From the
Interferon consensus sequence binding protein (ICSBP) is a
member of the interferon regulatory factor (IRF) family of proteins
that include IRF-1, IRF-2, and ISGF3
Furthermore, we have tested
possible interactions between ICSBP and IRFs. The chimeric construct
GAL4-ICSBP inhibited the stimulated effect of IRF-1 on a reporter gene,
implying for a possible interaction between IRF-1 and ICSBP.
Electromobility shift assays, demonstrated that ICSBP can associate
with IRF-2 or IRF-1 in vitro as well as in vivo.
Thus, ICSBP contains a third functional domain that enables the
association with IRFs. These associations are probably important for
the fine balance between positive and negative regulators involved in
the interferon-mediated signal transduction pathways in cells of the
immune system.
The activity of interferons (IFNs)
The IFN consensus sequence binding protein (ICSBP) was isolated by
exploiting its ability to bind to ICS. ICSBP is expressed exclusively
in cells of the immune system such as monocytes, B-cells, and T-cells
(5, 6). The expression of this protein is enhanced following exposure
of the cells to IFN-
The N-terminal 120 amino acids
of the ICSBP demonstrates limited homology with three other
transcription factors; IRF-1, IRF-2, and
ISGF3
Each individual member of this family
exerts distinct biological effects. IRF-1 functions as a
transcriptional activator and as a tumor suppressor gene. It is also
capable of exerting antiproliferative effects and
apoptosis
(11, 12, 13, 14) . Conversely,
IRF-2 functions as a negative regulator and when overexpressed, it
induces oncogenic transformation
(11, 12) . ISGF3
In this presentation, we studied
functional domains of ICSBP using fusion constructs. First, the DBD of
ICSBP was fused to the herpes simplex virus transcriptional activation
domain VP16 (ICSBP-VP16). Second, the DBD of ICSBP was replaced with
that of the yeast transcriptional activator GAL4 (GAL4-ICSBP). The data
demonstrate that ICSBP contains at least two distinct functional
domains, a DBD and a transcriptional repressing domain. In addition,
the GAL4-ICSBP chimeric construct inhibited the enhanced activation of
PRDI containing promoter by IRF-1. This indicates a possible
interaction between IRF-1 and ICSBP that was further confirmed by
electromobility shift assays (EMSA) not only for IRF-1 but also for
IRF-2. Therefore, we conclude that a third domain responsible for the
association with other members of IRFs is present.
Each set of
experiments was conducted at least three times and yielded similar
results and statistical significance was determined by the t test with a probability value (p) < 0.05.
Antiserum against
ICSBP was described previously
(6) . Anti-peptide antibodies
against portions of ICSBP, IRF-1, and IRF-2 were generously obtained
from Dr. K. Ozato (NIH) and were recently described
(25) .
Previously, we
demonstrated that ICSBP specifically represses PRDI driven CAT
construct
(6, 8) . Fig. 2demonstrates the negative
effect of pICSBP on the reporter plasmid (compare the first and third bars). In contrast to pICSBP, the plasmid
pICSBPVP promoted a very strong CAT signal which indicates that VP16
functions as a strong activator in the context of ICSBP binding domain
and binding sites. The low basal CAT levels observed with the reporter
construct are the result of the reduced amounts of the reporter gene
incorporated in this assay. In these experiments we used less than of
the CAT plasmid concentration we usually use (0.2 µg instead of 4
µg)
(6, 8) in order to facilitate the detection of
CAT activity in the linear range of both the repressor, pICSBP, and the
activator, pICSBPVP. The strong activation of ICSBP-VP16 fusion was
mediated through interaction of the ICSBP DBD with the PRDI elements
since cotransfection of GAL4-VP16 fusion construct, pSGVP, did not
affect CAT activity (Fig. 2). Further, IFN-
We have tested the effect of pICSBPVP transfected into VAD12.79
on cell surface expression of MHC class I. VAD12.79 cells were
transfected with 15 µg of pICSBPVP and control constructs (pSGVP
and pUC19), and surface expression was analyzed 48 h later.
B summarizes the results of such an experiment. An
induction of MHC surface expression, similar to the one obtained
following IFN treatment, was observed in cells transfected with the
chimeric transcriptional activator ICSBP-VP16. This induction was not
observed in cells transfected with the transcriptional activator
GAL4-VP16 (pSGVP) which is incapable of interacting with the MHC
promoter, or with the control plasmid pUC19 (). Similar
results were also observed with the monocytic cell line U937. Upon
transfection with pICSBPVP, significant increase in the overall HLA
cell surface expression was detected by FACS analysis (data not shown).
Thus, ICSBP-VP16 can serve as a synthetic transcription factor that can
reproduce some of IFN actions.
