRF-5, or both of these proteins.
Surprisingly, the simultaneous expression of both IRF-5 and IRF-7 did
not result in an enhanced synthesis of biologically active IFN in
infected cells; to the contrary, expression of both factors resulted in a distinct repression of IFN. We also reveal that whereas IRF-5 can
interact with IRF-7, there is a mutual binding exclusion between these
two family members to IFNA promoters from the lysates of infected cells. These results indicate the lack of cooperative interaction between IRF-5 and IRF-7 and enhancement of IFNA
production but show that the relative levels of IRF-5 and
IRF-7 expressed in infected cells determine the distinct
profile of IFNA genes induced.
Cells and Virus--
2fTGH/IRF-5 and 2fTGH/IRF-7 overexpressing
cells were generated as described previously (21, 26). MDBK
bovine kidney cells and HeLa cells were obtained from ATCC, and 2fTGH
cells, obtained from G. Stark (Cleveland Clinic Foundation, Cleveland,
OH), were grown in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum. Sendai virus was purchased from Specific Pathogen-free Avian Supply (Preston, CT), and NDV was purchased from
ATCC (VR-699). Infections were conducted with 640 hemagglutinin units/100-mm plate (80% confluency) for a given period.
Plasmids and Antibodies--
GST-IRF-5,
GST-IRF-5N, GST-IRF-5C, GST-IRF-7N,
and GST-IRF-7C expression plasmids were described previously
(5, 38). Similarly, IRF-1, IRF-3,
IRF-5, and IRF-7 expression plasmids (20, 21, 27,
43); HuIFNA1 and -A14 soluble alkaline
phosphatase (SAP) reporter plasmids (44, 45); and polyclonal IRF-1,
IRF-3, and IRF-7 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). M2 anti-FLAG monoclonal antibody was obtained from Sigma.
GST Pull-down Assay--
GST fusion constructs were transformed
into BL21 bacteria and induced with 0.5 mM
isopropyl-1-thio- Immunoprecipitation--
2fTGH/IRF-5 cells were co-transfected
with the IRF-7 expression plasmid to examine interactions
between IRF-5/IRF-7. Cells were uninfected or infected with Sendai
virus or NDV for 6 h and then lysed in IP lysis buffer (20 mM Hepes, pH 7.9, 50 mM NaCl, 10 mM
EDTA, 2 mM EGTA, 0.1% Nonidet P-40, 10% glycerol, and 0.2 mM protease inhibitor mixture). Extracts (250 µg) were
incubated with 1 µg of anti-IRF-1 antibodies or 1 µg of
anti-IRF-7 antibodies cross-linked to 30 µl of protein
A-Sepharose beads, respectively, for 1 h at 4 °C. Precipitates
were washed 4 times with IP lysis buffer and eluted by boiling the
beads for 3 min in 1× SDS loading buffer. Eluted proteins were
separated by 10% SDS-PAGE and transferred, and IRF-5 was
detected by anti-FLAG antibodies.
Transfections and SAP Assay--
In the transient transfection
assay, 2 × 106 2fTGH or HeLa cells were transfected
with a constant amount of DNA (5 µg/60-mm plate) by using Superfect
transfection reagent (Qiagen). For the SAP assays, equal amounts (2.5 µg) of reporter plasmid and IRF-expressing plasmid were
co-transfected with the Oligonucleotide Pull-down Assay and
Immunoblot--
Double-stranded oligomers corresponding to the
IFNA1 and IFNA14 VRE region ( Reverse Transcription-PCR Analysis and Antiviral Assay--
One
microgram of total RNA isolated by the cesium chloride method was
reverse-transcribed to cDNA with oligo(dT) primers in a 30-µl
reaction. From this mixture of cDNAs, IFNA was amplified by PCR using primers that detect all IFNA genes, and
Chromatin Immunoprecipitation Assay--
The detailed assay
procedure was described previously (5, 21). Briefly, 2fTGH/IRF-5 or
2fTGH/IRF-7 cells (6 × 107) were infected with Sendai
virus or NDV for 6 h or left uninfected. The proteins bound to DNA
were cross-linked by addition of formaldehyde in aqueous buffer (0.1 M NaCl, 1 mM EDTA, 50 mM Hepes, pH
8.0) to a final concentration of 1% for 30 min at 37 °C. The
reaction was stopped by addition of 0.125 M glycine. The
cell pellets were washed with 5 ml of wash buffer (5 mM
PIPES, pH 8.0, 85 mM KCl, 0.5% Nonidet P-40, 0.2 mM PMSF), resuspended in sonication buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) on ice, and
lysed by sonication for 10 s. Samples were diluted 10-fold with
dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM
EDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl)
and pre-cleared with protein A-Sepharose. Following the pre-clearing,
equal amounts of proteins (as determined by the Bio-Rad protein assay
reagent) were immunoprecipitated with 1 µg of anti-IRF-1, anti-IRF-3,
anti-IRF-7, or anti-FLAG antibodies for 4 h at 4 °C.
Immunocomplexes were extensively washed and treated with RNase A (50 µg/ml), 0.5% SDS, and proteinase K (500 µg/ml). The cross-linked
DNA-protein complexes were reverted by heating at 65 °C for 6 h, and the DNA was recovered by phenol/chloroform extraction. DNA
purified by precipitation with 2 M ammonium acetate/ethanol was used as a template for PCR amplification with universal primers corresponding to the regions of human endogenous IFNA genes
that are conserved in all subtypes (5, 25).
Interaction between IRF-5 and Other Members of the IRF
Family--
Previous work (5, 21) has shown that IRF-5 interacts with
IRF-3 forming a functional heterodimer involved in the induction of
IFNA and IFNB genes in infected cells. However,
even in the cells where IRF-3 expression was nearly
eliminated, IRF-5 was still capable of inducing low levels
of biologically active IFN
Taken together, these data suggest that the binding capacity of IRF-1
to GST-IRF-5 is not significantly altered by viral infection. On the
other hand, binding of IRF-7 to IRF-5 is enhanced in infected cells
suggesting that virus-induced post-translational modification of IRF-7
plays a role in its ability to interact with IRF-5. We therefore
examined the association between IRF-5 and IRF-1 or IRF-7 in cells by
immunoprecipitation. In these experiments, 2fTGH/IRF-5 cells were
transfected with IRF-7, and 24 h post-transfection, cells were either left uninfected or infected with Sendai virus or NDV
for 8 h. Cell lysates were then immunoprecipitated with anti-IRF-1
or anti-IRF-7 antibodies, and the presence of IRF-5 in precipitates was
detected by immunoblotting with anti-FLAG antibodies. The relative
level of IRF proteins expressed in transfected cells was estimated by
immunoblot analysis. Data in Fig. 1C shows that association
of IRF-1 with IRF-5 is not dependent on viral infection, whereas IRF-7
binds to IRF-5 only in infected cells. These results would indicate
that in infected cells expressing both IRF-5 and
IRF-7, heterodimer formation is likely.
Distinct Activation of IFNA1 by IRF-5 and Its
Heterodimers--
The results shown in Fig. 1 indicate that
formation of an IRF-5/IRF-1 heterodimer is not specific to infected
cells, whereas the IRF-5/IRF-7 heterodimer forms specifically after
virus infection. In order to determine what is the functional outcome
of the association between IRF-5 and IRF-1 or IRF-5 and IRF-7, we
examined the ability of these IRF family members to activate the
IFNA1 promoter using the reporter plasmid containing SAP
under the regulation of IFNA1 VRE (Fig.
2). As shown in Fig. 2A,
overexpression of IRF-1, IRF-5, or
IRF-7 in HeLa cells led to transcriptional activation of the IFNA1 SAP reporter. By immunoblot analysis, all three IRF
were expressed to a similar level (data not shown). Whereas
IRF-5 activated the SAP reporter in both uninfected and
NDV-infected cells, activation by IRF-7 occurred only
in infected cells. In contrast to these two IRF, activation by
IRF-1 in infected cells was low, as shown previously (15).
Consequently, co-transfection of IRF-1 and IRF-5
with the IFNA1 SAP reporter plasmid to HeLa cells infected with NDV yielded similar activation levels to those observed with IRF-5 alone. Although the IFNA1 promoter was
activated in uninfected HeLa cells co-transfected with both
IRF-5 and IRF-7, upon infection with NDV, the
activity of the IFNA1 SAP reporter was suppressed compared
with levels induced by either of these factors alone. These results
suggest that IRF-5/IRF-7 heterodimer formation in infected cells (as
shown in Fig. 1C) does not lead to activation of the
IFNA1 promoter.
We have shown previously (19) that IRF-1, IRF-3, and IRF-7 are part of
the transcriptional complex-enhanceosome binding to the
IFNA1 promoter in infected cells. We therefore investigated the functional differences between IRF-1/IRF-5 and IRF-1/IRF-7 heterodimers. For these experiments, increasing amounts of the IRF-1 expression plasmid were co-transfected together with
IRF-5 or IRF-7 and the IFNA1 SAP
reporter plasmid to HeLa cells. Levels of protein expressed were
determined by immunoblot analysis and observed to be equivalent to the
amount of transfected cDNA (data not shown). Results in Fig.
