| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Department of Food Engineering and Biotechnology, Technion, Haifa, Israel; and
Department of Gene Regulation and Differentiation, Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-induced factor
explains why some of the EIREs are also induced by type II IFN. Our
results lay the molecular basis for the unique regulation of ISGs,
harboring EIRE, in immune cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
or 
) confer an antiviral state, affect cell
growth and differentiation, and modulate the immune system. Type I IFNs
initiate a signaling cascade that results in the induction of a subset
of genes termed IFN-stimulated genes
(ISGs).4 This
induction is mediated by a specific DNA binding elements designated
IFN-stimulated response elements (ISREs), which are located at their
promoters (1). The ISRE element serves as the binding site
for a family of transcription factors termed IFN regulatory factors
(IRFs). Nine cellular IRFs that execute part of the effects of IFN have
been identified to date. Some of the IRFs, such as IRF-1, IRF-2, IRF-3,
and IRF-7, bind directly to ISRE elements, while others, such as IRF-4
and IRF-8/IFN consensus sequence binding protein (ICSBP), do not bind
effectively to the ISRE element alone (for review, see Ref.
2). However, IRF-8\/ICSBP can associate with either IRF-1
or IRF-2, forming heterocomplexes that bind efficiently to the DNA
(3, 4). Further, both IRF-4 and IRF-8\/ICSBP also
associate with non-IRF members such as PU.1 or E47 on a DNA composite
element, half of which is an IRF binding site and half of which is the
binding site of the interacting partner (5, 6). These
protein-protein interactions are mediated through a conserved domain
termed the IRF association domain (IAD) (7). The IAD motif
was found in all seven IRFs, excluding IRF-1 and IRF-2, which associate
with IRF-8\/ICSBP. In contrast, a domain rich in Pro, Glu, Ser,
and Thr (PEST) mediates the interaction of PU.1,E47, IRF-1, and IRF-2
with IRF-8\/ICSBP. Further, the IAD and the PEST domains also mediate
the interactions between IRF-4 and PU.1 or E47 (6, 7, 8). The expression of both IRF-4 and IRF-8\/ICSBP is limited to immune cells (5, 9, 10, 11). Studies with knockout mice demonstrated that IRF-4 plays a crucial role in controlling the activation and homeostasis of immune responses and is critical for proper maturation of B cells (12). IRF-8\/ICSBP is essential for myeloid cell differentiation toward mature macrophages (13). Both IRFs possess a dual role in lymphoid cells; binding to ISRE elements leads to a repression of ISG expression, while interaction with lymphoid/myeloid essential transcription factors (PU.1 and E47) leads to transcriptional activation (5, 9, 10, 11). PU.1 is predominately expressed in cells of the hemopoietic lineage and is a critical regulator of myeloid/lymphocyte differentiation. PU.1 can bind alone to a core motif (A/GGAA) that is shared by most ETS proteins (14); however, additional flanking sequences define the ultimate binding specificity: for example, the ETS/IRF composite element (EICE) to which IRF-4 and IRF-8\/ICSBP bind following interaction with PU.1. Such composite DNA motifs were identified in numerous genes essential for proper macrophage and B cells function (15).
The ISG15 gene, which contains ISRE element in its promoter, is one of
the most strongly induced proteins in cells following IFN type I
treatment or viral infection (16, 17, 18). Deletion or
mutations in this ISRE element lead to the inability to respond to
these signals (16). ISG15 is a 17-kDa ubiquitin-like
protein produced as a precursor protein. Correct processing is
essential for its conjugation to cellular target and for its
extracellular cytokine function (19, 20). ISG15 is
secreted from monocytes, and the secreted form leads to NK
proliferation and augmentation of non-MHC-restricted cytotoxicity.
Thus, ISG15 may be considered a cytokine responsible for augmenting and
amplifying the immunomodulatory effects of IFN- or IFN-.
Here we show that ISG15 contains a unique ISRE element that enables the binding not only of IRFs, but also of PU.1. This ISRE represents a subtype of ISREs that, in addition to IRFs, recruits PU.1. This composite ISRE element is synergistically activated by a heterocomplex formed between PU.1 and either IRF-8\/ICSBP or IRF-4. These interactions lead to specific regulation of ISG15 in immune cells, laying the molecular basis for its unique role in these cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
NIH-3T3, U937, Namalwa, and K562 cells were obtained from American Type Culture Collection (Manassas, VA). NIH-3T3 were maintained in DMEM, while the other cell lines were maintained in RPMI 1640. All media were supplemented with 10% FCS, except for Namalwa cells, which were supplemented with 7.5% FCS.
Plasmids
Mammalian expression vectors, which are also suitable for in vitro transcription (IVT), corresponding to IRF-1, IRF-4, PU.1, IRF-8\/ICSBP, ICSBPL331P, and ICSBPS260A, were previously described (7, 21).
The plasmids pCB6PU.1
PEST and pCB6PU.1S148A were obtained from Dr.
M. Atchison (University of Pennsylvania, Philadelphia, PA)
(22). Site-directed mutagenesis of tyrosine residues Y23
and Y48 within the DNA binding domain (DBD) of IRF-8\/ICSBP (ICSBPY23F
and ICSBPY48F) to phenylalanine were generated using the plasmid
pTarget-ICSBP (7) and the GeneEditor kit (Promega,
Madison, WI) with the primers Y23F
(5'-GACAGTAGCATGTTTCCAGGCCTGATTTGGG-3') and Y48F
(5'-GGAAACACGCCGGCAAGCAAGATTTTAAT-3').