Fig. 3
describes the
indirect effect of GAL4-ICSBP fusion construct on CAT activity driven
by the mouse mammary tumor virus (MMTV) long terminal repeats bearing
13 repetitions of GAL4 binding sites (pMCM13) that is activated by
GAL4-VP16
(19) (illustrated in Fig. 1). When pMCM13 was
transfected into HeLa cells, low levels of CAT activity were detected.
As previously reported
(19) , cotransfection of pMCM13 with the
GAL4-VP16 fusion construct, pSGVP, resulted in very high levels of CAT
activity. However, a profound decrease in CAT activity was observed
when equimolar amounts of pSGICSBP were cotransfected with pSGVP. On
the other hand, cotransfection of pSGPBSCI, in which the ICSBP open
reading frame was fused in an antisense orientation, did not induce any
significant change in the very strong expression of the CAT reporter
gene (Fig. 3) albeit its ability to bind DNA (data not shown).
These results indicate that the effect of pSGICSBP does not result from
competition with pSGVP at the binding site (squelching effect), but
rather is due to a specific inhibition mediated by the GAL4-ICSBP
fusion construct. To show that the inhibitory effect depends upon
interaction with specific DNA elements, the effect of intact ICSBP
driven by an heterologous promoter (pICSBP) was tested in the
cotransfection experiments. Cotransfection of ICSBP (which is not
capable of interacting with GAL4 binding sites) did not affect
significantly the expression of the reporter gene.
In
nuclear extracts prepared from HL-60 cells, which do not express ICSBP,
we could not detect any band that could be eliminated by antiserum
directed against ICSBP. On the other hand, addition of anti-IRF-1 or
anti-IRF-2 antibodies to the gelshift reactions resulted in a
supershift of the IRF-1 band or the elimination of the IRF-2 band,
respectively (data not shown).
The various antibodies used were
specific and did not demonstrate any cross-reactivity as measured by
Western analysis
(25) . These results demonstrate further that
ICSBP is a component of a multimeric complex in the IFN transduction
pathway that also includes IRF-1 or IRF-2.
ICSBP is a trans-acting negative regulator that belongs to a
relatively new family of DNA binding proteins, the IRFs. In this
communication we show that ICSBP contains three functional domains: DNA
binding domain, repression domain, and a domain that enables the
association with other IRFs. The DBD is separable from the repressor
domain and from the association domain.
When connected to VP16, the
DBD of ICSBP promotes induction of PRDI containing promoters and can
mimic some of IFN action which suggests that the DBD of ICSBP by itself
does not account for its repressor activity. This was also supported by
the fact that expression construct containing only the DBD of ICSBP
(pICSBPDBD) did not affect the activity of the reporter gene when
transfected at molar ratios not exceeding 2:1 (respectively). Recently,
Lin et al.(26) reported detailed analysis of IRF-1 and
IRF-2 DBDs. It was demonstrated that IRF-1 DBD has no significant
effect on PRDI containing promoter while IRF-2 DBD still maintained its
repressor activity. This was attributed to differences in DNA binding
affinities between the two transcription factors. Since ICSBP binds to
DNA at low affinity
(25) , it may account for the insignificant
repression activity of its DBD. However, one can not exclude the
possibility that the observed repression activity of the DBD of IRF-2
is a result of squelching effect.
The synthetic transcriptional
factor GAL4-VP16 functions as a strong activator of GAL4 containing
promoters and this transcriptional activation is the result of a direct
interaction with the transcriptional initiation complex. Recently, it
has been demonstrated that acidic activators, such as VP16, directly
associate with the general transcription factor TFIIB and result in an
elevated level of transcription by increasing the stability of the
preinitiation complex (27-29). Thus, the fact that the GAL4-ICSBP
fusion construct is capable of inhibiting GAL4-VP16-mediated
transcriptional activation is intriguing. We demonstrate that this
inhibition does not result from the occupation of binding sites by a
nonfunctional DNA binding protein because either the DBD of GAL4 alone
(pSG424) or GAL4 connected to nonsense ICSBP (pSGPBSCI) has no effect
on GAL4-VP16 activation (Fig. 3). This suggests two possible
explanations: (a) ICSBP repressing domain directly interacts
with the preinitiation complex and competes with VP16 or (b)
ICSBP functions through a direct association with VP16. The latter
possibility can be excluded because cotransfection of intact ICSBP with
GAL4-VP16 has no effect on the strong transcriptional activity
(Fig. 3). However, one can not exclude the possibility that the
effect of ICSBP on the initiation complex is mediated through
association with other proteins (as discussed below) rather than as a
result of its direct interaction with this complex.