2B demonstrated that increasing levels of IRF-1
repressed the IRF-5-mediated transactivation of the
IFNA1 promoter in both uninfected and NDV-infected cells. In
comparison, transfection of increasing amounts of IRF-1 with
IRF-7 led to a concentration-dependent increase
in transcription of the IFNA1 promoter. These results
indicate that IRF-1 may compete for binding of
IRF-5 to the IFNA1 VRE, whereas increasing levels
of IRF-1 enhanced the transcriptional activity of
IRF-7 in infected cells suggesting a cooperative interaction
between these two factors. Thus, in the context of the IFNA1
promoter, IRF-1 can act both as a repressor or enhancer
depending on its interaction with distinct IRF family members.
Co-transfection of IRF-5 and IRF-7 did not
activate the expression of the IFNA1 promoter. Because both
IRF-5 and IRF-7 were able to transactivate the
IFNA1 promoter when expressed alone in the cells, the low
transactivation activity of the IRF-5/IRF-7 heterodimers in infected
cells was unexpected. To determine whether the transactivating
potential of IRF-5 could be rescued, we co-transfected the
IFNA1 SAP with a constant amount of IRF-7 and
increasing amounts of IRF-5. The results shown in Fig.
2C indicate that the transactivation potential of
IRF-5 could be increased when the levels of IRF-5 in the
cells were significantly higher than IRF-7 levels, as detected by
immunoblot analysis (data not shown). Similarly, activation of the
IFNA promoter was restored in cells that expressed higher levels of IRF-7 than IRF-5 (data not shown).
These data indicate that the IRF-5/IRF-7 heterodimer is unable to
stimulate transcription of the IFNA1 gene in infected cells,
yet either of these IRF could stimulate IFNA gene
transcription when its relative level of expression predominated.
Competition between Binding of IRF-5 and IRF-7 to IFNA VRE Is
Dependent on the Relative Levels of IRF Expressed--
The data from
the SAP reporter assay revealed that IRF-5/IRF-7 heterodimers repressed
IFNA1 gene transcription. We have therefore examined the
binding of IRF-5 and IRF-7 to IFNA VRE from lysates of
infected cells that express these IRF. Because IRF-5 can form heterodimers with IRF-3 (5), we have also examined whether binding of
IRF-5 to IFNA VRE is dependent on the binding of IRF-3. We
have shown previously (21) that IRF-3 binds to the IFNA1 but
not IFNA14 VRE; therefore, we have examined the binding of IRF-5 (21) or IRF-7 to these two VRE using the oligonucleotide pull-down assay. Lysates of 2fTGH/IRF-5 and 2fTGH/IRF-7 cells infected
either with Sendai virus or NDV were utilized for these experiments.
The top two panels in Fig.
3A show the binding of IRF-5
and IRF-7 to the IFNA1 and -A14 VREs. Binding of
IRF-7 to IFNA1 or IFNA14 VRE was detected only
from lysates of infected cells and was higher from lysates of Sendai
virus-infected cells than NDV-infected cells. In contrast, binding of
IRF-5 to IFNA1 and IFNA14 VRE was detected both
from lysates of uninfected and infected cells. However, although the
binding of IRF-5 from uninfected and Sendai virus infected cells was
about the same, it was greatly enhanced after infection with NDV.
We next compared binding of IRF-5 and IRF-7 to IFNA VREs from lysates
of 2fTGH cells expressing both IRF-5 and IRF-7. For these studies, we
either transfected the 2fTGH/IRF-7 cells with IRF-5
(2fTGH/IRF-7 + IRF-5) or the 2fTGH/IRF-5 cells with IRF-7 (2fTGH/IRF-7 + IRF-5), and the lysates were then prepared from infected
and uninfected cells. Results from immunoblot analysis of the input
lysates (Fig. 3A) show that the cells expressed the endogenous IRF at higher levels than the transfected IRF; IRF-3 was
expressed at about the same levels in both lysates. As shown in Fig.
3A, IRF-3 was bound only to the IFNA1
VRE from lysates of infected cells, which is in correlation with
previous reports (19, 21). In lysates of cells that expressed higher
levels of IRF-7 than IRF-5 (2fTGH/ IRF-7 + IRF-5), binding of IRF-7
to both IFNA1 and IFNA14 VRE could be detected
from lysates of infected cells. However, binding of IRF-5 to
IFNA1 and -A14 VRE was drastically reduced in
these cells. The reduction in IRF-5 binding could not be related to the
absence of IRF-3 because binding to both IFNA1 and
-A14 VRE was greatly reduced. In the cells expressing higher levels of IRF-5 than IRF-7 (2fTGH/IRF5 + IRF-7), binding of IRF-5 to
IFNA1 and -A14 VRE was detected both from the
lysates of infected and uninfected cells; however, IRF-5 bound more
efficiently from the lysates of NDV-infected cells. The binding of
IRF-7 to these VRE was insignificant, and no difference between binding
from infected and uninfected lysates could be detected. Taken together, these results indicate that the binding of IRF-5 and IRF-7 to IFNA VRE
reflects the relative levels of expression in the cells and that these
two IRF probably do not bind to the IFNA VRE as a heterodimer.
In order to determine whether IRF-5 and IRF-7 are competing for binding
to the IFNA1 VRE, we used the oligonucleotide pull-down assay to examine the binding profiles of one IRF, in the presence of
increasing amounts of the second IRF. As shown in Fig. 3B, increasing amounts of IRF-5 expressed in the 2fTGH/IRF-7 cells competed
the binding of IRF-7 to the IFNA1 VRE. In a similar manner, when IRF-7 levels were incrementally increased in 2fTGH/IRF-5 cells,
binding of IRF-5 was diminished. Interestingly, the binding affinity of
IRF-5 for IFNA1 VRE seems to be higher than that of IRF-7
binding to IFNA1 VRE. By graphing the level of binding of IRF-5 or IRF-7 as a function of the amount of transfected plasmid, it
became apparent that the binding of IRF-5 to the IFNA1 VRE occurred at a 2-3-fold lower concentration than binding of IRF-7. These results indicate that IRF-5 and IRF-7 do not bind as a
heterodimer; instead their binding is dependent on the relative levels
of expression and affinity for the IFNA promoter.
Induction of Biologically Active IFN
We next examined whether co-expression of IRF-5 and
IRF-7 also affected the profile of induced IFNA
subtypes. We have shown previously that NDV-infected 2fTGH/IRF-5
cells preferentially induced the IFNA8 subtype (Table
I, values in parentheses, column 3),
whereas 2fTGH/IRF-7 cells preferentially expressed the IFNA1 subtype (Table I, column 5, values in parentheses). In order to
determine the predominant subtype in 2fTGH cells expressing both
IRF-5 and IRF-7, we have amplified the IFNA
mRNA and cloned the fragments from NDV-infected cells, as described
previously (25, 40). We have found that this method yields a more
accurate identification of the IFNA subtypes expressed than the
previously used RNA protection assay (48). Randomly selected clones
were then identified by sequencing (21, 25). As shown in Table I,
2fTGH/ IRF-5 + IRF-7 and 2fTGH/IRF-7 + IRF-5 transfected cells expressed either IFNA8 or IFNA1, respectively, as
the predominant subtype. However, co-expression of IRF-5 or
IRF-7 introduced two major changes: 1) although the
2fTGH/IRF-7 cells did not express IFNA8, the presence of
IRF-5 in these cells conferred expression of
IFNA8 and IFNA2; and 2) in the 2fTGH/IRF-5 cells,
co-expression of IRF-7 decreased expression of
IFNA8 and IFNA7 while enhancing expression of
IFNA1. These data indicate that co-expression of IRF-5 and IRF-7 in infected cells modulates the
levels of the IFNA subtypes conferred by either IRF alone.
IRF-5 and IRF-7 Compete for Binding to the Endogenous IFNA
Promoters in 2fTGH Cells--
We have shown previously (19) that
IRF-1, IRF-3, and IRF-7 are components of the enhanceosome binding to
IFNA1 VRE in infected cells. Because the binding of IRF-5
and IRF-7 to IFNA1 VRE, as analyzed by the oligonucleotide
pull-down assay, shows a competitive binding exclusion, we sought to
determine the binding of IRF-5 and IRF-7 to the endogenous
IFNA promoters in vivo. To this end, we used the
chromatin immunoprecipitation assay (ChIP). The 2fTGH/IRF-5 or
2fTGH/IRF-7 cells utilized for these studies (Fig.
5A) were transiently
transfected either with IRF-7 or IRF-5,
respectively (Fig. 5B), and then infected with Sendai virus
or NDV. Proteins were cross-linked to DNA, and the protein-DNA
complexes were precipitated with antibodies specific for IRF-1, IRF-3,
IRF-5 (FLAG), or IRF-7 (Fig. 5A), as described under
"Experimental Procedures." The analyzed IFNA promoters
were first amplified from DNA-protein complexes before
immunoprecipitation to ensure that the amount of DNA used for
precipitation with different antibodies was constant (data not shown).