The plasmid p(ISRE)4-LUC, obtained from Dr. K. Ozato (National Institutes of Health, Bethesda, MD), is a reporter gene plasmid in which the luciferase gene (firefly) is driven by the thymidine kinase basal promoter connected to four repeats of the ISG15-ISRE in the basic plasmid pBL-2 (Promega). In the plasmid p1.4Ld-LUC, 1.4 kb of the promoter region of the Ld gene from the murine MHC class I is driving the luciferase (firefly) reporter gene (obtained from Dr. K. Ozato, National Institutes of Health). To monitor transfection efficiency, a dual luciferase reporter assay was used with the plasmid pMDISRluc, in which the SV40 promoter drives the expression of Renilla luciferase.
DNA transfections and reporter gene analyses
NIH-3T3 cells were transfected by the calcium phosphate-DNA coprecipitation method as described previously (7, 23). Cells were plated in a six-well dish and transfected with 400 ng to 1 µg of the various expression plasmids and reporter plasmids (firefly luciferase), 600 ng pMDISRluc (SV40 promoter driving the expression of Renilla luciferase to monitor transfection efficiencies), and pUC19 serving as carrier DNA up to total of 3 µg. The expression plasmids coding for the various transcription factors and reporter genes are indicated in the text. The cells were harvested 48 h later and lysed using the lysis buffer with the Dual Luciferase assay kit (Promega), and luciferase activities were determined according to the manufacturer’s instructions using a TD-20/20 luminometer (Turner Design; Promega). Reporter gene activities were normalized for protein concentration and transfection efficiencies as previously described (24). Western blot analyses were performed with each transfected expression vector to ensure the expected level of ectopic protein expression (data not shown). Each set of transfection experiments was repeated at least three times, generating similar results.
U937 cells were diluted to 106 cells/ml 16–24 h
before transfection. At the time of transfection the cells were washed
twice in PBS and suspended at 2.5 x
107cells/ml in RPMI medium lacking FCS and
antibiotics. Part (0.4 ml) of the cell suspension was placed into a
0.4-cm electroporation cuvette (Bio-Rad, Richmond, CA), and up to 30
µg plasmid DNA suspended in 
20 µl distilled water was added.
Cells and plasmids DNA were incubated for 10 min at room temperature
before electroporation at 500 µF and 300 V (Bio-Rad gene pulser).
Following the electric shock the cells were left at room temperature
for an additional 15 min and then diluted into 10 ml RPMI
containing 10% FCS. Forty-eight hours after electroporation the cells
were harvested and analyzed as described above for NIH-3T3 cells.
In vitro transcription and translation
The assays were performed as described previously (7). Plasmids containing the gene of interest under the T7 promoter were linearized downstream of the coding region with the appropriate restriction enzyme. Five micrograms of linearized plasmids were in vitro transcribed by T7 RNA polymerase using a commercial kit (Stratagene, La Jolla, CA). Proteins were translated in vitro using the rabbit reticulocyte lysate system (Promega) according to the manufacturer’s instructions. To monitor translation efficiency, small scale reactions containing [35S]methionine were performed each time, and the labeled proteins were separated on 10% SDS-PAGE and subjected to autoradiography.
EMSA
Nuclear extracts and gel-shift reactions were conducted as previously described (24, 25). A typical reaction contained 1–5 µl IVT proteins or 5–8 µg nuclear extracts that were incubated in binding buffer (10 mM HEPES (pH 8), 5 mM MgCl2, 50 mM KCl, 0.025% bromophenol blue, 0.005% xylene cyanole, 10% Ficoll, 3% glycerol, and 1 µg sonicated polyd(IC)) for 20 min. One microgram of sheared salmon sperm DNA was added only with IVT proteins. At least 50,000 cpm of the labeled probe (ISG15-ISRE) was added for an additional 10 min on ice. The two synthetic oligonucleotides corresponding to the ISG15-ISRE (5'-GATCCTCGGGAAAGGGAAACCGAAACTGAAGCC-3') were annealed and labeled with Klenow fragment. The samples were loaded on a pre-run 6.5% polyacrylamide gel. The dried gels were exposed to x-ray films. For supershift reaction, 1–2 µl Abs was added, and the reactions were incubated for 60–90 min on ice before addition of the labeled probe. Abs directed against PU.1 were purchased from BD PharMingen (San Diego, CA), and Abs against IRF-4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-peptide Abs against IRF-8\/ICSBP (anti-pepC) were obtained from Dr. K. Ozato. Antipeptide Abs against IRF-1 (pep300) and IRF-2 (pep311) were previously described (26).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ISGs are regulated by type I IFNs through the binding of IRFs to
the ISRE located at their promoter region. IRF-1 is a prototype IRF,
which transcriptionally activates such promoters. We have previously
shown that IRF-8\/ICSBP can associate with IRF-1, and this association
leads to repression of IRF-1 action on either the ISRE of MHC class I
or the positive regulatory domain I (PRDI) element (a core sequence of
ISRE) located on the promoter of IFN- (24, 27). Here we
show that the same effect of IRF-8\/ICSBP on IRF-1 also takes place on
the ISRE element of ISG15. Fig. 1
A shows that IRF-1 can
activate the expression of a reporter gene driven by four ISRE repeats
from the promoter of ISG15 (Fig. 1A, lane 2).