Intact ICSBP
represses ICS driven CAT activity and similarly, the GAL4-ICSBP fusion
construct also represses GAL4 driven CAT activity. However, ICSBP
repression can be attenuated by exposing the cells to
IFNs
(6, 8) . The attenuation of repression can be
explained by two possible mechanisms: (a) the induction of a
transcriptional activator that functions to replace ICSBP and
(b) the induction of a protein that associates with ICSBP
through a protein-protein interaction enabling formation of
heteroduplex that is sequence-dependent. Our data suggest that the
IFN-mediated attenuation of ICSBP repression may involve either
replacement of the repressor (ICSBP) by an activator (such as IRF-1) or
through formation of heteroduplexes that are sequence specific. This
idea is based on the fact that IFNs do not affect repression mediated
by GAL4-ICSBP. Additional support for this interpretation comes from
experiments where the introduction of an antisense construct to IRF-1
in IFN-
In conclusion, the data presented here enabled the
assignment of three functional domains to ICSBP. This transcription
factor is distinct among the IRFs in its restricted expression (cells
of the immune system) and its unique ability to form heterocomplexes
with this family of transcription factors. The fact that different
complexes with IRFs were detected in different cells of the immune
system (B-cells versus monocytic cells) suggest that ICSBP has
a unique role in the regulation of ISGs in these cells. These
characteristics of ICSBP may lead to a more delicate and tunable
balance in response to IFN stimulation between positive and negative
regulators on one hand, or to a new transcriptional activities and DNA
binding specificities on the other hand.
We thank Drs. J. Kasik and H. Hauser for critical
reading of the manuscript and A. Gilboa for the technical assistance.
We are grateful to Dr. K. Ozato for providing us with antipeptide
antibodies and for sharing the results prior to publication.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
which share sequence
similarity at the putative DNA binding domain (DBD). ICSBP is expressed
exclusively in cells of the immune system and acts as a repressor of
interferon consensus sequence (ICS) containing promoters that can be
alleviated by interferons. In this communication, we have searched for
functional domains of ICSBP by dissecting the DBD from the repression
activity. The putative DBD of ICSBP (amino acids 1-121) when
fused in frame to the transcriptional activation domain of the herpes
simplex VP16 (ICSBP-VP16) is a very strong activator of ICS-containing
promoters. In addition, ICSBP-VP16 fusion construct transfected into
adenovirus (Ad) 12 transformed cells enabled cell surface expression of
major histocompatibility complex class I antigens as did treatment with
interferon. On the other hand, the DBD of the yeast transcriptional
activator GAL4 was fused in frame to a truncated ICSBP in which the DBD
was impaired resulting in a chimeric construct GAL4-ICSBP. This
construct is capable of repressing promoters containing GAL4 binding
sites. Thus, ICSBP contains at least two independent domains: a DBD and
a transcriptional repressor domain.
(
)
is
mediated through specific cell surface receptors. Upon binding of IFNs
to these receptors, a signal is transduced to the nucleus which results
in the transcriptional induction of specific genes to confer cellular
response. This induction is mediated through the interaction of DNA
binding proteins with specific sequences contained in the promoter of
these genes. These sequences have been termed either interferon
consensus sequences (ICS) or alternatively the interferon stimulated
response elements (ISRE)
(1, 2, 3, 4) .
and to a lesser degree following exposure to
IFN-
. In addition to ICS element, ICSBP also binds to repeats of
hexamer motif termed PRDI. This motif is contained in the promoter
regions of type I IFNs and it has sequence similarity to the ICS.
Recently, it was shown that ICSBP functions as a repressor on promoters
containing the ICS/PRDI element
(6, 7) and that this
repression can be attenuated by exposing the cells to either type I or
type II IFNs
(6, 8) .
(5, 6, 9, 10) . Because of the
presence of unique sequence characteristics such as tryptophan repeats,
it has been postulated that this area of limited homology is restricted
to the DBD portion of these transcription
factors
(5, 6, 9, 10) . Thus, these
factors constitute a new family of DNA binding proteins termed the IFN
regulatory factors (IRFs).
functions to confer DNA binding specificity to a multi-polypeptide
complex termed ISGF3
. Following the exposure of cells to
IFN-
, this complex is rapidly translocated from the cytoplasm to
the nucleus where it then functions as a transcriptional activator,
ISGF3
(15, 16) .
Cell Culture
HeLa cells (human cervical carcinoma), U937 cells
(promonocytic cells), HL-60 cells (promyelocytic cells) and Namalwa
cells (B-cells) were obtained from ATCC (Rockville, Maryland). The cell
line VAD12.79 was derived following transformation of C57Bl/10 mouse
embryonal fibroblasts expressing a miniature swine class I transgene
(PD1) with the human adenovirus 12
(17) . HeLa and VAD12.79 cell
lines were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, gentamicin, and glutamine
(Biological Industries, Kibbutz Bet-Haemek, Israel). U937, HL-60, and
Namalwa cell lines were maintained in RPMI 1640 medium supplemented as
above except for Namalwa cells that were grown in the presence of 7.5%
fetal calf serum.
Plasmids
The reporter plasmid pMCM13 and the expression plasmids
pSG424 and pSGVP (all illustrated in Fig. 1) were provided by Dr.