As described previously (5), the conditions of PCR were optimized to
give rise to a linear amplification response of the endogenous
IFNA promoter region from the immunoprecipitated DNA-protein
complexes. Amplification of IFNA promoters from the DNA-protein complexes immunoprecipitated with IRF-1, IRF-3, or IRF-5
antibodies from 2fTGH/IRF-5 cells is shown in Fig. 5A,
top three panels. The fragment containing the
IFNA promoter region was amplified only from DNA-protein
complexes immunoprecipitated by IRF-1 and IRF-3 antibodies from
infected cells, but no amplification was observed in immunoprecipitates
from uninfected cells. However, the endogenous IFNA promoter
was amplified from complexes immunoprecipitated with anti-FLAG
antibodies (IRF-5) in both uninfected and NDV-infected cells (Fig.
5A). These data indicate that IRF-1, as well as IRF-3 and
IRF-5, are components of the IFNA enhanceosome in
NDV-infected cells, but neither IRF-1 nor IRF-3 binds to the
IFNA promoters in uninfected cells. In contrast, IRF-5 binds
to IFNA promoters in uninfected cells (5, 21). In the
2fTGH/IRF-7 cells, the IFNA promoter region was amplified
from complexes immunoprecipitated by IRF-7 antibodies in virus-infected
cells but not in uninfected cells.
To examine the effect of IRF-5 and IRF-7 co-expression on their binding
to the endogenous IFNA promoter region, DNA-protein complexes from 2fTGH/IRF-5 or 2fTGH/IRF-7 cell lines transfected with
either IRF-7 or IRF-5, respectively (i.e. 2fTGH/IRF-5 + IRF-7), were immunoprecipitated with anti-IRF-3, anti-IRF-5 (FLAG), or anti-IRF-7 antibodies (Fig. 5B). In the NDV-infected
2fTGH/IRF-5 cells expressing IRF-7 (levels of IRF-5 expression were
greater than IRF-7), the endogenous IFNA promoter was
amplified from DNA precipitated by anti-IRF-3 and anti-IRF-5
antibodies, but nearly undetectable levels were amplified from the
anti-IRF-7 immunoprecipitates. Although NDV infection induced binding
of IRF-3 and IRF-5 to the IFNA promoter, amplification was
not observed in anti-FLAG immunoprecipitates from Sendai virus-infected
cells. These results indicate that in the presence of high levels of
IRF-5, binding of IRF-7 to the IFNA promoter was below the
levels of detection. Interestingly, in 2fTGH/IRF-7 cells expressing
higher levels of IRF-7 than IRF-5, the IFNA promoter could
be amplified from anti-IRF-3, IRF-7, and FLAG immunoprecipitates of
DNA-protein complexes from NDV-infected cells. However, the
IFNA promoter was amplified only from anti-IRF-3 and IRF-7
immunoprecipitates of DNA-protein complexes from Sendai virus-infected
cells. These results confirm the previous observation that Sendai virus
infection does not stimulate transcriptional activity of IRF-5 (5, 21).
Taken together, these data indicate that whereas IRF-3, IRF-5, and
IRF-7 bind to the IFNA promoter in NDV-infected cells
expressing high levels of IRF-7, IRF-5 has a greater binding affinity
than IRF-7 and out-competes the binding of IRF-7 when these factors are
expressed in the cells at about the same levels.
Characterization of the IFNA Enhanceosome Complex--
In order to
determine whether IRF-5 is a component of the enhanceosome containing
IRF-3, IRF-7, and IRF-1 which binds to IFNA promoters in
cells that express all of these IRF, such as dendritic cells,3 we performed a sequential ChIP assay (19). The
DNA-protein complexes from uninfected or NDV-infected 2fTGH/IRF-5 cells
transfected with IRF-7 that express endogenous IRF-1 and IRF-3 (19)
were first precipitated with anti-IRF-3 (Fig.
6A), anti-FLAG (Fig. 6B), or anti-IRF-7 (Fig. 6C) antibodies, and the
IFNA promoter region was then amplified from these
precipitates. The supernatants of these precipitates were then again
precipitated with anti-IRF-1, anti-IRF-3, anti-FLAG, or anti-IRF-7
antibodies, and the IFNA promoter region was amplified from
the second immunoprecipitates. Results in Fig. 6A show that
the IFNA promoters could be amplified from the anti-IRF-3
precipitates but not from the subsequent anti-IRF-1, IRF-3, or IRF-7
precipitates indicating that the complex binding to the IFNA
promoter contains IRF-3, IRF-1, and IRF-7 (19). However, the
IFNA promoter could be amplified when the second precipitation was performed with anti-FLAG antibodies that detect IRF-5. This result would suggest that in both uninfected and
NDV-infected cells, IRF-5 can bind as a homodimer in the absence of
IRF-3 and the other IRF family members. The fact that no amplification
of the IFNA promoter could be detected from a second
precipitation with anti-IRF-3 antibodies indicates that the first
precipitation was quantitative.
In the case where the initial precipitation was performed with
anti-FLAG (IRF-5) antibodies (Fig. 6B), the IFNA
promoter could be amplified from both infected and uninfected cells.
Low levels of the IFNA promoter could also be amplified from
second precipitations with anti-IRF-1, IRF-3, and IRF-7 antibodies from
infected cells, supporting the previous observation that binding of
these three IRF is independent of IRF-5 binding. Finally, the
IFNA promoter was amplified when the DNA-protein complexes
from infected cells were first precipitated with IRF-7 antibodies and
from the subsequent precipitates with anti-IRF-3 or IRF-5 antibodies
(Fig. 6C). However, in the absence of IRF-7, no binding of
IRF-1 to the IFNA promoter was detected. Thus, although we
did not detect binding of IRF-7 in the absence of IRF-3 (Fig.
6A), indicating that IRF-7 binds as a heterodimer with IRF-3
(19, 38), precipitation with anti-IRF-7 antibodies did not completely
remove IRF-3 protein revealing that IRF-3 can also bind in the absence
of IRF-7. When the DNA-protein complexes from NDV-infected 2fTGH cells
overexpressing IRF-5 and IRF-7 were first precipitated with IRF-1
antibodies, the IFNA promoter was efficiently amplified
(data not shown). Interestingly, secondary precipitations with
anti-IRF-1, IRF-3, IRF-5, or IRF-7 antibodies revealed that PCR
amplification of the IFNA promoter could only be detected
from IRF-3 or IRF-5 precipitates indicating again that binding of IRF-3
and IRF-5 but not IRF-7 to this promoter can occur in the absence of
IRF-1. Thus, IRF-1 is an important part of the IFNA
enhanceosome complex in cells expressing IRF-7 but not in cells
expressing IRF-3 and IRF-5. These results would indicate that although
IRF-5 can bind to the endogenous IFNA promoter in the
absence of IRF-1, IRF-3, or IRF-7, its binding is greatly enhanced in
the presence of IRF-3 (5). We therefore conclude that IRF-5 may not be
part of the IFNA enhanceosome containing IRF-1, IRF-3, and
IRF-7.
Mapping the IRF-5/IRF-7 Interaction Domain--
By the
GST pull-down and co-immunoprecipitation assays, we have shown that
IRF-5 and IRF-7 form heterodimers in infected cells. It appears that
these heterodimers have a lower DNA binding capacity than either IRF
alone. In order to fully discern the reason why the IRF-5/IRF-7
heterodimer has a lower DNA binding capacity, we have mapped the
interacting regions between IRF-5 and IRF-7. To this effect, cell
lysates from NDV-infected 2fTGH/IRF-7 and 2fTGH/IRF-5 were incubated
with either immobilized amino-terminal or carboxyl-terminal GST-IRF
fusion proteins, and the specifically bound proteins were detected by
immunoblot analysis with the appropriate serum. As shown in Fig.
7A, IRF-5 interacts with both
amino- and carboxyl-terminal GST-IRF-7 fusion proteins, whereas IRF-7
interacts only with the amino terminus of IRF-5 (Fig. 7B).
This is in contrast to IRF-3, which interacts with the carboxyl
terminus of IRF-5 (Fig. 7B) (5). These results indicate that
formation of IRF-5/IRF-7 heterodimers results in masking of the
IRF-5 and IRF-7 DNA binding domains.
It has been generally accepted that the transcription factors
IRF-3 and IRF-7 play a critical role in the induction of type I IFN
genes in infected cells. The expression of IRF-3 is sufficient to
support virus-mediated activation of IFNB genes (31),
whereas IRF-7 is required for the transcriptional activation of
IFNA genes (24, 25). A somewhat surprising finding was that
ectopic expression of IRF-5 could rescue the transcription of
IFNA genes in the absence of IRF-7 (9, 21). The expression
of IRF-5 and IRF-7 in cells is not exclusive, and the presence of
IRF-7 and IRF-5 transcripts can be detected in uninfected monocytes and
dendritic cells, as well as precursor dendritic cells (pDC2) that are
high producers of IFN By addressing the molecular mechanism resulting in the IRF-5
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (PRDII)-binding site (4).
