A truncated IFN-regulatory factor-8\IFN consensus sequence-binding protein acts as dominant-negative, interferes with endogenous proteinprotein interactions and leads to apoptosis of immune cells
Sharon Hashmueli1,
Merav Gleit-Kielmanowicz1,
David Meraro1,
Aviva Azriel1,
Doron Melamed2 and
Ben-Zion Levi1
Departments of 1 Food Engineering and Biotechnology and 2 Medicine, Technion, Haifa 3200, Israel
The first two authors contributed equally to this work
Correspondence to: B.-Z. Levi; E-mail: blevi{at}tx.technion.ac.il
Transmitting editor: D. Wallach
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Abstract
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IFN consensus sequence-binding protein (ICSBP) is a member of the IFN-regulatory factors (IRF) and is thus also called IRF-8. Its expression is restricted to hematopoietic cells and IRF-8\ICSBP/ mice are defective in myeloid cell differentiation. This factor exerts its transcriptional activity through interaction with other transcription factors, which leads to either repression or activation. In this paper, we describe the use of a dominant-negative (DN) mutant of IRF-8\ICSBP designed to serve as a molecular tool to dissociate the role of the various proteinprotein interactions. This DN-ICSBP is truncated at the DNA-binding domain and can still associate with other factors, but the heterocomplexes produced are incapable of binding to the DNA. We show that the DN-ICSBP is able to compete for the interaction of IRF-8\ICSBP with either IRF or non-IRF members such as PU.1. Accordingly, this DN construct was able to inhibit the PU.1-dependent expression of the IgL
in the plasmacytoma cell line J558L. However, stable expression of this DN-ICSBP led to apoptosis of only hematopoietic cells. The data suggests that DN-ICSBP can form heterocomplexes with an as-yet unidentified survival factor for hematopoietic cells.
Keywords: IFN-regulatory factor, PU.1, transcriptional regulation
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Introduction
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IFN-regulatory factors (IRF) constitute a family of transcription factors with nine cellular members. This family of transcription factors mediates in part IFN signaling, and thus modulates the immune system, affects cell growth and differentiation, and elicits anti-viral activity. All nine members share significant homology at the first 115 amino acids, which comprise the DNA-binding domain (DBD). Consequently, these factors bind to a similar DNA motif termed the IFN simulated response element (ISRE) found mainly in the promoters of IFN type I responding genes [for review, see (1)]. Further, seven members, excluding IRF-1 and IRF-2, demonstrate sequence similarity towards the C-terminus termed the IRF association domain (IAD) (2). This module facilitates proteinprotein interaction for IRF-8\IFN consensus sequence-binding protein (ICSBP), IRF-4 and IRF-9\ISGF3
, and is probably essential for some of the interactions described for IRF-3 and IRF-7. This module has a predicted structural similarity with the MH2 domain of the Smad family of transcription factors, which mediate transforming growth factor-ß signaling through the formation of either homo- or heterocomplexes (2,3).
IRF-4 and IRF-8\ICSBP are expressed in cells of hematopoietic origin, and demonstrate high sequence similarly. IRF-4 is an essential factor for B cell maturation and Ig production. As such, IRF-4 is found in transcriptional activation complexes that also include PU.1 on the E
24 enhancer of the IgL
and E47 on the IgL
3' enhancer (4,5). IRF-8\ICSBP is essential for myeloid progenitor cell differentiation toward mature macrophages while blocking differentiation toward granulocytes (6). IRF-8\ICSBP exerts it transcriptional activity through interaction with other factors. Interactions with IRF-1 or IRF-2 on ISRE lead to the formation of a heterocomplex with repression activity. Like its closest homologue, IRF-4, IRF-8\ICSBP also interacts with PU.1 and E47, and these interactions result in the formation of transcriptional activation heterocomplexes. The DNA-binding sites for these heterocomplexes are composite elements that include both IRF and the interacting partner binding sites, and are found on various promoters, including those that are essential for macrophage activity (7,8). In addition, both IRF-4 and IRF-8\ICSBP were identified in multi-protein complexes that also include IRF-1, PU.1 and CBP/p300 (9). Using the IAD of IRF-8\ICSBP as bait in yeast two-hybrid screens, other interacting partners were identified among which was Trip15/CSN2 (10).
The association modules of factors that interact with IRF-8\ICSBP were characterized and do not exhibit any sequence similarity, but were identified as PEST domains (2,11). These domains, enriched with proline, glutamic acid, serine and threonine, were originally identified in proteins that are prone to fast degradation by the proteasome system (12). Our studies indicate that these modules are essential for the interaction with IRF-8\ICSBP (7, 11).