M. Ptashne (Harvard University) through Dr. I. Sadowski (University of
British Columbia, Canada)
(18, 19) . The reporter plasmid
pG5E472CAT was obtained from Dr. J. Lillie through Dr. H. Manor
(Technion, Israel) (20). The reporter plasmid p128I4r was provided by
Dr. T. Maniatis (Harvard University)
(21) . The fusion plasmids
pSGICSBP and pSGPBSCI were constructed by cloning a BamHI
fragment of H-ICSBP corresponding to amino acids 33-425
(6) to the BamHI site of pSG424 in sense and antisense
orientations, respectively (plasmids are illustrated in Fig. 1).
Figure 1:
Schematic illustration of the various
expression plasmids (A) and CAT constructs
(B).
To construct pICSBPVP, an EcoRI restriction site located at
a multiple cloning site downstream to VP16 on the plasmid pSGVP was
removed by digesting with neighboring restriction sites,
HindIII and XbaI. The digested plasmid was made blunt
end by Klenow fragment reaction, and self-ligated to generate pSGVP1. A
BglII-EcoRI fragment corresponding to the GAL4 DBD of
pSGVP1 was replaced with a PCR amplified fragment, corresponding to
amino acids 1-121 of H-ICSBP, to which two restriction sites,
BglII and EcoRI respectively, were added (see
illustration in Fig. 1). The primers corresponding to the 5` end
and to the 3` end respectively were: H-25 TCTAGATCTCCATGGCTGACCGGAATG
and H-26 GTGATATCAGAATTCCACGCCTAGTTTGC. In order to clone the ICSBP DBD
under the SV40 promoter, the plasmid pICSBPVP was digested with
EcoRI and HindIII to remove the VP16 portion and
following fill-in reaction with Klenow fragment, the plasmid was
self-ligated to generate pICSBPDBD (see Fig. 1). The mammalian
IRF-1 expression plasmid that also contain T7 promoter, pMT7-IRF-1, was
described previously
(13) . A 1.4-kilobase XhoI fragment
containing the coding sequence of human IRF-2 (10) was cloned in
pBluescript under the T7 promoter that enabled in vitro transcription. Human ICSBP was cloned under T7 promoter of the
plasmid pET3d (Novagen, Madison, WI).
DNA Transfections and CAT Assays
Plasmid DNA was transfected by either electroporation or by
calcium phosphate-DNA precipitation as follows.
Electroporation Procedure
Cells were diluted to a
concentration of 10 cells/ml 16-24 h prior to
transfection. Just before transfection, HeLa cells were harvested,
washed twice with phosphate-buffered saline, resuspended in
Dulbecco's modified Eagle's medium containing 10% fetal
calf serum at a concentration of 8
10
cells/ml and
left on ice for 30 min. The cell suspension (0.1 ml) was then placed
into a 0.4-cm electroporation cuvette (Bio-Rad), and 10 µg of
plasmid DNA (suspended in no more than 20 µl of distilled water)
were added. Following a 10-min incubation at room temperature, the
cells were electroporated at 250 µF and 290 V and left at room
temperature for an additional 20-40 min. The cells were then
resuspended in 10 ml of Dulbecco's modified Eagle's medium
containing 10% fetal calf serum, the medium was replaced with fresh
medium after 24 h, and the cells were harvested after an additional 24
h. In some experiments, human IFN-
(2500 units/ml, Frone®,
kindly provided by Interpharm Laboratories Ltd., Rehovot, Israel) was
added 24 h after electroporation. CAT assays were performed and
normalized for protein concentration and transfection efficiency as
described
(6) .
Plasmids
Plasmids used for cotransfection
experiments were pG5E472CAT (4 µg), p128I4r (4 µg), or pMCM13
(0.2 µg) reporter plasmids and 0.2-0.4 µg of the
expression plasmids pSGICSBP, pSGPBSCI, pSGVP, pSG424, and pICSBPVP or
2 µg of pICSBP (an expression vector for ICSBP in mammalian cells
obtained from Dr. K. Ozato (NIH)
(7) . The plasmid pUC19 was
added as carrier DNA up to a total of 10 µg of DNA per assay. Other
cotransfection conditions are described in the text.
Calcium Phosphate
VAD12.79 cells were transfected
by the DNA coprecipitation method as described previously
(22) with 15 µg of various plasmid DNA, washed 24 h
following transfection, harvested 24 h later, and taken for
fluorescence-activated cell sorter (FACS) analyses.
FACS Analysis
Cells were harvested by mild trypsinization followed by
washing with medium supplemented with 5% fetal calf serum.
Approximately 10 cells were incubated at 4 °C with the
appropriate concentration of the first antibody for 30 min, washed, and
further incubated in the dark for an additional 30 min with the second
antibody. The cells were washed with phosphate-buffered saline and
analyzed by a Becton Dickinson cell sorter. The various monoclonal
antibodies directed against MHC class I molecules were previously
described (see Ref. 23 and references therein). As indicated in the
text, cells were treated with either 600 units/ml of murine
IFN-
/
(Lee Bioresearch Inc., San-Diego, CA) for 48 h or 100
units/ml of murine IFN-
(Boehringer Mannheim) for 72 h before
analysis.