/
mice expressed normal levels of
IFNA and -B (16, 17), it was observed that IRF-1
associates with the VRE of both human IFNA and
IFNB promoters in vivo, thus suggesting that
IRF-1 may contribute to the transcriptional regulation of
the human IFNA genes (18, 19). Three members of the IRF
family (IRF-3, IRF-5, and IRF-7) were
shown to be direct transducers of virus-mediated signaling and to play
a crucial role in the expression of type I IFN genes
(19-26). Whereas IRF-3 is constitutively expressed in all
cell types (27), constitutive expression of IRF-7 was observed only in lymphoid cells but could be induced by type I IFN in other cell types (20, 28). Expression of
IRF-5 was detected primarily in B cells and dendritic cells
and was further enhanced by type I IFN (5, 9, 21).
in the isolated mouse
embryo fibroblasts was completely abolished, whereas the expression of
IFN
was significantly reduced (30). These results are consistent
with the observations in human cells, where expression of
IRF-3 conferred only virus-mediated induction of the
IFNB gene (31), and IRF-7 was required for expression of IFNA genes. In both the infected mouse and
human cells, expression of IFNA could be rescued upon
reconstitution of IRF-7 expression (25). Surprising,
however, was the observation that in the absence of IRF-7,
induction of IFNA genes could be rescued by overexpression
of IRF-5 (5, 21). However, in contrast to IRF-7,
activation of IRF-5 was virus-specific and resulted in the
expression of different IFNA subtypes than were identified in infected cells expressing IRF-7 (21).
, both IRF-7 and IRF-5
are expressed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside for 3 h. The
GST fusion proteins were purified from bacterial lysates by affinity
chromatography on a glutathione-agarose column (Sigma). Coomassie Blue
staining of the GST fusion proteins was used to quantify the amount of
protein bound to beads. Equal amounts of GST fusion proteins and beads
were then applied in each pull-down assay. The 35S-labeled
proteins were synthesized in vitro using the coupled TNT T7 transcription/translation system (Promega) according
to the manufacturer's instructions. Each reaction mixture contained 1 µg of nonlinearized expression plasmid and was incubated (90 min,
30 °C) in the presence of 4 µl of Translabel (DuPont) amino acid
mixture. GST fusion proteins (0.5 µg) bound to glutathione-agarose beads were incubated with 10-µl reaction mixture aliquots of
35S-labeled proteins in 250 µl of binding buffer (10 mM Tris-Cl, pH 7.6, 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 1 mM dithiothreitol, 0.1% Nonidet P-40, 500 µg of bovine
serum albumin, and 8% glycerol) at 4 °C for 2 h. After three
washes (10 min at room temperature) with binding buffer, the proteins
bound to the beads were solubilized in sample buffer and resolved by
8% SDS-PAGE. The gel was dried and exposed to film. In addition,
binding of IRF from whole cell lysates of uninfected or virus-infected
2fTGH/IRF-5 cells was performed using lysis buffer (20 mM
Tris, pH 8.0, 1 mM EDTA, 200 mM NaCl, 0.5%
Nonidet P-40, and 0.2 mM protease inhibitor mixture). GST
fusion protein immobilized on beads and whole cell extract were
incubated in 300 µl of binding buffer at 4 °C for 2 h. The beads were then washed 3 times with binding buffer, and the bound proteins were eluted and resolved by 10% SDS-PAGE, transferred to
Hybond transfer membranes (Amersham Biosciences), and incubated with
anti-IRF-1 antibodies or anti-IRF-7 antibodies. Immunocomplexes were
detected by using the ECL system.
-galactosidase expression plasmid (50-100
ng). The transfected cells were divided 14 h later and infected
with NDV for 16 h. The alkaline phosphatase (SAP) assays were
performed as described previously (44, 46). Each experiment was
repeated 3 times. The
-galactosidase expression levels were used to
normalize the difference in transfection efficiency.
110 to
53 bp)
were synthesized and biotin-labeled at the 5' end of the antisense
strand. Two micrograms of the double-stranded oligomers were incubated
with streptavidin magnetic beads (200 µl) (Dynal Inc.) for 1 h
in TEN buffer (20 mM Tris, pH 8.0, 1 mM EDTA,
0.1 M NaCl), and the unbound DNA was removed by extensive washing with same buffer. IRF from whole cell extracts were bound to
DNA on magnetic beads as described previously (21). Beads were then
washed, and the bound proteins were analyzed on SDS-PAGE. IRF were
identified by immunoblot with the respective antibodies as described
(5, 21). Blots were stripped (200 ml of 10% SDS in 1000 ml, 62.5 mM Tris, pH 6.7, 0.7% 2-mercaptoethanol) by incubating for
10 min at 65 °C and then rotating for 10 min at room temperature.
Blots were then washed 3-5 times with TBST and re-probed with the
respective antibodies.
-actin was amplified as an internal control (25). Individual
IFNA subtypes were identified by cloning and subsequent
sequencing of the PCR-amplified fragments as described recently (25). A
one-way analysis of variance
(faculty.vassar.edu/~lowry/VassarStats.html) was used to analyze the
significance of observed differences in IFNA subtype expression. Levels of biologically active IFN
in the medium were determined by the antiviral assay (47).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and was able to bind to the
IFNA VRE (21). To what extent IRF-5 interacts with other IRF
family members and the functional consequences of these interactions on
type I IFN gene expression have not yet been determined. Here we
examined whether IRF-5 could associate with IRF-1 or
IRF-7, both of which have been shown to be present in the
enhanceosome assembled on the promoters of IFNA1 and
IFNB genes (19, 33). The in vitro interactions
between IRF-5 and 35S-labeled IRF-1, IRF-3,
IRF-5, or IRF-7 was examined by the GST pull-down assay. As shown in
Fig. 1A, all examined IRF
bound efficiently to GST-IRF-5, whereas they did not bind to
GST-agarose bead. These results were further confirmed by the GST-IRF-5
pull-down assay with whole cell lysates from uninfected or
virus-infected 2fTGH/IRF-7
expressing cells (Fig. 1B) (5).
Immunoblot analysis of cell extracts has shown that both IRF-5 and
IRF-7 were expressed in these cells, although at different levels.
Because we have shown previously (5, 21) both physical and functional
interactions between IRF-5/IRF-5 and IRF-5/IRF-3, we have focused
on the interactions between IRF-5 and IRF-1 or IRF-7. Fig.
1B shows that endogenous IRF-1 binds to GST-IRF-5 both from
lysates of uninfected cells and Sendai virus or NDV
infected
2fTGH/IRF-7 cells. However, no binding of IRF-1 to GST-agarose beads
could be detected. In contrast to the IRF-1 binding profile, IRF-7
bound to GST-IRF-5 much more efficiently from cell lysates of Sendai
virus and NDV
infected 2fTGH/IRF-7 cells than from the lysates of
uninfected cells. The low level of IRF-7 detected as bound to the
GST-agarose beads in this experiment was not reproducible.
View larger version (36K):
[in a new window]
Fig. 1.
Interaction between IRF-5 and other IRF
family members. A, interactions between GST-IRF-5
fusion protein and in vitro translated IRF-1, IRF-3, IRF-5,
or IRF-7. 35S-Labeled IRF-1 (lanes 1 and
2), IRF-3 (lanes 3 and 4), IRF-5
(lanes 5 and 6), and IRF-7 (lanes 7 and 8) input (25%) and their binding to GST-agarose beads
(lanes 1, 3, 5, and 7)
or GST-tagged IRF-5 agarose beads (lanes 2, 4,
6, and 8) are shown. B, binding
of IRF-1 or IRF-7 from cell lysates of infected and uninfected
2fTGH/IRF-7 cells to GST-IRF-5 was analyzed by the GST pull-down assay.
10% input from whole cell extracts of uninfected
(cont, lane 1), Sendai virus
(SeV, lane 2), or NDV
infected cells (lane
3) are shown. Cell extracts were applied to GST-agarose beads or
GST-tagged IRF-5
agarose beads, and specifically bound
proteins were detected by Western blotting (WB) with
anti-IRF-1 or anti-IRF-7 polyclonal antibodies (Ab) as
described under "Experimental Procedures." C,
heterodimerization of IRF-5 with IRF-1 or IRF-7 in infected and
uninfected cells. 2fTGH/IRF-5
expressing cells were transfected with
IRF-7 and then left either uninfected (cont),
infected with Sendai virus (SeV), or NDV for 6 h. Whole
cell extracts (250 µg) were immunoprecipitated (IP) with
either anti-IRF-1 or anti-IRF-7 polyclonal antibodies as described
under "Experimental Procedures." The immunoprecipitated complexes
were separated on SDS-10% PAGE gels, and the presence of IRF-5 in
immunoprecipitates was detected by immunoblotting with anti-FLAG
monoclonal antibodies. Relative levels of endogenous IRF-1, IRF-5, and
transfected IRF-7 in cell lysates are shown (20% input).
View larger version (26K):
[in a new window]
Fig. 2.
Transcriptional activation of the
IFNA1 promoter by IRF-5 heterodimers.
A, the IRF-5/IRF-7 heterodimers repressed transactivation of
the IFNA1 SAP reporter in infected cells. Expression
plasmids encoding IRF-1, IRF-5, or
IRF-7 were co-transfected with the IFNA1 SAP
reporter plasmid together with the -galactosidase-expressing plasmid
to HeLa cells, as described under "Experimental Procedures." Cells
were infected with NDV for 16 h or left uninfected, and SAP
activity was measured as described under "Experimental Procedures."