Recently, it was shown that the activation of a specific subset of IFN-
activation site elements is mediated through the assembly of an unidentified DNA-binding heterocomplex to which ICSBP is associated indirectly via proteinprotein interactions (13).
Altogether, it is clear that IRF-8\ICSBP is involved in numerous proteinprotein interactions. Each interaction may render the complex different biological activities. We used a dominant-negative (DN) mutant of IRF-8\ICSBP designed to serve as a molecular tool to dissociate the role of the various interactions. Our results show that the DN-ICSBP is able to compete for the interaction of both IRF-8\ICSBP and IRF-4 with PU.1. In addition, this DN construct was able to inhibit IgL
expression when transduced to the plasmacytoma cell line J558L. However, stable expression of this DN-ICSBP only in hematopoietic cells leads to cell death (apoptosis). The data suggests that DN-ICSBP can form heterocomplexes with a factor (most probably PU.1) which is essential for the survival of immune cells only.
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Methods
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Cell culture
NIH 3T3 and U937 were obtained from ATCC (Manassas, Virginia). The plasmacytoma cell line J558L was from the collection of Dr Melamed (Technion) and the retroviral packaging cell line, 293gp, was a gift from Dr Verma (The Salk Institute) through Dr Bengal (Technion). NIH 3T3 and 293gp cells were maintained in DMEM, while the other cell lines were maintained in RPMI 1640. All media were supplemented with 10% FCS and antibiotics.
Plasmids
The plasmids pGAL4-DN-ICSBP and pGAL4-DN-L331P-ICSBP were generated by cloning a
1300-bp BamHI fragment from the plasmids pTarget-ICSBP-HA and pTarget-L331P-ICSBP-HA (10) into the BamHI site of the plasmid pSG424 (14) in which the DBD of the yeast transcription factor is cloned under the SV40 promoter. These two plasmids were digested with HindIII and NotI, and subcloned into the corresponding restriction sites in the mammalian expression vector pcDNA3.1 (Invitrogen, Purchase, NY) generating the plasmids pcDNA3.1-GAL4-ICSBP and pcDNA3.1L331P-ICSBP, both tagged with HA. In addition, the GAL4 DBD was cloned into pcDNA3.1 following digestion of pSG424 with the corresponding restriction enzymes HindIII and BamHI, generating the plasmid pcDNA3.1-GAL4. These three pcDNA3.1-derived plasmids were digested with NheI and BglII, and cloned into the corresponding restriction sites in pIRES2-EGFP (Clontech, Palo Alto, CA). pIRES2-EGFP contains a bicistronic cassette in which the second cistron, enhanced green fluorescent protein (EGFP), is translated through an internal ribosome entry site (IRES) of the encephalomyocarditis virus. This vector permits the co-expression a gene of interest and EGFP, which serves as a reporter gene for the expression of this bicistronic cassette. To generate bicistronic retroviral vectors (RVV), the bicistronic cassette from the pIRES2-EGFP derivative plasmids were subcloned to pLNCX (Clontech), a retroviral expression vector created from MMLV. In this vector the expression of a gene of interest is under the internal CMV long terminal repeat that also contains the packaging signal
and neomycin resistance gene. Thus, pLNCX-EGFP (no gene inserted in the first cistron), pLNCX-GAL4, pLNCX-DN-ICSBP and pLNCX-L331P-DN-ICSBP were generated. pLNCX-L331P-DN-ICSBP harbors a point mutation within the IAD of IRF-8\ICSBP in which Leu331 was mutated to Pro. This mutation leads to defective IRF-8\ICSBP, which is incapable of interacting with other factors through the IAD.
Retroviral packaging
293gp cells (1.5 x 106) were plated on gelatin-coated 10-cm plates. After 24 h, cells were co-transfected by the calcium phosphate precipitation method (15) with 5 µg of pVSV-G (Clontech) and 1015 µg of RVV, and incubated for 18 h. The media was replaced with 6 ml fresh media and 48 h later, retrovirus-containing supernatant (RV-SN) was collected and filtered through a 45-µm filter and stored in aliquots at 70°C.