In Vitro Transcription and Translation
Plasmids containing ICSBP, IRF-1, and IRF-2 under the T7
promoter were linearized downstream to the coding region with the
appropriate restriction enzyme. 3 µg of linearized plasmids were
in vitro transcribed by T7 RNA polymerase using commercial kit
(Stratagene, La Jolla, CA). Proteins were translated in vitro using rabbit reticulocyte lysate (RRL) system (Promega, Madison,
WI) according to the manufacturer instructions.
EMSA
Gel shift reactions were carried out as described previously
(13). In vitro translated factors were prepared as described
above. Nuclear extracts were prepared according to Dignam et
al.(24) . A typical reaction contained either 5 µg of
nuclear extract or up to 6 µl of RRL containing either IRF-1 or
IRF-2 or ICSBP or combinations. The various protein preparations were
incubated in binding buffer (10 mM HEPES, pH 8.0, 5
mM MgCl, 50 mM KCl, 10% Ficoll, 3%
glycerol, 2 µg of sonicated poly(dI-dC), 1 µg of sheared salmon
sperm DNA, 0.025% bromphenol blue, and 0.025% xylene cyanole) with at
least 50,000 cpm of labeled trimer of PRDI ((AAGTGA)
) for
15 min on ice. In supershift reactions, antiserum was incubated for 1 h
with extracts and the labeled probe in the binding buffer was added at
the last 15 min. The samples were loaded on a pre-run 7% polyacrylamide
gel. The dried gels were exposed to x-ray films.
Construction of ICSBP-VP16 and GAL4-ICSBP Fusion
Plasmids
To study functional domains of ICSBP, fusion constructs
(illustrated in Fig. 1) were prepared to segregate the DBD from
the module responsible for repression. First, the DBD of ICSBP (amino
acids 1-121) was either cloned under the SV40 early promoter
(pICSBPDBD) or fused in-frame upstream to the transcriptional
activating domain of herpes simplex VP16 (amino acids 412-490)
generating pICSBPVP (Fig. 1). Second, to characterize the
repressor domain, we have used a truncated ICSBP that is incapable of
binding to ICS/PRDI motifs because the first 33 amino acids were
deleted
(6) . Such a truncated sequence should contain only the
repression activity and thus was fused downstream to the DBD (amino
acids 1-147) of the GAL4 transcriptional activator generating
pSGICSBP (Fig. 1). As a control, the same truncated ICSBP DNA
fragment was fused in an antisense orientation to the GAL4 DBD
(pSGPBSCI) (Fig. 1). In addition, reporter plasmids and
expression constructs used for transfections and CAT assays (described
under ``Materials and Methods'') are illustrated in
Fig. 1
.
The DBD of ICSBP Is Independent of Its Repressor
Activity
Domain swap analyses were performed to determine
whether the DBD of ICSBP is independent of its repressor activity. For
that purpose, a pICSBPVP fusion construct was generated in which the
first 121 N-terminal amino acids of ICSBP were fused in frame to the
transcriptional activation domain of VP16 (Fig. 1). The effect of
pICSBPVP on the expression of a reporter plasmid, p128I4r (illustrated
in Fig. 1), containing 4 PRDI repeats connected to the
-globin basal promoter was tested. Since GAL4-VP16 fusion was
reported as toxic to cells when expressed at high levels
(19) ,
we first tested the effect of varying amounts of pICSBPVP transiently
transfected into HeLa cells. When pICSBPVP was transfected at
4-10 µg/plate, very low levels of CAT activity were detected.
However, maximal CAT activities were obtained at concentrations of
0.2-0.4 µg/plate, and a significant CAT signal was still
detected at plasmid levels as low as 0.01 µg/plate (data not
shown). Other transcriptional activators (such as IRF-1) or
transcriptional repressor (such as IRF-2 or ICSBP) did not exhibit any
significant effect on a reporter gene when cotransfected at such low
levels as 0.01 µg/plate (data not shown).
treated cells
which were cotransfected with the chimeric construct had no effect on
the very strong activation of the reporter gene (data not shown). We
have also analyzed the effect of just the DBD of ICSBP in these
cotransfection studies. The DBD when cloned under the SV40 promoter
(pICSBPDBD in Fig. 1) had no significant effect on CAT levels
driven by the PRDI containing promoter when transfected at molar ratio
not exceeding 2:1 (pICSBPDBD:p128I4r, respectively, data not shown). In
addition, partially purified polypeptide corresponding to the DBD from
lysate of expressing bacteria was tested by EMSA and demonstrated
binding capability to a trimer of PRDI motif (not shown). Therefore,
This indicate that the repression domain of ICSBP is not restricted to
this domain.