Cotransfection of IRF-8\/ICSBP leads to repression (Fig. 1A,
lane 8). Previously, we have shown that this repression is
mediated through association between IRF-1 and the IAD module of
IRF-8\/ICSBP. This was demonstrated by mutating the conserved leucine
331, which is located in the IAD in a predicted -helix structure to
proline. This point mutation interferes with this structured domain,
leading to a mutant IRF-8\/ICSBP incapable of interacting with other
factors (7). It is clear from Fig. 1A that this
mutation also affects the ability of IRF-8\/ICSBP to repress IRF-1 on
the ISG15-ISRE element (Fig. 1A, compare lane 9
to lane 2). Further, we have also shown that serine 260 in
IRF-8\/ICSBP is essential for protein-protein interaction
(21). Similar results are demonstrated here; mutation of
this serine residue within the IAD of IRF-8\/ICSBP eliminates its
ability to negate IRF-1 (Fig. 1A, lane 10). In
contrast, mutating tyrosine residues within the DBD of IRF-8\/ICSBP to
phenylalanine (Y48F and Y23F) have no effect on the repression activity
of IRF-8\/ICSBP (Fig. 1A, lanes 11 and
12, respectively).
|
B shows that unlike IRF-8\/ICSBP, IRF-4 elicits weak
transcriptional activation on a reporter gene driven by ISG15-ISRE
(Fig. 1B, lane 3). Cotransfection of IRF-1 and
IRF-4 at equimolar levels does not lead to synergistic repression, but,
rather, to a mid-level expression, which is probably a result of
co-occupancy of the ISRE elements by both IRF-1 and IRF-4 (Fig. 1B, lane 4). Cotransfection of IRF-1 with
IRF-8\/ICSBP and IRF-4 leads to an additional decrease in the reporter
gene level (Fig. 1B, lane 5). These results are
further supported by EMSA, as shown in Fig. 1C. It is clear
that unlike IRF-1, neither IRF-8\/ICSBP nor IRF-4 binds to the ISRE
element alone (Fig. 1C, lanes 2, 4,
and 5, respectively). Yet IRF-1 and ICSBP form a
heterocomplex (Fig. 1C, lane 3) that was
supershifted with Abs directed against each of the interacting factors
(data not shown). In contrast, no heterocomplex band was observed for
IRF-1 and IRF-4 or IRF-8\/ICSBP and IRF-4 (Fig. 1C,
lanes 6 and 7, respectively). The same phenomenon
was also observed with IRF-2 (data not shown). These results are
consistent with our previous data showing that only IRF-8\/ICSBP can
interact with either IRF-1 or IRF-2, and this interaction leads to
synergistic repression (7, 24). Such an effect was not
detected with IRF-4. PU.1 and either IRF-8\/ICSBP or IRF-4 elicit synergistic activation of ISG15-ISRE
The ISG15 expression is dysregulated in macrophages from
IRF-8ICSBP knockout mice, demonstrating its unique role in these cells
(28). This prompted us to determine whether this
dysregulated expression can be attributed to lack of heterocomplex
formation between PU.1 and IRF-8\/ICSBP, which are both essential for
myeloid cell differentiation (14). To test this
possibility, transient transfection studies were performed with PU.1,
IRF-8\/ICSBP and their mutants (Fig. 2).
Cotransfection of IRF-8\/ICSBP and PU.1 leads to the activation of a
reporter gene driven by the ISG15-ISRE (Fig. 2A, compare
lanes 2 and 3 to lane 8). These
results are in contrast to the results observed with IRF-1 (Fig. 1).
This activation is dependent on protein-protein interaction, because
cotransfection of mutant IRF-8\/ICSBP expression constructs defective
in the IAD (mutation of the conserved leucine 331 or the nonconserved
serine 260) leads to elimination of this transcriptional synergy (Fig. 2A, compare lane 8 to lanes 9 and
10). In addition, this interaction is dependent on the
presence of tyrosine residues 23 and 48 in IRF-8\/ICSBP, which are
conserved in IRF-4. Mutating these residues to phenylalanine diminished
the synergistic activity with PU.1 (Fig. 2A, lanes
11 and 12). It is possible that the phosphorylation
state of these residues is essential for the interaction with only
PU.1, because these mutations on tyrosine residues had no effect on the
interaction with IRF-1 (Fig. 1) and IRF-2 (data not shown). This
suggests that the interaction of PU.1 with IRF-8\/ICSBP is dependent
upon its intact IAD as well as DBD. Synergistic activation of the
ISG15-driven reporter gene by IRF-8ICSBP and PU.1 was also observed
with the promonocytic cell line U937, which constitutively expresses
these two factors (data not shown). To avoid endogenous background
expression of these genes, which may interfere with the ectopic
expression, additional experiments were performed with NIH-3T3 cells,
which do not express these genes.
|
B clearly show that
the synergistic effect of PU.1 and IRF-8\/ICSBP on the ISRE of ISG15 is
lost when either serine 148 is mutated to alanine or the whole PEST
domain is deleted (Fig. 2B, compare lanes 6,
7, and 8, respectively).
IRF-4 was the first IRF member that demonstrated transcriptional
synergy with PU.1 on EICE. Therefore, we tested whether such a
synergistic effect also takes place on the classical ISRE element of
ISG15. The data in Fig. 2C clearly show a synergistic effect
of PU.1 and IRF-4 on the expression of an ISRE-driven reporter gene
(Fig. 2C, lane 5). This synergistic effect is
also dependent upon intact IRF-4-IAD, because mutation of the conserved
leucine 386 to proline (corresponding to leucine 331 in IRF-8\/ICSBP),
which interferes with the predicted -helix structure, also results
in a lack of synergistic activity of this mutant IRF-4 with PU.1 (Fig. 2C, lane 6).