Determination of retroviral titer
NIH 3T3 cells were seeded in 24-well plates at 2.2 x 104 cells/well. After 24 h, the media was removed, and the cells were infected with infection cocktail containing 10100 µl RV-SN, 4 µg/ml Polybrene (Sigma, St Louis, MO) and growth medium (DMEM with serum) to a final volume of 200 µl. After 12 h, 300 µl DMEM (without Polybrene) was added to each well. After 24 h, the media was replaced with 0.5 ml DMEM without Polybrene and the cells were incubated for an additional 24 h. The cells were then trypsinized, counted and analyzed by FACS since all viral vectors harbor an EGFP in the bicistronic cassette. Viral titer (in duplicates) was calculated as the percentage of fluorescent cells multiplied by absolute the number of cells divided by the RV-SN dilution factor.
Infection of target cells with RVVs
NIH 3T3 cells were plated at a density of 3 x 106/10-cm plate 1218 h before infection. Cells were infected by removing media and addition of infection cocktail containing 4 ml RV-SN, 8 mg/ml Polybrene and DMEM to a final volume of 7 ml. The cells were grown in this infection cocktail for an additional 24 h and media was replaced with 10 ml fresh media. Cells were analyzed or further treated 48 h later. For FACS analysis the cells were detached from plates with Trypsin and suspended in fresh DMEM for analysis.
J558L and U937 cells from an exponentially growing culture were plated at a density of 3 x 106/10-cm plate and infected by addition of infection cocktail containing 6 ml RV-SN, 8 mg/ml Polybrene and RPMI media to a final volume of 7 ml. The cells were cultivated in this infection cocktail for 46 h. Fresh media was added to 10 ml and the cells were grown for an additional 1618 h. Cells were harvested and resuspended in 10 ml fresh media for an additional 48 h. Prior to FACS analysis the cells were collected and suspended in fresh media.
Analysis of genomic integration of RVV by genomic PCR
J558L, U937 and NIH 3T3 cells were transfected with the various RVV, and selected with G-418 for 3 weeks. Cells (2 x 107) cells were harvested and genomic DNA was extracted with EZ-DNA Genomic DNA Isolation Reagent (MD Biosciences, Israel). The amount and quality of DNA was assessed by DNA gel electrophoresis and by optical density at 260/280 nm. PCR was preformed on 100 ng of genomic DNA with 20 pmol of each primer, 30 mM MgCl2, 10 mM dNTP and Taq polymerase. Primers H3 (5'-TGG TGT GAG GCA CAA ATA TC-3') and H6 (5'-CTT CCA GAC TGG TGG GCG CA-3') were used to monitor the integration of DN-ICSBP; primers R3 (5'-GAG CAA GGG CGA GGA GCT GTT A-3') and R4 (5'-GCG GTC ACG AAC TCC AGC AGG AC-3') were used to monitor the integration of EGFP.
Fluorescent staining of cell-surface markers
Cells (5 x 1051 x 106) were harvested for 2 min at 1500 g, the media was removed, and the cells were washed once with 1 ml cold PBS buffer and once with 0.5 ml staining buffer (1% BSA/0.05% sodium azide in PBS). Cells were then incubated with primary antibody diluted in staining buffer to a final volume of 100 µl for 20 min at 4°C. The cells were then washed twice with staining buffer and incubated with fluorescent-biotinylated secondary antibody (Caltag, Burlingame, CA) diluted in 100 µl staining buffer for 20 min at 4°C followed by staining with streptavidinTriColor conjugate (Caltag; dilution 1:200) for 20 min at 4°C in the dark. The cells were then washed twice with staining buffer, and suspended in 300 µl staining buffer and 300 µl fixation buffer (2% paraformaldehyde in PBS).
Fluorescent staining of cytoplasmic antigens
Cells (5 x 1051 x 106) were harvested for 2 min at 1500 g, the media was removed, and the cells were washed once with 1 ml cold PBS buffer and once with 0.5ml fixation buffer (4% paraformaldehyde in PBS). Cells were then incubated with 1:200 dilution of primary antibody in a staining buffer (1% BSA/0.1% saponin in PBS) to a final volume of 100 µl for 40 min at room temperature. The cells were then washed twice with staining buffer and incubated with 1:200 dilution of fluorescent-biotinylated secondary antibody (Caltag) in 100 µl staining buffer for 40 min at 4°C and following two washes in staining buffers cells were stained with 1:200 dilution of streptavidinTriColor conjugate (Caltag) in the same buffer for 20 min at 4°C in the dark. The cells were then washed twice in staining buffer, suspended in 300 µl of PBS and used for FACS analysis.