Figure 2:
ICSBP-VP16 (pICSBPVP) fusion is a
strong activator of PRDI-containing promoter. HeLa cells were
transfected with p128I4r alone and with either pICSBPVP, pICSBP, or
pSGVP expression vector (see ``Materials and Methods'') and
CAT activity was determined as percentage of conversion of
chloramphenicol to monoacetylated form. The actual CAT assay spots
corresponding to the faster migrating form of monoacetylated
chloramphenicol are placed under the relevant
bar.
The Chimeric Transcriptional Activator pICSBPVP, Can
Bypass Some of IFN Activities
Transient transfection assays
revealed that the chimeric factor ICSBP-VP16 is a stronger activator
than IRF-1 (data not shown). Thus, we have tested its ability to mimic
some of IFN activities. For that purpose, we have used primary
embryonal fibroblasts transformed by Ad12 (VAD12.79 cells). This cell
line was derived from transgenic mice carrying the miniature swine MHC
class I gene, PD1. In these cells, surface expression of class
I MHC antigens is generally very low
(17) . However, treatment
with IFN-/
(600 units/ml) or IFN-
(100 units/ml) results
in elevated expression of MHC class I antigens on the surface of the
cells (A). MHC expression is demonstrated by the increase
in the number of class I positive cells in the population and by the
increase in the mean fluorescence per cell (, numbers in
parentheses). Treatment with IFNs resulted in a significant increase in
cell surface expression of PD1 transgene and H-2D antigen. The increase
in the expression of the H-2K was less dramatic than that of H-2D and
PD1.
GAL4-ICSBP Fusion Construct Represses GAL4 Driven
Reporter Constructs
To further characterize the repressor
activity of ICSBP, we tested whether this activity is restricted to
promoters containing ICS elements or is determined by its structure
regardless of the DBD connected to it. For that purpose, the ability of
the GAL4-ICSBP fusion construct (pSGICSBP) to either repress the CAT
reporter gene driven by GAL4 binding sites (direct approach) or to
negate the stimulatory effect of GAL4-VP16 on similar reporter
construct (indirect approach) was tested.
Figure 3:
Indirect
effect of GAL4-ICSBP fusion construct (pSGICSBP) on
transcriptional activation mediated by GAL4-VP16 (pSGVP). HeLa
cells were transfected with pMCM13 alone or in combination with just
pSGVP or pSGVP in the presence of either pSGICSBP, pSGPBSCI, or pICSBP
(see ``Materials and Methods''). CAT assays were performed as
described under Fig. 2. The actual CAT assay spots corresponding to the
faster migrating form of monoacetylated chloramphenicol are placed
under the relevant bar.
Fig. 4
describes the direct effect of GAL4-ICSBP fusion on
construct bearing GAL4 binding sites. Among three reporter construct
tested, the construct pG5E472CAT that contains five GAL4 binding sites
connected to the adenovirus early gene E4 (illustrated in
Fig. 1
) was the only one that promoted moderate levels of CAT
activity when transfected into HeLa cells (Fig. 4). Therefore,
this construct was employed to study the direct effect of GAL4-ICSBP on
CAT expression. Fig. 4shows that the basal CAT activity of
pG5E472CAT was markedly repressed (3.5-fold) when pSGICSBP was
cotransfected with the reporter construct. Conversely, the control
constructs pSGPBSCI or pSG424, containing only the GAL4 binding domain,
had no significant effect on the basal CAT activity. These results
further support that the repressor effect mediated by GAL4-ICSBP was
not due to a squelching effect resulting from occupation of the DNA
binding site in a nonfunctional manner. As a positive control, we used
in the same cotransfection assay the strong artificial transcriptional
activator GAL4-VP16 (pSGVP) resulting, as expected, in very high levels
of CAT activity. Thus, using both direct and indirect approaches, we
demonstrate that ICSBP is a ``true'' repressor and its effect
occurs only in the presence of the appropriate DBD.
Figure 4:
Direct repression effect of GAL4-ICSBP.
HeLa cells were transfected with pG5E472CAT alone or with either
pSGICSBP or pSGPBSCI or pSG424 or pSGVP (see ``Materials and
Methods'') and CAT assays were performed as described under Fig.
2.
Previously, we
have demonstrated that IFNs attenuate the repression effect of
ICSBP
(8) . To test if IFNs can affect the repression activity of
GAL4-ICSBP, the last set of cotransfection experiments (direct
approach) was also performed with IFN- treated cells.
Surprisingly, these experiments yielded similar results (data not
shown) implying that the attenuating effect of IFNs must be specific to
ICS/PRDI elements because the presence of IFN did not alter the
negative effect exhibited by the GAL4-ICSBP fusion protein.
ICSBP Can Associate with IRF-1 or IRF-2
To
investigate possible interactions between ICSBP and other members of
the IRF family of proteins, we tested the ability of GAL4-ICSBP to
affect the enhanced activity of IRF-1 on PRDI containing promoters.