SAP levels were normalized to the constant level of
-galactosidase
expressed. B, distinct effect of IRF-1 on the
transactivation potential of IRF-5 and IRF-7.
Constant amounts of the IFNA1 SAP reporter,
IRF-5, or IRF-7 (2 µg) were co-transfected with
increasing amounts of IRF-1 (2, 4, and 8 µg) to HeLa
cells. Cells were left uninfected or infected with NDV for 16 h,
and SAP activity was measured. C, repression of the
IFNA1 SAP reporter by IRF-5/IRF-7 heterodimers was overcome
in a dose-dependent manner by IRF-5. Constant
amounts of the IFNA1 SAP reporter and IRF-7 (2 µg) were co-transfected with increasing amounts of IRF-5
(2, 4, and 8 µg) to HeLa cells. Cells were left uninfected or
infected with NDV for 16 h, and SAP activity was measured.
View larger version (46K):
[in a new window]
Fig. 3.
IRF-5 and IRF-7 compete for binding to IFNA
VRE. A, binding of IRF-7 or IRF-5 from cell lysates
(250 µg) of infected or uninfected 2fTGH/IRF-7 expressing cells or
2fTGH/IRF-5
expressing cells to IFNA1 and IFNA14
VRE. 2fTGH/IRF-7 or 2fTGH/IRF-5 cells were then transfected with
FLAG-tagged IRF-5 (2fTGH/IRF-7 + IRF-5) or IRF-7
(2fTGH/IRF-5 + IRF-7), respectively. The relative levels of IRF-5,
IRF-7, and endogenous IRF-3 in cell lysates were estimated by
immunoblot analysis (10% input). The same extracts were used for the
oligonucleotide pull-down assay as described under "Experimental
Procedures," and the levels of IRF-5, IRF-7, and IRF-3 bound to the
respective IFNA1 or -A14 VRE were consequently
detected by immunoblotting. Blots were stripped and re-probed with each
antibody in the order shown. Ab, antibody; SeV,
Sendai virus. B, IRF-7 and IRF-5 compete for binding to
IFNA1 VRE. 2fTGH/IRF-7 or 2fTGH/IRF-5 cells were transfected
with increasing amounts of FLAG-tagged IRF-5 or
IRF-7, respectively, and infected with NDV. The relative
levels of IRF-5 and IRF-7 in cell lysates were determined by
immunoblotting (10% input). The extracts were used for the
oligonucleotide pull-down assay where the levels of IRF-5 and IRF-7
bound to the IFNA1 VRE were detected by immunoblotting.
Relative binding levels were quantitated (binding affinity) to show the
relative intensity of IRF-5 or IRF-7 bound as a function of the amount
of transfected plasmid.
in Virus-infected 2fTGH
Cells Expressing IRF-5and IRF-7--
It was shown
previously (21) that induction of IFNA genes by IRF-5
occurred only in cells infected by NDV but not Sendai virus. However,
the induction of IFNA genes in cells that express both IRF-5
and IRF-7 has not yet been examined. We have therefore analyzed the expression of IFNA genes and proteins in 2fTGH
cells expressing both ectopic IRF-5 and IRF-7.
For these studies, we have used either NDV-infected 2fTGH/IRF-5 cells
transfected with IRF-7 or 2fTGH/IRF-7 cells transfected with
IRF-5. Fig. 4A
illustrates that in the absence of IRF-7, NDV induced high
levels of IFN
(1500 units/ml) in 2fTGH/IRF-5 cells; yet these levels
were drastically reduced when cells also expressed IRF-7
(320 units/ml). Furthermore, whereas Sendai virus did not induce
production of IFN
in 2fTGH/IRF-5 cells, it induced low levels of
IFN
(80 units/ml) when the 2fTGH/IRF-5 cells were also expressing
IRF-7. The level of IRF-5 and IRF-7 protein expression in
2fTGH cells, as determined by immunoblot analysis, indicated that the
levels in these cells were comparable. Immunoblot analysis of cell
lysate from 2fTGH/IRF-7 cells transfected with IRF-5 showed
significantly higher levels of IRF-7 than IRF-5. In 2fTGH/IRF-7 cells,
IFN
was effectively induced both by Sendai virus (800 units/ml) and
NDV (350 units/ml) infection. However, in the presence of
IRF-5, the levels of IFN
produced were significantly decreased in Sendai virus-infected cells (10-fold reduction), whereas
in NDV-infected cells, the decrease was only about 2-fold. The relative
levels of IFNA mRNA present in Sendai virus and NDV-infected cells
correlated with the levels of biologically active IFN
. These data
indicate that in cells expressing both IRF-5 and
IRF-7, the production of biologically active IFN
was
lower than in cells expressing only IRF-5 or
IRF-7; expression of IRF-5 significantly decreased IFN
production in Sendai virus-infected cells.
View larger version (40K):
[in a new window]
Fig. 4.
Expression of the endogenous IFNA
genes and production of biologically active
IFN in infected 2fTGH cells is a result of
IRF-5 and IRF-7 competition.
A, the relative levels of IFNA transcripts in
infected 2fTGH/IRF-5 cells (left panels) and 2fTGH/IRF-5
cells transfected with IRF-7 (right panels). IFNA
cDNAs were amplified using primers corresponding to the conserved
regions of all human IFNA genes. Conditions of amplification
were optimized to achieve a linear response. The levels of biologically
active IFN
synthesized in these cells were determined by antiviral
assay using bovine trachea cells, and the values in international units
per ml are shown at the bottom. The relative levels of IRF-5
and IRF-7 proteins in cell lysates were determined by immunoblotting
with anti-FLAG or anti-IRF-7 antibodies (Ab), respectively.
SeV, Sendai virus. B, analysis of IFNA
transcripts in infected 2fTGH/IRF-7 cells (left panels) and
2fTGH/IRF-7 cells transfected with IRF-5 (right
panels). The levels of biologically active IFN
were determined
as described above. The relative levels of ectopic IRF-5 and endogenous
IRF-7 in cell lysates are shown.
IFNA subtypes induced by NDV infection in 2fTGH cells
expressing ectopic IRF-5 and IRF-7
View larger version (31K):
[in a new window]
Fig. 5.
IRF-5 and IRF-7 compete for
binding to the endogenous IFNA promoters in 2fTGH
cells. A, in vivo binding of IRF-1, IRF-3,
IRF-5, and IRF-7 to IFNA promoters in infected 2fTGH/IRF-5
or 2fTGH/IRF-7 expressing cells. A ChIP of DNA-protein complexes was
performed as described under "Experimental Procedures" with
anti-IRF-1, IRF-3, or IRF-7 polyclonal antibodies (Ab).
IP, immunoprecipitation; SeV, Sendai virus. The
IRF-5 binding was detected by precipitation with anti-FLAG monoclonal
antibodies. IFNA promoters were amplified from the
precipitates by using primers that detect promoters of all
IFNA genes. The relative levels of amplified fragments of
IFNA promoters are shown. The IFNA promoters were
also amplified from the DNA-protein complexes before the precipitation
to normalize the amount of DNA used for precipitation (data not shown).
B, 2fTGH/IRF-5
expressing cells or 2fTGH/IRF-7
expressing
cells transfected with IRF-7 and FLAG-tagged IRF-5, respectively, were
infected with virus or left uninfected. The ChIP assay was performed as
described above and fragments of IFNA promoters were
amplified from the precipitates.
View larger version (22K):
[in a new window]
Fig. 6.
In vivo characterization of the
endogenous IFNA enhanceosome complex in 2fTGH cells by the
ChIP assay. A, the cross-linked DNA-protein complexes
from NDV-infected 2fTGH/IRF-5 cells transfected with IRF-7 were first
precipitated with anti-IRF-3, IRF-5, or IRF-7 antibodies. The
precipitates were collected by centrifugation, and the supernatants
were divided and subjected to a second round of precipitation with
either IRF-1, IRF-3, IRF-5, or IRF-7 antibodies, respectively. The
first and second precipitates were dissolved in 60 µl of water, and
15 µl of these samples were used for PCR amplification using primers
that detect all IFNA promoters, as described under
"Experimental Procedures." The amplified IFNA promoter
fragments are shown.
View larger version (21K):
[in a new window]
Fig. 7.
Mapping the IRF-5/IRF-7 interaction
domains. A, formation of the IRF-5/IRF-7 heterodimer is
mediated through the IRF-5 DNA binding domain. Whole cell lysate (250 µg) from NDV-infected 2fTGH/IRF-5 expressing cells was used for
mapping the IRF-7 interaction domain. The relative levels of IRF-5 in
cell lysates were determined by immunoblotting (10% input).
Extracts were applied to GST-agarose beads (lane 1),
amino-terminal IRF-7 fused to GST-agarose beads (lane 2), or
carboxyl-terminal IRF-7 fused to GST-agarose beads (lane 3).