Annexin-V detection assay
Annexin-Vbiotin apoptosis detection assay (R & D Systems, Minneapolis, MN) was performed as directed by the manufacturer. In principle, 1 x 1051 x 106 cells were harvested for 5 min at 500 g at room temperature. The media was removed and cells were washed once with 0.5 ml PBS and once with PBS + 1% FCS. Cells were gently suspended in Annexin-Vbiotin reaction mix, prepared as directed (Annexin-V dilution up to 1:200) and incubated for 15 min at room temperature in the dark. Cells were harvested and resuspended in buffer containing streptavidin bound to fluorescent TriColor marker (dilution 1:200) for 15 min in the dark at room temperature. After 15 min, 0.4 ml binding buffer was added to cells and FACS analysis took place within 1 h.
FACS analysis
FACS analysis was preformed with a Becton Dickinson FACSCalibur machine with WinMDI 2.8 software. Calibration was preformed before each analysis with an appropriate negative control (cells stained with non-relevant or secondary antibody only), and the negative peak was adjusted between 1 and 10 on a logarithmic scale (FL-1 or FL-3). Sample volume was at least 0.6 ml and analysis was performed at a medium or high flow rate (3060 ml/min) unless high resolution was needed (slow, 15 ml/min). Analysis of FL-1 (FITC, 530 nm) and FL-3 (TriColor, 670 nm) did not require compensation adjustments.
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Results
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Previously, we have shown that IRF-8\ICSBP binds to target DNA only following interaction with IRF and non-IRF transcription factors (11). Two intact DBD, that of IRF-8\ICSBP and that of the interacting partner, are essential for the formation of DNA-binding heterocomplexes. In addition, two intact association domains are essential, e.g. the IAD of IRF-8\ICSBP and the PEST domain of the interacting partner. When the DBD of IRF-8\ICSBP is either missing or defective, proteinprotein association still occurs, but the heterocomplex produced lacks DNA-binding capacity.
Construction of a bicistronic RVV for the expression of DN-ICSBP
Based on these findings, we have generated a DN mutant of IRF-8\ICSBP (DN-ICSBP) which competes for the activity of the endogenous factor by interfering with proteinprotein interaction. To generate such a protein, we deleted the first 33 amino acids of IRF-8\ICSBP, which are part of the conserved DBD, and fused instead the yeast GAL4 DBD. This mutant will be able to promote proteinprotein interactions with other transcription factors, via its IAD, but will inhibit DNA binding of the associated factor, leading to inhibition of their transcriptional activity. The fused DBD of the yeast transcription factor GAL4 serves as a nuclear localization element, which lacks target DNA sequences in mammalian cells and thus is inactive. The chimeric DN-ICSBP was cloned in the bicistronic expression vector pIRES2, which also allows the translation of EGFP from the same transcript due to the presence of the EMCV IRES. This bicistronic cassette was subcloned to the RVV pLNCX (Promega, San Luis Obispo, CA) under the control of the CMV promoter. In addition, the vector harbors G418 selection marker (for details, see Fig. 1A and Methods). As a control we used an IRF-8\ICSBP mutant in which Leu331 was mutated to Pro. Such a DN-L331P-ICSBP is defective not only in its DNA-binding capacity, but also in its ability to be engaged in proteinprotein interactions (11). The DBD of GAL4 alone served as an additional control to ensure that this DNA-binding moiety is indeed inactive in mammalian cells.

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Fig. 1. DN-ICSBP inhibits transcriptional synergy between PU.1 and IRF. (A) Schematic illustration of the various clones in the RVV pLNCX. For details, see text and Methods. NIH 3T3 cells were infected with the RVV encoding to GAL4, DN-L331P-ICSBP and DN-ICSBP as indicated. After 96 h, the cells were co-transfected with 60 ng of the vectors encoding for PU.1 and IRF-8\ICSBP (B) or PU.1 and IRF-4 (C), and with the reporter plasmid encoding for luciferase under the control of the B enhancer element. At 48 h post-transfection, the cells were harvested and luciferase activity was measured. The luciferase activity was normalized by the transfection efficiency and number of cells. Each set of transfection experiments was repeated at least 3 times generating similar results.