Cotransfection of IRF-1 expression construct (pMT7-IRF-1) with PRDI-CAT
reporter plasmid (p128I4r) resulted in enhanced CAT activity
(Fig. 5). When GAL4-ICSBP construct (pSGICSBP) was also
cotransfected at equimolar amount relative to the IRF-1 expression
plasmid, a significant decrease of 50% in the activity of the reporter
gene was observed. On the other hand, no such effect on the IRF-1
induced CAT activity was detected when similar amounts of either
pSGPBSCI in which ICSBP was cloned in the opposite orientation or
pSG424 containing only GAL4 DBD were employed (Fig. 5). As
expected, the constructs pSG424, pSGICSBP, and pSGPBSCI, which are
incapable of interaction with PRDI motif, did not promote any effect on
the reporter plasmid (Fig. 5). This data suggest a possible
interaction of IRF-1 with the fusion protein GAL4-ICSBP resulting in a
significant reduction in the effect of IRF-1 on PRDI containing CAT
construct.
Figure 5:
GAL4-ICSBP
fusion construct can inhibit IRF-1 action on PRDI-CAT reporter
construct. HeLa cells were cotransfected with 0.25 µg of the
reporter plasmid p128I4r and 0.5 µg of the various constructs:
pMT7-IRF-1, pSGICSBP, pSGPBSCI, and pSG424 as indicated in the figure.
CAT activity was determined as described under Fig. 2. The actual CAT
assay spots corresponding to the faster migrating form of
monoacetylated chloramphenicol are placed under the relevant
bar.
To further investigate possible association of ICSBP with
IRF-1 and IRF-2 in a more direct manner, EMSA analyses were performed.
For that purpose, IRF-1, IRF-2, and ICSBP were translated in vitro using RRL (for details see Materials and Methods) and reacted with
the labeled trimer of PRDI. Fig. 6shows the ability of ICSBP to
associate with IRF-2 in vitro. As expected, ICSBP did not bind
the PRDI motif (Fig. 6, lane 1)
(25) . On the
other hand, a shifted band was obvious in gel-shift reaction containing
in vitro translated IRF-2 (Fig. 6, lane 2,
indicated by an arrowhead). When both ICSBP and IRF-2 were
mixed together a new band appeared on the gel (Fig. 6, lane
3, indicated by an arrowhead). This band represents an
heteroduplex between the two factors since antibodies against IRF-2
eliminated both bands; the fast migrating band that corresponds to
IRF-2 and the slow migrating band that corresponds to ICSBP
(Fig. 6, lane 5). In addition, antiserum against ICSBP
supershifted only the upper band thus indicating that it is composed of
both ICSBP and IRF-2 (Fig. 6, lane 6). Preimmune
antiserum did not interfere with the association of ICSBP with IRF-2
(Fig. 6, lane 4). Similar results were also obtained
with IRF-1 (data not shown) although it seemed that the association of
ISCBP with IRF-2 was stronger.
Figure 6:
In
vitro association of ICSBP with IRF-2. EMSA analyses were
performed with in vitro translated ICSBP (lane 1) and
IRF-2 (lane 2) or both (lanes 3-6) with
P-labeled trimer of PRDI. To some of the reactions
preimmune serum (lane 3) or antisera directed against IRF-2 or
ICSBP (lanes 4 and 5, respectively) were added.
Samples were separated on 7% polyacrylamide gels that were dried under
vacuum and exposed to x-ray films (for details see ``Materials and
Methods''). Arrowheads indicate IRF-2 bands or
ICSBP/IRF-2 heteroduplex (Hetero.)
bands.
To determine whether such
heteroduplexes exist in vivo, nuclear extracts were prepared
from the promonocytic cell line U937 and from the B-cell line Namalwa
which express constitutive levels of ICSBP at both mRNA and protein
levels and from the premyelocytic cells HL-60 which do not express
ICSBP
(6) .(
)
The extracts were analyzed by
EMSA with radioactive labeled trimer of PRDI in the presence of
antiserum directed against either ICSBP, IRF-1, or IRF-2. Discrete
shifted bands were detected in U937 nuclear extracts which were
specifically competed by 100-fold excess of unlabeled ICS
oligonucleotide (Fig. 7A, lanes 1 and
2, respectively) but not by a nonspecific competitor (data not
shown). When the extracts were incubated with preimmune serum the
binding pattern was not changed significantly (Fig. 7A,
lane 3). However, when antisera directed against either IRF-1
or ICSBP were included in the EMSA, the same band was eliminated
(Fig. 7A, lanes 4 and 5, respectively,
indicated by an arrowhead). Furthermore, in the presence of
anti-ICSBP antibodies a new band appeared which was absent in the
preimmune or anti-IRF-2 lanes and was fainter in the anti-IRF-1 band
(Fig. 7A). The nature of this band is unclear. The
anti-IRF-2 lane shows identical band pattern to that of the preimmune
lane (Fig. 7A, lanes 3 and 6,
respectively). Thus, these results show direct association between
IRF-1 and ICSBP in U937 cells.
