©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Domain Analysis of Interferon Consensus Sequence Binding Protein (ICSBP) and Its Association with Interferon Regulatory Factors (*)

Rakefet Sharf (1), Aviva Azriel (1), Flavio Lejbkowicz (1)(§), Sigal S. Winograd (2), Rachel Ehrlich (2), Ben-Zion Levi (1)(¶)

From the (1) Department of Food Engineering & Biotechnology, Technion, Haifa 32000, Israel and (2) Department of Cell Research and Immunology, The George Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.

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.


INTRODUCTION

The activity of interferons (IFNs)() 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) .

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- 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) .

The N-terminal 120 amino acids of the ICSBP demonstrates limited homology with three other transcription factors; IRF-1, IRF-2, and ISGF3 (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).

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 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) .

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.


MATERIALS AND METHODS

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.

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.

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.

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) .


RESULTS

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).

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- 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.

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.

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.

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.


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.

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.


DISCUSSION

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--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.

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.

  
Table: 8.9 (2.0)


FOOTNOTES

*
This research was supported in part by grants from the Council for Tobacco Research-U.S.A., Inc., Israel Cancer Research Fund, The Israel Cancer Association and the German-Israeli Foundation for Scientific Research and Development (to B. Z. L.), and by the Israel Cancer Research Fund (RCDA) (to R. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Lady Davis Trust fellowship.

To whom correspondence should be addressed. Tel.: 972-4-293345; Fax: 972-4-320742; Bitnet: fobenzi@techunix.technion.ac.il.

The abbreviations used are: IFN, interferon; ICS, interferon consensus sequence; ICSBP, interferon consensus sequence binding protein; ISRE, interferon-stimulated response element; IRF, interferon regulatory factor; ISGF, interferon-stimulated gene factor; PRD, positive regulatory domain; RRL, rabbit reticulocyte lysate; CAT, chloramphenicol acetyl transferase; DBD, DNA binding domain; MHC, major histocompatibility complex; Ad12, adenovirus 12; FACS, fluorescence-activated cell sorter; EMSA, electromobility shift assays.

F. Lejbkowicz, unpublished data.

R. Sharf, unpublished data.


ACKNOWLEDGEMENTS

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.


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