To determine whether the synergistic effect between PU.1 and
IRF-8\/ICSBP or IRF-4 is unique to the ISRE of ISG15, we also tested
the MHC class I promoter containing characterized ISRE and the PRDI
element from the IFN- promoter (10, 27). No synergistic
effect between PU.1 and IRF-8\/ICSBP was observed on a luciferase
reporter gene driven by either MHC class I ISRE (Fig. 2D) or
the PRDI element of IFN- (data not shown). These results suggest
that the observed synergistic activity of PU.1 is not common to all
ISREs.
The ISRE of ISG15 supports association between PU.1 and either IRF-8\/ICSBP or IRF-4
The above data demonstrate that the ISRE of ISG15 is
synergistically activated by PU.1 and either IRF-8\/ICSBP or IRF-4. To
demonstrate the physical interaction between these factors, EMSA was
performed. PU.1 as well as IRF-8\/ICSBP and various mutants of
IRF-8\/ICSBP were in vitro-translated and reacted with the labeled ISRE
element of ISG15. Neither PU.1 nor IRF-8\/ICSBP binding to this ISRE
element could be detected in this assay (Fig. 3A, lanes 2 and
3, respectively). However, when the two proteins were mixed
together a new band corresponding to the heterocomplex band was
observed (Fig. 3A, lane 4). This heterocomplex
band was supershifted with Abs directed against IRF-8\/ICSBP and was
eliminated by Abs against PU.1 (data not shown). This interaction was
dependent upon intact IRF-8\/ICSBP-IAD, because the mutations in
leucine 331 to proline and serine 260 to alanine lead to either loss of
the heterocomplex band (Fig. 3A, lane 11, for
ICSBPL331P) or a significant reduction in its intensity (Fig. 3A, lane 10, for ICSBPS260A). The EMSA results
with ICSBP-IAD are in agreement with the cotransfection studies, which
demonstrated that these mutants lost their ability to synergize with
PU.1 (Fig. 2). We also tested mutants of ICSBP in tyrosine residues
within the DBD, which are conserved in IRF-4 (Y23F and Y48F). It is
clear that mutation at tyrosine 48 lead to reduced interaction as
demonstrated by a weaker heterocomplex band, while mutation at tyrosine
23 leads to increased interaction (Fig. 3A, lanes
8 and 6, respectively).
|
B demonstrates that a similar heterocomplex band is
formed between PU.1 and IRF-4 (lane 3) on the ISRE
from the ISG15 promoter. This band was not detected with IRF-4L368P
mutant (Fig. 3B, lane 4). Further, no
heterocomplex was detected when IRF-8\/ICSBP and IRF-4 were reacted
with the ISRE of ISG15 (Fig. 3B, lane 5).
Interestingly, when all three proteins were mixed together, a much
stronger heterocomplex band was observed (Fig. 3B,
lane 6). This suggests that the heterocomplex might be
composed of all three components (PU.1, IRF-8\/ICSBP, and IRF-4) in an
unknown stoichiometry.
No heterocomplex bands were observed with PU.1, IRF-8ICSBP, and
IRF-4 when the ISRE of MHC class I or the PRDI element of IFN- was
used as a probe in the EMSA (data not shown).
Interaction between PU.1 and either IRF-8\/ICSBP and/or IRF-4 is also detected in hemopoietic cells
We next wanted to determine whether heterocomplexes between PU.1
and either IRF-8\/ICSBP and IRF-4 also occur in vivo. For that purpose,
nuclear extracts were prepared from the promonocytic cell line U937 and
the B lymphocyte cell line Namalwa, which both constitutively express
all three factors. In addition, nuclear extract was prepared from the
erythroleukemia cell line K-562, which expresses PU.1, but does not
express IRF-8\/ICSBP or IRF-4. Five major bands (I, II, II, IRF-2, and
PU.1) were observed in EMSA using labeled ISRE from ISG15 and U937 cell
extract (Fig. 4, upper panel).
Abs against either PU.1 or IRF-2 enabled us to detect the bands
corresponding to the binding of only these factors compared with cell
extract reacted with preimmune serum (Fig. 4, upper panel,
lanes 3, 5, and 1, respectively). In
addition, these two Abs led to a reduction in the intensity of
all the three upper bands (I, II, and III). However, the decrease in
the intensity of band II was most prominent. This suggested that the
band marked as II might represent the major heterocomplex bands between
PU.1 and either IRF-8\/ICSBP and IRF-4 or the heterocomplex band
between IRF-2 and only IRF-8\/ICSBP. Accordingly, the addition of Abs
directed against IRF-8\/ICSBP and IRF-4 mainly eliminated this band
(band II, Fig. 4, upper panel, lanes 2 and
4, respectively). Abs against IRF-2, IRF-8\/ICSBP, and IRF-4
led to the appearance of a shifted band, while Abs directed against
PU.1 resulted in band elimination (Fig. 4, upper
panel, compare lanes 2, 4, and
5 to lane 3). In contrast, Abs directed against
IRF-1 did not lead to any change in the band pattern compared with that
in the control (Fig. 4, upper panel, compare
lane 6 to lane 1), but, rather, led to a stronger
signal. Similar results were observed with Namalwa cell extract (Fig. 4, middle panel). When K562 cell extract, which does not
express IRF-8\/ICSBP and IRF-4, was used, there was no effect on the
bands pattern with Abs against IRF-8\/ICSBP, IRF-4, PU.1, and IRF-1. A
supershifted band is clearly seen with Abs directed against IRF-2,
resulting in a significant reduction in the intensity of band II.