Figure 7:
In
vivo association of ICSBP with IRF-1 and IRF-2. EMSA analyses were
performed with nuclear extracts prepared from U937 cells (Panel
A) or Namalwa cells (Panel B) with P-labeled
trimer of PRDI in the presence of either 100-fold excess of ICS
oligomer (lane 2) or preimmune serum (lane 3) or
antisera directed against ICSBP or IRF-1 or IRF-2 (lanes 4,
5, and 6, respectively). Samples were separated on 7%
polyacrylamide gels that were dried under vacuum and exposed to x-ray
films (for details see ``Materials and Methods'').
Arrowheads indicate ICSBP/IRF-1 or ICSBP/IRF-2
heteroduplexes.
Similarly, nuclear extracts from
Namalwa cells generated discrete bands in EMSA that were competed with
excess of 100-fold ICS (Fig. 7B, lanes 1 and
2, respectively) but not by nonspecific competitor (data not
shown). With Namalwa extracts, antisera against IRF-2 or ICSBP
eliminated the same band (Fig. 7B, lanes 4 and
6, respectively, indicated by an arrowhead), while
antiserum against IRF-1 did not demonstrate similar change
(Fig. 7B, lane 5). The preimmune lane was
composed of six bands as follows: a strong slow migrating band followed
by a doublet and three more bands (Fig. 7B, lane
3). The lower part of the doublet in the anti-IRF-2 lane
disappeared, probably indicating the IRF-2 band, while the upper part
of the doublet was strengthened (Fig. 7B, lane
6). In the anti-ICSBP lane, a strong band was detected in place of
the doublet and the other two adjacent fast migrating bands were
fainter than the corresponding bands in the preimmune lane
(Fig. 7B, lanes 4 and 3,
respectively). The upper band in the anti-IRF-1 lane was weaker than
the corresponding band in the preimmune lane and the upper portion of
the double band was missing indicating the position of IRF-1 band while
the lower part (IRF-2 band) was more prominent (Fig. 7B,
lane 5). These results indicate that in Namalwa cells, unlike
U937 cells, association between ICSBP and IRF-2 is more abundant.
-treated cells blocks the alleviation of ICSBP repression
on ICS/PRDI containing promoters
(8) . This suggests that
attenuation of ICSBP-mediated repression is due to the IFN-
induction of IRF-1 which then either replaces ICSBP by binding to the
same DNA motif or by direct association with ICSBP thus affecting its
repressor activity
(8) . The results obtained with the IRF-1
antisense construct led us to test this last hypothesis by analyzing
the ability of GAL4-ICSBP chimeric construct to affect IRF-1 mediated
activation of PRDI-containing promoter. Indeed our data present further
support for this hypothesis; GAL4-ICSBP fusion has no effect on
PRDI-containing promoters (Fig. 5), but it significantly inhibits
the IRF-1-mediated activation of PRDI-CAT reporter construct. These
results further support the existence of physical interaction between
ICSBP and IRF-1. A direct demonstration of such interactions was
obtained by the EMSA analyses. Interactions between ICSBP and IRFs were
demonstrated by mixing in vitro translated IRFs with ICSBP
(Fig. 6). Recently, the presence of heteroduplexes between ICSBP
and either IRF-1 or IRF-2 was also demonstrated both in vitro and in vivo by Bovolenta and colleagues
(25) that
also presented evidence for possible interaction of ICSBP with another
member of IRFs, ISGF3
. In our work, we also show that such
association can be detected in cell lines that are constitutively
expressing ICSBP. Furthermore, the type of interactions observed in
extracts from various cells differs; in the promonocytic cell line U937
the main association is with IRF-1 while the association detected in
the B-cell line Namalwa, is mainly with IRF-2 (Fig. 7).
Consistent with these conclusions is the fact that in HL-60 cells which
do not express ICSBP no multimeric complexes are observed. This
suggests that ICSBP might have different effects in different cells of
the immune system depending upon the milieu of IRFs. Further, it may
render IRF-1 or IRF-2 different activities or affinities to DNA
sequences. Preliminary data
(
)
indicate that
in vitro post-translational modifications such as
phosphorylation on tyrosine residues can prevent ICSBP from binding to
DNA but at the same time promote formation of heteroduplexes with IRFs.
This implies that signals that can affect the phosphorylation state of
ICSBP can affect its activities. Furthermore, using chimeric constructs
we provide evidence that such interactions may have biological
relevance. Our results also imply that other heteroduplexes of ICSBP
with yet unidentified factors are also possible
(Fig. 7B, lane 4). The biological significance
of the various heterocomplexes and their distinct distribution in
different cells is still unclear. It has to be kept in mind that the
in vivo interactions demonstrated here are from nuclear
extracts of tumor cell lines and further experiments have to be
performed in order to demonstrate such different interactions in normal
immune cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.