Specifically bound proteins were detected by immunoblotting with
anti-FLAG antibodies (Ab). B, whole cell lysate
(250 µg) from NDV-infected 2fTGH/IRF-7
expressing cells was used for
mapping the IRF-5 interaction domain. The relative levels of IRF-7 and
IRF-3 in cell lysates were determined by immunoblotting (10%
input). Extracts were applied to GST-agarose beads (lane
1), amino-terminal IRF-5 fused to GST-agarose beads (lane
2), or carboxyl-terminal IRF-5 fused to GST-agarose beads
(lane 3). Specifically bound proteins were detected by
immunoblotting with anti-IRF-7 or anti-IRF-3 antibodies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
.3 Moreover, type I IFN can further
stimulate expression of both of these factors. It has therefore become
important to determine the contribution of IRF-5 to the activation of
IFNA genes in the context of IRF-7. In the present study, we
show that although IRF-5 and IRF-7 can form heterodimers, this
interaction is not cooperative and results in an inhibition of
IFNA gene transcription.
mediated
suppression of IRF-7 activity, we have characterized the interaction
between IRF-5 and other IRF family members known to be involved in the
activation of IFN genes, and we have observed that IRF-5
interacts with both IRF-3 and IRF-7 (summarized in Fig.
8). The presence of IRF-5/IRF-7
heterodimers could be detected in infected but not uninfected cells. We
have shown previously (5, 21) that NDV but not Sendai virus infection
activates IRF-5 by phosphorylation in the carboxyl terminus of the
protein, whereas IRF-7 and IRF-3 are activated by both of these
viruses. The fact that we could detect binding between IRF-5 and IRF-7 in lysates of cells infected with both Sendai virus and NDV indicates that phosphorylation of IRF-7, but not IRF-5, is required for heterodimer formation. It was shown that IRF-3 and IRF-7 homodimers, as
well as IRF-3/IRF-7 heterodimers, could be detected only in infected
cells (22, 37, 38). Consequently, it was proposed that the
virus-mediated phosphorylation of IRF-3 and IRF-7 resulted in
allosteric changes that allowed for dimerization of these proteins and
their interactions with the other members of the IRF family, as well as
CBP/p300 and PCAF. Phosphorylation of IRF-3 and IRF-7 is also
necessary for their retention in the nucleus (22, 37, 49). Because the
virus-induced phosphorylation of the serine-rich carboxyl-terminal
region of IRF-5 (5) does not seem to be required for its ability to
form homodimers or heterodimers with other IRF, this indicates that in
the IRF-5 polypeptide, unlike the other two IRF (IRF-3 and IRF-7), the
interaction domain is not masked. Furthermore, the carboxyl-terminal
phosphorylation of IRF-5 may not be a prerequisite for its nuclear
retention because the IRF-5 polypeptide contains two nuclear
localization signals that facilitate its nuclear transport even in
uninfected cells (5). These data indicate that the IRF-5 polypeptide
shows several unique features that distinguish this transcription
factor from IRF-3 and IRF-7, and the functions of IRF-5 and IRF-7 are
not redundant.
View larger version (28K):
[in a new window]
Fig. 8.
IRF-5 dimerization and promoter binding.
A, schematic representation of IRF-5 dimerization and
binding to the endogenous IFNA promoters in uninfected
cells. IRF-5 is present in the cytoplasm and nucleus of uninfected
cells due to low levels of constitutive phosphorylation and the
presence of an unmasked carboxyl-terminal nuclear localization
signals (5). In uninfected cells, IRF-5 can form homodimers (5) and
IRF-5/IRF-1 heterodimers; yet only IRF-5 homodimers can bind to the
endogenous IFNA promoters. No heterodimerization between
IRF-5 and IRF-3 or IRF-7 can be detected in uninfected cells.
B, schematic illustration of IRF-5 dimerization and binding
to endogenous IFNA promoters in virus-infected cells.
Phosphorylation (P) of Ser/Thr residues in the carboxyl
terminus leads to conformational changes in IRF-3, IRF-5 (5), and IRF-7
that further relieves carboxyl-terminal autoinhibition and exposes both
DBD and interaction domain regions (IAD). The open
conformations of IRF-3 and IRF-7 are now able to heterodimerize with
IRF-5. Translocation to the nucleus leads to DNA binding at ISRE- and
PRD-like (LE) containing promoters. Whereas IRF-5 interacts
with IRF-3 through the carboxyl-terminal interaction domain forming a
functional heterodimer, IRF-5/IRF-7 heterodimerization occurs through
the DBD and thus does not lead to a functional complex binding to the
IFNA promoters.
Overexpression of IRF-5 rescued IFN production in the absence of
IRF-7, and in the transient transfection assay, IRF-5 enhanced IRF-3
mediated transcriptional activity of the
IFNA1 and IFNB promoters in infected
cells (5). However, in contrast to the cooperative interaction between
IRF-3/IRF-5, the interaction between IRF-5 and IRF-7 resulted in the
repression of IFNA1 transcriptional activity. The
fact that the basal activity of IFNA promoters was not
affected in uninfected cells expressing both IRF-7 and IRF-5 revealed
that the IRF-5/IRF-7 heterodimers do not block transcription by
interaction with basal transcription factors. The block in transcription was limited to infected cells, suggesting that the transactivation potential of IRF-5 and IRF-7 is lost when these factors
heterodimerize. This inhibition is likely a result of heterodimer
formation that masks the IRF-5 or IRF-7 DNA binding domain and their
ability to bind to the IFNA promoters (Fig. 8B). When both IRF-5 and IRF-7 were present in the lysates of infected cells, their binding to IFNA1 and
IFNA14 VRE was mutually exclusive, whereas the
binding of IRF-3 to IFNA1 VRE was not modulated
by the presence of IRF-5 or IRF-7. A similar competition of IRF-5 or
IRF-7 binding to the endogenous IFNA promoters was observed in infected cells expressing both IRF-5 and IRF-7 by the ChIP assay.
These data further show that in the cells expressing high levels of
IRF-5, IRF-5 but not IRF-7 was the major component of the
IFNA enhanceosome. In contrast, when the relative levels of IRF-7 were higher than IRF-5, IRF-7 bound to the IFNA
promoters, yet the binding of IRF-5 was not completely excluded. It is
important, however, to remember that analysis of IRF bound to the
endogenous IFNA promoters gives an overall picture of IRF
binding to the majority of IFNA promoters. This assay does
not allow one to distinguish quantitatively IRF binding to the
promoters of individual endogenous genes. Thus, the possibility that
binding of IRF-5 and IRF-7 to the endogenous IFNA promoters
detected in these assays represents binding to distinct IFNA
promoters cannot yet be excluded.
The analysis of the expression of IFNA subtypes in cells overexpressing either IRF-5 or IRF-7 does indicate that IRF-5 and IRF-7 target different IFNA promoters (21, 25). We have shown previously that viral infection of 2fTGH/IRF-7 cells induced predominantly expression of IFNA1, and no expression of IFNA2 or IFNA8 genes could be detected in these cells. However, when IRF-5 was co-expressed in these cells, both IFNA2 and IFNA8 genes were expressed. Similarly, IRF-7 expression in 2fTGH/IRF-5 cells resulted in the enhanced expression of IFNA1 and decreased expression of the IFNA8 and IFNA7 genes, whereas the 2fTGH/IRF-5 cells expressed predominantly IFNA8 and only low levels of IFNA1. These results indicate that the relative levels of IRF-5 and IRF-7 in the cells significantly modulate the expressed levels of IFNA subtypes. In addition, we have shown previously that the relative levels of IRF-3 and IRF-7 also affect the expression of IFNA subtypes (25, 26). Taken together, these results suggest that despite the close sequence homology between the subtypes of IFNA promoters, the transcriptional activity of these individual promoters shows a distinct activation response to IRF-3, IRF-5, IRF-7, and their combinations. Thus, it is unlikely that the level of IRF-3 and IRF-7 DNA binding alone determines the specificity and activation of individual IFNA genes in infected cells.