Altogether these results demonstrate that all the factors, PU.1,
IRF-8\/ICSBP, IRF-4, and IRF-2 (with the exception of IRF-1), interact
with the ISRE of ISG15. These interactions are dependent upon the
composition of transcription factors in the cells, because such
interactions were not observed in K562 cell extract.
|
C), but also through interactions with non-IRF
members, e.g., PU.1. The core motif for PU.1 DNA binding is GGAA
(29), and the ISRE of ISG15 contains two GGAA motifs. To
define the region essential for the interaction observed in U937 cells,
five mutant oligonucleotides (illustrated in Fig. 5A) were synthesized and used
as competitors in the EMSA. It is clear that the M1 oligonucleotide, in
which the 5'-positioned GGAA motif was mutated, competed for the
binding as well as the wild-type oligonucleotide (Fig. 5B,
compare lanes 2 and 3 to lane 1). In
contrast, the M2 oligonucleotide, in which the 3'-positioned GGAA motif
was mutated, did not compete for the binding (Fig. 5B,
lane 4). Like the M2 oligonucleotide, the M5
oligonucleotide, in which both putative PU.1 binding sites were
mutated, did not elicit any competition. This suggests that the
3'-positioned GGAA motif, which is closer to the IRF binding site, is
essential for protein-protein interaction. The M3 and the M4
oligonucleotides were mutated in the two possible IRF binding sites,
ACCGAAA and ACTGAA. The data show that only partial competition was
achieved with each oligonucleotide, suggesting that both are essential
for IRF binding.
|
demonstrate that the M2 mutant had the
strongest effect on PU.1 binding and the interaction with IRF-8\/ICSBP
and IRF-4 (similar to the pattern seen with M5). Further, when M4 was
used as a probe, no heterocomplex band with IRF-8\/ICSBP and IRF-4 was
observed, although the binding of IRF-2 was still detectable.
These experiments show that the 3'-positioned GGAA and the 3' IRF
binding site (AACTGA) are essential for the formation of
heterocomplexes between PU.1 and either IRF-8\/ICSBP or IRF-4 on the
ISRE element of ISG15. Both IRF binding sites were able to recruit
IRF-2, because its binding was not lost with any of the mutant
oligonucleotides. Taken together our data suggest that the ISG15-ISRE
allows not only the binding of IRF members, but also the binding of
PU.1, and should be considered a DNA composite element.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(in the context of ISGF3) to bind this
element was demonstrated by EMSA, and the effect on transcriptional
regulation was shown in many cell types and tissues (16, 32, 33). Further, while using this ISRE it was shown that both IRF-1
and IRF-2 can associate with IRF-8\/ICSBP to form heterocomplexes, and
these complexes possess repression activity in immune cells (3, 4).
In this communication we provide evidence that the ISRE of ISG15 is
synergistically activated by the myeloid/lymphocyte-restricted factors
PU.1 and either IRF-8\/ICSBP or IRF-4. This synergistic activity is
further supported by the EMSA results showing that heterocomplexes
between these factors are detectable with in vitro-translated proteins
or in nuclear extract from cell lines of monocytic and lymphocytic
origin. These results demonstrate that this ISG15-ISRE element is able
to recruit not only IRFs, but also a non-IRF member such as PU.1, which
belong to the ETS family of transcription factors. Interaction among
IRF-8\/ICSBP, IRF-4, and PU.1 was reported for numerous enhancer
elements in many genes that are essential for proper biological
activities of either macrophages or B cells. These interactions occur
on EICE, which were identified on enhancer elements of Ig L chain,
CD20, IL-1, Toll-like receptor 4, and more (for review, see Ref.
15). This is consistent with the restricted expression of
these factors in cells of myelocyte/lymphocyte origin. Our results
clearly show that the classical ISG15-ISRE can also recruit PU.1 and
therefore can also be defined as EICE. Sequence alignment of numerous
ISRE elements displayed in Table II
clearly shows that classical ISREs can be divided into two categories.
The first category (subtype) consists of ISRE elements that recruit
only IRFs (Table II, upper section). The lower
part of Table II shows classical EICE elements in which the PU.1
binding site (GGAA) is shaded, and the IRF binding site is in the boxed
area adjacent to the PU.1 element. The middle part of Table II shows classical ISREs from many ISGs that display sequence
similarity to the ISG15-ISRE. The PU.1 binding site is shaded, and the
IRF binding site is in the boxed area. The formation of heterocomplexes
between IRF-8ICSBP and either IRF-1 or IRF-2 was reported for ISG15
such as ISREs (ISG15 and ISG54). This may reflect the difference
between classical EICEs, which are characterized by the formation of
heterocomplexes with only PU.1 and either IRF-8\/ICSBP or IRF-4. The
major difference between these two subtypes is an extra adenosine
residue just downstream from the PU.1 binding site. The extra base
results in the formation of two IRF binding sites characterized by
GAAANN. This explains the difference between EICEs, which have only one
PU.1 binding site and one IRF binding site and ISG15 such as ISREs that
contain, in addition to the PU.1 binding site, two putative ISREs. This
fits with the fact that these elements allow the formation of IRFs
heterocomplexes as well as PU.1/IRF heterocomplexes, thus designated
here as ETS/IRF response element (EIRE). The data presented here
support this duality of the ISRE of ISG15 with respect to heterocomplex
formation. Similar results were obtained with regulatory
elements from CYBB and NCF2 genes, which encode for the
phagocyte respiratory burst oxidase subunits
gp91phox and p67phox,
respectively. These results suggest that the CYBB and NCF2 enhancer
elements are EIRE and as such can be engaged by IRFs heterocomplexes as
well as PU.1IRFs heterocomplexes as reported recently
(34).
|
as harboring EIREs
also possess a unique role in immune cells. For example, the induced
expression of ISG15 by type I IFNs in many cell types and tissues is
well documented. However, ISG15 is also secreted from monocytes and
lymphocytes and modulates the immune system. Its ability to activate NK
cells is dependent on cocultivation with T cells (19).
Similarly, the induced expression of the IFN-induced and
dsRNA-activated kinase (PKR) in many cell types and tissues, resulting
in antiviral and antiproliferative effects, is well established
(35). Yet, recent studies also implicated PKR in the
growth control of mature T lymphocytes (36). IFN-inducible
protein 10 is induced and secreted by many cell types.
Costimulation of macrophages by LPS and IFN- leads to its expression
and subsequent effect on Th1 cells harboring the target receptor,
CXCR3 (37). The 9–27 gene is also a prototypic ISG
that is expressed in many cell types as a cell surface protein.
However, it has a unique role in B cells, being a component of a larger
B cell CD81 complex involved in signal transduction and cell adhesion
(38).
The fact that these EIRE elements can recruit IRF-8\/ICSBP or IRF-4
suggests that these elements will respond not only to IFN type I, but
also to IFN-, which leads to transcriptional induction of
IRF-8\/ICSBP and to antigenic stimulation resulting in the induction of
both IRF-8\/ICSBP and IRF-4. Hence, PKR, IFN-inducible protein 10,
9–27, CYBB, and NCF2 are also induced by IFN- or antigenic
stimulation or both (34, 36, 37, 38).
We show that the interaction of PU.1 with either IRF-8\/ICSBP or IRF-4 is dependent upon intact interaction modules, e.g., PEST and IAD, respectively. Our data also show that serine 260, which is unique to the IAD of IRF-8\/ICSBP, is essential for this interaction, suggesting that the phosphorylation state of this residue is essential (for more details see Ref. 21). Tyrosine phosphorylation is also essential for proper activity of IRF-8\/ICSBP. Specific tyrosine phosphorylation prevents ICSBP from binding alone to target DNA; nevertheless, this phosphorylation is essential for efficient protein-protein interaction (24). Here we show that mainly tyrosine residue 48, which is shared only with IRF-4, affects the ability of IRF-8\/ICSBP to synergize and form an efficient heterocomplex with PU.1. Tyrosine residue 23, which is also conserved in IRF-4, IRF-5, and IRF-7, was not essential for the formation of heterocomplex with PU.1. These mutations of tyrosine residues in the DBD affect only the interaction with PU.1 and not that with IRF-1. These results support our published data demonstrating that the presence of target DNA is necessary for an efficient interaction of IRF-8\/ICSBP with PU.1, but not with IRF-1 (7). In addition, tyrosine residues 110, within the DBD, and tyrosine residue 211, within the IAD, are conserved in all IRFs, and mutating it to phenylalanine leads to a defective IRF-8\/ICSBP that does not interact effectively with either IRF-1/2 and PU.1 (D. Meraro and B. Z. Levi, unpublished observations) (39). These results suggest that modulation of the phosphorylation state of some of the tyrosine residues in both the DBD and the IAD might have a role in the ability of the modified factor to form DNA heterocomplexes, thus affecting gene regulation. Accordingly, SHP1 protein tyrosine phosphatase inhibits the ability of IRF-8\/ICSBP to interact with PU.1 and IRF-1, leading to reduced expression of the myeloid-specific genes CYBB (gp91phox) and NCF2 (p67phox) (39).
The multicomplexes formed on the EIRE elements of CYBB and NCF2 are
composed of PU.1, IRF-8\/ICSBP, and IRF-1 and also recruit the cAMP
response element binding protein (34). The latter harbors
histone acetyltransferase activity (40). Here we show that
the ISRE element of ISG15 recruits PU.1, IRF-8\/ICSBP, IRF-4, and
IRF-2. However, in all these studies the stoichiometry of the
multisubunit complexes is not clear. In vitro studies showed that these
EIRE can engage both IRFs heterocomplexes (e.g., IRF-8ICSBP/IRF-2 or
IRF-8\/ICSBP/IRF-1) and EtsIRFs heterocomplexes (e.g.,
PU.1/IRF-8\/ICSBP or PU.1/IRF-4). EMSA performed with nuclear extracts
prepared from U937 cells or Namalwa cells did not allow us to
distinguish between these two options. When in vitro-translated
proteins were reacted in EMSA it was clear that the interaction with
the DNA was much stronger when PU.1, IRF-8ICSBP, and IRF-4 were
incorporated (Fig. 3B, lane 6). This suggests
that such multicomplexes are formed through interaction between IADs
located on both IRF-8\/ICSBP and IRF-4 and PEST domains that were
identified on PU.1, IRF-1, and IRF-2. Our results presented here
demonstrate that both modules (IAD and PEST) were essential
for association and transcriptional synergy. Recently, a direct role
for IRF-8\/ICSBP in the regulation of ISG15 was demonstrated in
IRF-8\/ICSBP knockout mice (28). It was demonstrated that
the heterocomplex formed between IRF-2, IRF-8ICSBP, and IRF-4 was
detected in extracts from B cells, while a heterocomplex between IRF-4
and IRF-8\/ICSBP was detected in cell extracts of macrophages. However,
the presence of PU.1 in the heterocomplexes was not considered. In this
communication we provide evidence showing that PU.1 is also a major
component in the multicomplex formed on the ISRE of ISG15, which is
essential for transcriptional activation.