Interestingly, in the context of the IFNA1 promoter, IRF-5 can function as an activator when present as homodimers or heterodimers with IRF-3 and as a repressor when interacting with IRF-7. IRF-5 is not the only member of the IRF family that can exert both activation and repression activity. Thus, as shown in this study, high levels of IRF-1 result in a suppression of IFNA1 transactivation activity when complexed with IRF-5, yet IRF-1 and IRF-7 cooperatively activate this promoter. Previously it has been shown that IRF-2 represses the transcriptional activity of IRF-1 (13) and inhibits transcriptional activation of the IFNB gene (14), whereas it activates transcription of the VCAM gene (50) and histone 4 (51, 52). Furthermore, IRF-7 was initially discovered for its ability to repress the Q promoter of the EBV-encoded EBNA genes but can effectively activate IFNA and IFNB genes (28). Finally, IRF-8 acts as an activator upon binding to PU.1 (53-57), yet in association with another member of the ETS family, Tel, it acts as a strong repressor of promoters containing ISRE sites (58). The molecular mechanism by which IRF-5 suppresses IRF-7 activation of the IFNA1 promoter is not yet clearly established. Although IRF-7 interacts with both IRF-3 and IRF-5 in a phosphorylation-dependent manner, the interaction with IRF-3 activates the transcription of the IFNA1 promoter, whereas the interaction with IRF-5 represses it. The ability of IRF-5 to interact with IRF-3 and IRF-7 differs in several ways. Whereas IRF-7 and IRF-5 independently bind to the IFNA1 VRE both in vitro and in vivo, there is competition between IRF-7 and IRF-5 binding to this VRE element in vivo. Results from the GST pull-down assay suggest that interaction of IRF-5 with IRF-7 and formation of heterodimers occur through association with the amino-terminal regions of these proteins, thus masking their DNA binding domains (Fig. 8B). Furthermore, whereas IRF-7, IRF-5, and IRF-3 can all bind to the IFNA promoters in vivo, IRF-7 is a part of an enhanceosome that contains IRF-3 and IRF-1, whereas IRF-5 does not seem to be a part of this enhanceosome complex. All of these IRF interact with CBP/p300 and/or PCAF, and these interactions are essential for the transactivation by IRF-3 and IRF-7 (59-62). The histone transacetylases are also a part of the enhanceosome containing IRF-3, IRF-1, and IRF-7 (19). Recent data (63-65) indicate that both IRF-3 and IRF-7 are acetylated, and acetylation modulates their function. Thus, there are at least two other possibilities by which IRF-5 can repress activation by IRF-7. The first is by quelching the interaction of IRF-7 with the histone transacetylase PCAF, as was shown for the E1A adenovirus-encoded protein (66). The second is by its ability to recruit HDAC. The selective recruitment of HDAC to the neighborhood of the IRF-3 and IRF-7 enhanceosome may result in an alteration of chromatin structure and block the binding of IRF-7 to the enhanceosome. Although we do not have any evidence that either of these mechanisms is operative, it is not without interest that we have observed association between IRF-5 and histone transacetylases CBP/p300, PCAF, and the repressors SMRT, Skip, and mSin3a that are components of the HDAC co-repressor complex.2 The repression of IRF-3- and IRF-7-mediated activation of some murine IFNA promoters by interaction with the homeoprotein Pitx1 binding to an upstream region of murine IFNA promoters was recently observed (67). Whether this protein plays any role in the repression of transcriptional activity of human IFNA genes is not known. Altogether, these data indicate that IRF-5 expression may be a part of the regulatory network that ensures timely expression of the immediate early genes. In infected cells, the expression of IRF-7 is strongly enhanced by type I IFN produced in the context of viral infection and thus the IRF-5-mediated repression may be overcome by high levels of IRF-7 and result in the consequent stimulation of IFNA gene expression. Additional experiments are needed to address these possibilities.
In summary, we have described that IRF-5 cannot only activate and
cooperate with IRF-3 in the stimulation of IFNA gene
transcription, but it can also mediate the suppression of IRF-7-induced
expression of IFNA genes in response to viral infection.
Thus, it appears that in the context of an individual promoter, IRF-5
can exert both stimulating activity in association with IRF-3 and
suppressing function when associated with IRF-7 (Fig. 8). One of the
mechanisms by which the suppressive role is acquired is a result of
IRF-5/IRF-7 heterodimer formation and inhibition of IRF-5 and IRF-7
binding to the VRE of IFNA promoters. On the other hand, in
infected cells, binding of IRF-5 to IFNA promoters appeared
to be separate from the binding of the IRF-1, IRF-3, and IRF-7
containing enhanceosome, suggesting that IRF-5 may not be a component
of this complex. Considering the fact that an IRF-3/IRF-7-mediated
activation is dependent on the recruitment of CBP/p300, further
experiments are underway to determine whether IRF-5-mediated
suppression is also coupled to the recruitment of the HDAC
co-repressor complex to the IFNA promoter.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank members of the Pitha laboratory for helpful discussion during the course of the study. We also thank to Louisa Petrosillo for computer and formatting assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants R21AI19737-19 and R01AI/CA19737-19A1 (to P. M. P.) and an Anticancer Drug Development Pharmacology-Oncology Training Grant (to B. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence and reprint requests should be addressed: The Johns Hopkins University, Oncology Center, 1650 Orleans St., Baltimore, MD 21231. Tel.: 410-955-8900; Fax: 410-955-0840; E-mail: barnebe@jhmi.edu.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M212609200
2 B. J. Barnes, unpublished data.
3 B. J. Barnes and P. F. Bocarsly, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IFN, interferon; IRF, interferon regulatory factors; GST, glutathione S-transferase; VRE, virus-responsive elements; DBD, DNA binding domain; ChIP, chromatin immunoprecipitation assay; SAP, soluble alkaline phosphatase; NDV, Newcastle disease virus; PIPES, 1,4-piperazinediethanesulfonic acid; PCAF, p300/CREB-binding protein (CBP)-associated factor; CREB, cAMP-response element; HDAC, histone deacetylase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Muller, U., Steinhoff, U., Reis, L. F., Hemmi, S., Pavlovic, J., Zinkernagel, R. M., and Aguet, M. (1994) Science 264, 1918-1921[Medline] [Order article via Infotrieve] |
2. | Hiscott, J., Nguyen, H., and Lin, R. (1995) Semin. Virol. 6, 161-173[CrossRef] |
3. | Pitha, P. M., Au, W. C., Lowther, W., Juang, Y. T., Schafer, S. L., Burysek, L., Hiscott, J., and Moore, P. A. (1998) Biochimie (Paris) 80, 651-658[CrossRef] |
4. | Nguyen, H., Hiscott, J., and Pitha, P. M. (1997) Cytokine Growth Factor Rev. 8, 293-312[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Barnes, B. J.,
Kellum, M. J.,
Field, A. E.,
and Pitha, P. M.
(2002)
Mol. Cell. Biol.
22,
5721-5740 |
6. |
Burysek, L.,
Yeow, W. S.,
Lubyova, B.,
Kellum, M.,
Schafer, S. L.,
Huang, Y. Q.,
and Pitha, P. M.
(1999)
J. Virol.
73,
7334-7342 |
7. | Burysek, L., Yeow, W. S., and Pitha, P. M. (1999) J. Hum. Virol. 2, 19-32[Medline] [Order article via Infotrieve] |
8. |
Gao, J.,
Morrison, D. C.,
Parmely, T. J.,
Russell, S. W.,
and Murphy, W. J.
(1997)
J. Biol. Chem.
272,
1226-1230 |
9. | Barnes, B. J., Lubyova, B., and Pitha, P. M. (2002) J. Interferon Cytokine Res. 22, 59-71[CrossRef][Medline] [Order article via Infotrieve] |
10. | Escalante, C. R., Yie, J., Thanos, D., and Aggarwal, A. K. (1997) FEBS Lett. 414, 219-220[CrossRef][Medline] [Order article via Infotrieve] |
11. | Escalante, C. R., Yie, J., Thanos, D., and Aggarwal, A. K. (1998) Nature 391, 103-106[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Fujii, Y.,
Shimizu, T.,
Kusumoto, M.,
Kyogoku, Y.,
Taniguchi, T.,
and Hakoshima, T.
(1999)
EMBO J.
18,
5028-5041 |
13. | Fujita, T., Kimura, Y., Miyamoto, M., Barsoumian, E. L., and Taniguchi, T. (1989) Nature 337, 270-272[CrossRef][Medline] [Order article via Infotrieve] |
14. | Harada, H., Fujita, T., Miyamoto, M., Kimura, Y., Maruyama, M., Furia, A., Miyata, T., and Taniguchi, T. (1989) Cell 58, 729-739[Medline] [Order article via Infotrieve] |
15. | Miyamoto, M., Fujita, T., Kimura, Y., Maruyama, M., Harada, H., Sudo, Y., Miyata, T., and Taniguchi, T. (1988) Cell 54, 903-913[Medline] [Order article via Infotrieve] |
16. | Matsuyama, T., Kimura, T., Kitagawa, M., Pfeffer, K., Kawakami, T., Watanabe, N., Kundig, T. M., Amakawa, R., Kishihara, K., Wakeham, A., Potter, J., Furlonger, C. L., Narendran, A., Suzuki, H., Ohashi, P. S., Paige, C. J., Taniguchi, T., and Mak, T. W. (1993) Cell 75, 83-97[Medline] [Order article via Infotrieve] |
17. | Reis, L. F., Ruffner, H., Stark, G., Aguet, M., and Weissmann, C. (1994) EMBO J. 13, 4798-4806[Abstract] |
18. | Agalioti, T., Lomvardas, S., Parekh, B., Yie, J., Maniatis, T., and Thanos, D. (2000) Cell 103, 667-678[Medline] [Order article via Infotrieve] |
19. |
Au, W. C.,
and Pitha, P. M.
(2001)
J. Biol. Chem.
276,
41629-41637 |
20. |
Au, W. C.,
Moore, P. A.,
LaFleur, D. W.,
Tombal, B.,
and Pitha, P. M.
(1998)
J. Biol. Chem.
273,
29210-29217 |
21. |
Barnes, B. J.,
Moore, P. A.,
and Pitha, P. M.
(2001)
J. Biol. Chem.
276,
23382-23390 |
22. |
Lin, R.,
Mamane, Y.,
and Hiscott, J.
(2000)
J. Biol. Chem.
275,
34320-34327 |
23. |
Marie, I.,
Durbin, J. E.,
and Levy, D. E. S.