In conclusion, our data indicate that EIREs are unique in their ability
to recruit different factors in an assembled enhanceosomes. In
nonimmune cells the factors will mainly include IRF members. In immune
cells these EIRE elements will also recruit non-IRFs, such as PU.1, to
the assembled enhanceosome. Thus, EIREs may affect the transcriptional
capacity of different enhanceosomes in a cell type-restricted manner
that is dependent upon the milieu of transcription factors. This leads
to fine-tuning of the gene expression that is regulated in a spatial,
temporal, and restricted manner. It is intriguing to speculate that
myeloid cell genes that are specifically activated by IFN- or
inflammation are characterized by enhanceosomes with similar
characteristics. In this context, the PEST domains of PU.1, IRF-1, and
IRF-2 and the IADs of IRF-8ICSBP or IRF-4 may be used to bridge all
these elements together to the proximity of the RNA polymerase II
holoenzyme complex.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 D.M. and M.G.-K. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Ben-Zion Levi, Department of Food Engineering and Biotechnology, Technion, Haifa 32000, Israel. E-mail address: fobenzi{at}tx.technion.ac.il
4 Abbreviations used in this paper: ISG, IFN-stimulated gene; DBD, DNA binding domain; IRF, IFN regulatory factor; EICE, ETS/IRF composite element; EIRE, ETS/IRF response element; IAD, IRF association domain; PEST, Pro, Glu, Ser, and Thr; ICSBP, IFN consensus sequence binding protein; ISRE, IFN-stimulated response element; IVT, in vitro transcription/translation; PKR, dsRNA-activated kinase; PRDI, positive regulatory domain I.
Received for publication January 24, 2002. Accepted for publication April 17, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-regulated protein that binds the interferon-inducible enhancer element of major histocompatibility complex class I genes. Proc. Natl. Acad. Sci. USA 87:3743., , or using oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95:15623.-induced ubiquitin-like protein. J. Biol. Chem. 274:25061.-Interferon-induced transcription of HLA and metallothionein genes containing homologous upstream sequences. Nature 314:637.[Medline]
-induced p67phox and gp91phox expression. J. Immunol. 163:6095. chemoattractant) is an IFN-- and lipopolysaccharide-inducible glucocorticoid-attenuated response gene expressed in lung and other tissues during endotoxemia. J. Immunol. 164:6322.This article has been cited by other articles:
|
|
 |
|
  Y.-J. Lou, X.-R. Pan, P.-M. Jia, D. Li, S. Xiao, Z.-L. Zhang, S.-J. Chen, Z. Chen, and J.-H. Tong IFR-9/STAT2 Functional Interaction Drives Retinoic Acid-Induced Gene G Expression Independently of STAT1 Cancer Res., April 15, 2009; 69(8): 3673 - 3680. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Zimmerer, A. M. Lehman, A. S. Ruppert, C. W. Noble, T. Olencki, M. J. Walker, K. Kendra, and W. E. Carson III IFN-{alpha}-2b-Induced Signal Transduction and Gene Regulation in Patient Peripheral Blood Mononuclear Cells Is Not Enhanced by a Dose Increase from 5 to 10 Megaunits/m2 Clin. Cancer Res., March 1, 2008; 14(5): 1438 - 1445. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Yang, M. Thangaraju, D. D. Browning, Z. Dong, B. Korchin, D. C. Lev, V. Ganapathy, and K. Liu IFN Regulatory Factor 8 Mediates Apoptosis in Nonhemopoietic Tumor Cells via Regulation of Fas Expression J. Immunol., October 1, 2007; 179(7): 4775 - 4782. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Schroder, M. Lichtinger, K. M. Irvine, K. Brion, A. Trieu, I. L. Ross, T. Ravasi, K. J. Stacey, M. Rehli, D. A. Hume, et al. PU.1 and ICSBP control constitutive and IFN-{gamma}-regulated Tlr9 gene expression in mouse macrophages J. Leukoc. Biol., June 1, 2007; 81(6): 1577 - 1590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Zimmerer, G. B. Lesinski, S. V. Kondadasula, V. I. Karpa, A. Lehman, A. RayChaudhury, B. Becknell, and W. E. Carson III IFN-{alpha}-Induced Signal Transduction, Gene Expression, and Antitumor Activity of Immune Effector Cells Are Negatively Regulated by Suppressor of Cytokine Signaling Proteins J. Immunol., April 15, 2007; 178(8): 4832 - 4845. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Dror, N. Rave-Harel, A. Burchert, A. Azriel, T. Tamura, P. Tailor, A. Neubauer, K. Ozato, and B.-Z. Levi Interferon Regulatory Factor-8 Is Indispensable for the Expression of Promyelocytic Leukemia and the Formation of Nuclear Bodies in Myeloid Cells J. Biol. Chem., February 23, 2007; 282(8): 5633 - 5640. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. C. Huang, K. E. Duffy, L. R. San Mateo, B. Y. Amegadzie, R. T. Sarisky, and M. L. Mbow A pathway analysis of poly(I:C)-induced global gene expression change in human peripheral blood mononuclear cells Physiol Genomics, September 14, 2006; 26(2): 125 - 133. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Liu and X. Ma Interferon Regulatory Factor 8 Regulates RANTES Gene Transcription in Cooperation with Interferon Regulatory Factor-1, NF-{kappa}B, and PU.1 J. Biol. Chem., July 14, 2006; 281(28): 19188 - 19195. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Liu, M. Mendicino, Q. Ning, A. Ghanekar, W. He, I. McGilvray, I. Shalev, D. Pivato, D. A. Clark, M. J. Phillips, et al. Cytokine-Induced Hepatic Apoptosis Is Dependent on FGL2/Fibroleukin: The Role of Sp1/Sp3 and STAT1/PU.1 Composite cis Elements. J. Immunol., June 1, 2006; 176(11): 7028 - 7038. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wang, R. M. Baron, G. Zhu, M. Joo, J. W. Christman, E. S. Silverman, M. A. Perrella, R. J. Riese, and M. Cernadas PU.1 Regulates Cathepsin S Expression in Professional APCs J. Immunol., January 1, 2006; 176(1): 275 - 283. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. F. Eisenbeis, G. B. Lesinski, M. Anghelina, R. Parihar, D. Valentino, J. Liu, P. Nadella, P. Sundaram, D. C. Young, M. Sznol, et al. Phase I Study of the Sequential Combination of Interleukin-12 and Interferon Alfa-2b in Advanced Cancer: Evidence for Modulation of Interferon Signaling Pathways by Interleukin-12 J. Clin. Oncol., December 1, 2005; 23(34): 8835 - 8844. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Laricchia-Robbio, T. Tamura, T. Karpova, B. L. Sprague, J. G. McNally, and K. Ozato Partner-regulated interaction of IFN regulatory factor 8 with chromatin visualized in live macrophages PNAS, October 4, 2005; 102(40): 14368 - 14373. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Tamura, P. Thotakura, T. S. Tanaka, M. S. H. Ko, and K. Ozato Identification of target genes and a unique cis element regulated by IRF-8 in developing macrophages Blood, September 15, 2005; 106(6): 1938 - 1947. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. V. Pedchenko, G. Y. Park, M. Joo, T. S. Blackwell, and J. W. Christman Inducible binding of PU.1 and interacting proteins to the Toll-like receptor 4 promoter during endotoxemia Am J Physiol Lung Cell Mol Physiol, September 1, 2005; 289(3): L429 - L437. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Narasimhan, M. Wang, Z. Fu, J. M. Klein, A. L. Haas, and J.-J. P. Kim Crystal Structure of the Interferon-induced Ubiquitin-like Protein ISG15 J. Biol. Chem., July 22, 2005; 280(29): 27356 - 27365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Ho Sui, J. R. Mortimer, D. J. Arenillas, J. Brumm, C. J. Walsh, B. P. Kennedy, and W. W. Wasserman oPOSSUM: identification of over-represented transcription factor binding sites in co-expressed genes Nucleic Acids Res., June 2, 2005; 33(10): 3154 - 3164. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Gautier, M. Humbert, F. Deauvieau, M. Scuiller, J. Hiscott, E. E.M. Bates, G. Trinchieri, C. Caux, and P. Garrone A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells J. Exp. Med., May 2, 2005; 201(9): 1435 - 1446. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Tamura, P. Tailor, K. Yamaoka, H. J. Kong, H. Tsujimura, J. J. O'Shea, H. Singh, and K. Ozato IFN Regulatory Factor-4 and -8 Govern Dendritic Cell Subset Development and Their Functional Diversity J. Immunol., March 1, 2005; 174(5): 2573 - 2581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. D. Sgarlato, C. L. Eastman, and H. H. Sussman Panel of Genes Transcriptionally Up-regulated in Squamous Cell Carcinoma of the Cervix Identified by Representational Difference Analysis, Confirmed by Macroarray, and Validated by Real-Time Quantitative Reverse Transcription-PCR Clin. Chem., January 1, 2005; 51(1): 27 - 34. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. B. Lesinski, S. V. Kondadasula, T. Crespin, L. Shen, K. Kendra, M. Walker, and W. E. Carson III Multiparametric Flow Cytometric Analysis of Inter-Patient Variation in STAT1 Phosphorylation Following Interferon Alfa Immunotherapy J Natl Cancer Inst, September 1, 2004; 96(17): 1331 - 1342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Alter-Koltunoff, S. Ehrlich, N. Dror, A. Azriel, M. Eilers, H. Hauser, H. Bowen, C. H. Barton, T. Tamura, K. Ozato, et al. Nramp1-mediated Innate Resistance to Intraphagosomal Pathogens Is Regulated by IRF-8, PU.1, and Miz-1 J. Biol. Chem., November 7, 2003; 278(45): 44025 - 44032. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Chaussabel, R. T. Semnani, M. A. McDowell, D. Sacks, A. Sher, and T. B. Nutman Unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites Blood, July 15, 2003; 102(2): 672 - 681. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Hashmueli, M. Gleit-Kielmanowicz, D. Meraro, A. Azriel, D. Melamed, and B.-Z. Levi A truncated IFN-regulatory factor-8\IFN consensus sequence-binding protein acts as dominant-negative, interferes with endogenous protein-protein interactions and leads to apoptosis of immune cells Int. Immunol., July 1, 2003; 15(7): 807 - 815. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. O'Reilly, C. M. Quinn, T. El-Shanawany, S. Gordon, and D. R. Greaves Multiple Ets Factors and Interferon Regulatory Factor-4 Modulate CD68 Expression in a Cell Type-specific Manner J. Biol. Chem., June 6, 2003; 278(24): 21909 - 21919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Barnes, A. E. Field, and P. M. Pitha-Rowe Virus-induced Heterodimer Formation between IRF-5 and IRF-7 Modulates Assembly of the IFNA Enhanceosome in Vivo and Transcriptional Activity of IFNA Genes J. Biol. Chem., May 2, 2003; 278(19): 16630 - 16641. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. P. Gobin, P. Biesta, and P. J. Van den Elsen Regulation of human beta 2-microglobulin transactivation in hematopoietic cells Blood, April 15, 2003; 101(8): 3058 - 3064. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