(1998)
EMBO J.
17,
6660-6669 |
24. | Sato, M., Suemori, H., Hata, N., Asagiri, M., Ogasawara, K., Nakao, K., Nakaya, T., Katsuki, M., Noguchi, S., Tanaka, N., and Taniguchi, T. (2000) Immunity 13, 539-548[Medline] [Order article via Infotrieve] |
25. |
Yeow, W. S.,
Au, W. C.,
Juang, Y. T.,
Fields, C. D.,
Dent, C. L.,
Gewert, D. R.,
and Pitha, P. M.
(2000)
J. Biol. Chem.
275,
6313-6320 |
26. |
Yeow, W.-S.,
Au, W.-C.,
Lowther, W. J.,
and Pitha, P. M.
(2001)
J. Virol.
75,
3021-3027 |
27. | Au, W.-C., Moore, P. A., Lowther, W., Juang, Y.-T., and Pitha, P. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11657-11661[Abstract] |
28. | Zhang, L., and Pagano, J. S. (1997) Mol. Cell. Biol. 17, 5748-5757[Abstract] |
29. | Sato, M., Hata, N., Asagiri, M., Nakaya, T., Taniguchi, T., and Tanaka, N. (1998) FEBS Lett. 441, 106-110[CrossRef][Medline] [Order article via Infotrieve] |
30. | Taniguchi, T., Takaoka, H., Takayanagi, H., and Kenya, H. (2002) J. Interferon Cytokine Res. 22, 37[CrossRef] (Abstr. PL-1-4) |
31. |
Juang, Y.,
Lowther, W.,
Kellum, M.,
Au, W. C.,
Lin, R.,
Hiscott, J.,
and Pitha, P. M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9837-9842 |
32. |
Lin, R.,
Heylbroeck, C.,
Pitha, P. M.,
and Hiscott, J.
(1998)
Mol. Cell. Biol.
18,
2986-2996 |
33. | Wathelet, M. G., Lin, C. H., Parekh, B. S., Ronco, L. V., Howley, P. M., and Maniatis, T. (1998) Mol. Cell 1, 507-518[Medline] [Order article via Infotrieve] |
34. |
Weaver, B. K.,
Kumar, K. P.,
and Reich, N. C.
(1998)
Mol. Cell. Biol.
18,
1359-1368 |
35. |
Yoneyama, M.,
Suhara, W.,
Fukuhara, Y.,
Fukuda, M.,
Nishida, E.,
and Fujita, T.
(1998)
EMBO J.
17,
1087-1095 |
36. |
Lin, R.,
Heylbroeck, C.,
Genin, P.,
Pitha, P. M.,
and Hiscott, J.
(1999)
Mol. Cell. Biol.
19,
959-966 |
37. |
Marie, I.,
Smith, E.,
Prakash, A.,
and Levy, D. E.
(2000)
Mol. Cell. Biol.
20,
8803-8814 |
38. | Au, W. C., Yeow, W. S., and Pitha, P. M. (2001) Virology 280, 273-282[CrossRef][Medline] [Order article via Infotrieve] |
39. | Hiscott, J., Cantell, K., and Weissmann, C. (1984) Nucleic Acids Res. 12, 3727-3746[Abstract] |
40. | Megyeri, K., Au, W. C., Rosztoczy, I., Raj, N. B., Miller, R. L., Tomai, M. A., and Pitha, P. M. (1995) Mol. Cell. Biol. 15, 2207-2218[Abstract] |
41. | Thanos, D., and Maniatis, T. (1995) Cell 83, 1091-1100[Medline] [Order article via Infotrieve] |
42. |
Siegal, F. P.,
Kadowaki, N.,
Shodell, M.,
Fitzgerald-Bocarsly, P. A.,
Shah, K.,
Ho, S.,
Antonenko, S.,
and Liu, Y. J.
(1999)
Science
284,
1835-1837 |
43. |
Au, W.-C.,
Su, Y.,
Raj, N. B. K.,
and Pitha, P. M.
(1993)
J. Biol. Chem.
268,
24032-24040 |
44. | Dent, C. L., and Gewert, D. R. (1996) Eur. J. Biochem. 236, 895-903[Abstract] |
45. |
Raj, N. B. K.,
Au, W.-C.,
and Pitha, P. M.
(1991)
J. Biol. Chem.
266,
11360-11365 |
46. | Au, W. C., Raj, N. B., Pine, R., and Pitha, P. M. (1992) Nucleic Acids Res. 20, 2877-2884[Abstract] |
47. |
Cheung, S. C.,
Chattopadhyay, S. K.,
Hartley, J. W.,
Morse, H. C. I.,
and Pitha, P. M.
(1991)
J. Immunol.
146,
121-127 |
48. | Kelley, K. A., and Pitha, P. M. (1985) Nucleic Acids Res. 13, 825-839[Abstract] |
49. |
Lin, R.,
Mamane, Y.,
and Hiscott, J.
(1999)
Mol. Cell. Biol.
19,
2465-2474 |
50. |
Jesse, T. L.,
LaChance, R.,
Iademarco, M. F.,
and Dean, D. C.
(1998)
J. Cell Biol.
140,
1265-1276 |
51. | Aziz, F., van Wijnen, A. J., Vaughan, P. S., Wu, S., Shakoori, A. R., Lian, J. B., Soprano, K. J., Stein, J. L., and Stein, G. S. (1998) Mol. Biol. Rep. 25, 1-12[CrossRef][Medline] [Order article via Infotrieve] |
52. |
Vaughan, P. S.,
van der Meijden, C. M.,
Aziz, F.,
Harada, H.,
Taniguchi, T.,
van Wijnen, A. J.,
Stein, J. L.,
and Stein, G. S.
(1998)
J. Biol. Chem.
273,
194-199 |
53. |
Kim, Y. M.,
Kang, H. S.,
Paik, S. G.,
Pyun, K. H.,
Anderson, K. L.,
Torbett, B. E.,
and Choi, I.
(1999)
J. Immunol.
163,
2000-2007 |
54. | Marecki, S., and Fenton, M. J. (2000) Cell Biochem. Biophys. 33, 127-148[Medline] [Order article via Infotrieve] |
55. |
Marecki, S.,
Riendeau, C. J.,
Liang, M. D.,
and Fenton, M. J.
(2001)
J. Immunol.
166,
6829-6838 |
56. |
Meraro, D.,
Gleit-Kielmanowicz, M.,
Hauser, H.,
and Levi, B. Z.
(2002)
J. Immunol.
168,
6224-6231 |
57. |
Rehli, M.,
Poltorak, A.,
Schwarzfischer, L.,
Krause, S. W.,
Andreesen, R.,
and Beutler, B.
(2000)
J. Biol. Chem.
275,
9773-9781 |
58. |
Kuwata, T.,
Gongora, C.,
Kanno, Y.,
Sakaguchi, K.,
Tamura, T.,
Kanno, T.,
Basrur, V.,
Martinez, R.,
Appella, E.,
Golub, T.,
and Ozato, K.
(2002)
Mol. Cell. Biol.
22,
7439-7448 |
59. |
Caillaud, A.,
Prakash, A.,
Smith, E.,
Masumi, A.,
Hovanessian, A. G.,
Levy, D. E.,
and Marie, I. J.
(2002)
J. Biol. Chem.
277,
49417-49421 |
60. | Hiscott, J., Pitha, P., Genin, P., Nguyen, H., Heylbroeck, C., Mamane, Y., Algarte, M., and Lin, R. (1999) J. Interferon Cytokine Res. 19, 1-13[CrossRef][Medline] [Order article via Infotrieve] |
61. |
Kumar, K. P.,
McBride, K. M.,
Weaver, B. K.,
Dingwall, C.,
and Reich, N. C.
(2000)
Mol. Cell. Biol.
20,
4159-4168 |
62. |
Suhara, W.,
Yoneyama, M.,
Kitabayashi, I.,
and Fujita, T.
(2002)
J. Biol. Chem.
277,
22304-22313 |
63. | Lubyova, B., Simelyte, E., and Pitha, P. M. (2002) J. Interferon Cytokine Res. 22, 121[CrossRef][Medline] [Order article via Infotrieve] (Abstr. P-5-22) |
64. | Marie, I., Prakash, A., Smith, E., Hovanessian, A., and Levy, D. (2002) J. Interferon Cytokine Res. 22, 56 (Abstr. W-6-6) |
65. | Suhara, W., Yoneyama, M., Kitabayashil, I., and Fujita, T. (2002) J. Interferon Cytokine Res. 22, 131 (Abstr. P-6-13) |
66. | Nahle, Z., Polakoff, J., Davuluri, R. V., McCurrach, M. E., Jacobson, M. D., Narita, M., Zhang, M. Q., Lazebnik, Y., Bar-Sagi, D., and Lowe, S. W. (2002) Nat. Cell Biol. 4, 859-864[CrossRef][Medline] [Order article via Infotrieve] |
67. |
Island, M. L.,
Mesplede, T.,
Darracq, N.,
Bandu, M. T.,
Christeff, N.,
Djian, P.,
Drouin, J.,
and Navarro, S.
(2002)
Mol. Cell. Biol.
22,
7120-7133 |