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DN-ICSBP inhibits the transcriptional synergy between PU.1 and either IRF-8\ICSBP or IRF-4
To test the effectiveness of the constructed DN-ICSBP, we first tested its ability to inhibit the synergistic interaction between IRF-8\ICSBP and PU.1 (11). As seen in Fig. 1(B), co-transfection of IRF-8\ICSBP and PU.1 into NIH 3T3 led to synergistic induction of the luciferase reporter gene driven by the IgL chain
B element, which is composed of both IRF- and PU.1-binding sites (Fig. 1B, cf. lanes 1 and 2). Complete inhibition of this PU.1/IRF-8\ICSBP synergetic activation was observed when the cells were co-transfected with DN-ICSBP (Fig. 1B, lane 4). On the other hand, co-transfection of DN-L331P-ICSBP, which harbors a point mutation within the IAD, did not result in any inhibitory effect on the synergistic activity between PU.1 and IRF-8\ICSBP (Fig. 1B, lane 3). These results indicate that DN-ICSBP is able to compete with ICSBP on the interaction with PU.1 leading to the formation of a defective heterocomplex which is incapable of binding to the target DNA sequence.
IRF-4, the closest homologue of IRF-8\ICSBP, is an essential factor for Ig production in B cells through association with PU.1 (16). We next tested the ability of the DN-ICSBP to inhibit the transcriptional synergy elicited via the interaction between IRF-4 and PU.1. As seen in Fig. 1(C), co-transfection of IRF-4 and PU.1 into NIH 3T3 cells resulted in
40-fold increase in the activity of the luciferase reporter gene driven by the IgL chain
B element (Fig. 1C, lane 4). These cells were also co-transfected with increasing amounts of DN-ICSBP, and, as seen in Fig. 1, this led to a gradual decrease in the observed transcriptional synergy between IRF-4 and PU.1 (Fig. 1C, cf. lanes 4 and 1416). Excess expression of DN-ICSBP competed effectively for the interaction with PU.1, leading to the formation of a defective heterocomplex that was incapable of binding to the DNA. As expected, neither increasing amounts of co-transfected GAL4 construct nor a high amount of the DN-L331P-ICSBP construct had any significant effect on the transcriptional synergy between IRF-4 and PU.1 (Fig. 1C, cf. lanes 4 and 1113 and 17, respectively).
DN-ICSBP inhibits transcriptional synergism between IRF-4 and PU.1 through direct association with PU.1
The data presented in Fig. 1 suggested that, like IRF-4, the transfected DN-ICSBP also interacts with PU.1. However, an aberrant heterocomplex is formed, which is incapable of binding to the DNA. Thus, DN-ICSBP is competing with IRF-4 on the interaction with PU.1. This was not the case when just GAL4 or a mutated DN-ICSBP (DN-L331P-ICSBP) defective in its IAD were transfected. These factors did not compete with IRF-4 for the interaction with PU.1. To demonstrate that such interaction between DN-ICSBP and PU.1 took place, immunoprecipitation studies followed by western blot analysis were performed with extracts of transfected cells. To follow the expression of IRF-8\ICSBP we used an HA tag that was fused to the 3' end of the factor. As seen in Fig. 2(A), immunoprecipitation of DN-ICSBP, via the HA tag, led to co-precipitation of PU.1. On the other hand, immunoprecipitation of the DN-L331P-ICSBP did not lead to efficient co-precipitation of PU.1 (Fig. 2A, upper panel, cf. lanes 3 and 4). However, these two factors (DN-ICSBP and DN-L331P-ICSBP) were expressed at comparable levels in the transfected cells as determined by direct western blot analysis with antibodies directed against the DBD of GAL4 (Fig. 2A, lower panel, cf. lanes 3 and 4). Similarly, immunoprecipitation of PU.1 led to co-precipitation of mainly DN-ICSBP and, to a much lesser extent, DN-L331P-ICSBP as determined by western blot analysis with antibodies directed against the GAL4 moiety of these factors (Fig. 2B, upper panel, cf. lanes 3 and lane 2 respectively). Direct western blot analysis with antibodies against PU.1 indicated that the factor was expressed at comparable levels in the various transfection studies (Fig. 2B, lower panel, lanes 13). Altogether, these results indicate that DN-ICSBP is effectively competing with IRF-8\ICSBP and IRF-4 on the interaction with PU.1.

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Fig. 2. Co-immunoprecipitation of PU.1 and DN-ICSBP. 293gp (1. 5 x 106) cells were co-transfected with 10 µg PU.1, CX (empty vector), CX-DN-ICSBP and CX-DN-L331P-ICSBP as indicated. At 48 h post-transfection cells were harvested and immunoprecipitation (IP) was preformed with Protein A beads bound to anti-GAL4 antibodies (A) or anti-PU.1 antibodies (B). The precipitated proteins were separated on 10% SDSPAGE gel, transferred to a nitrocellulose membrane and western blotting was preformed with the indicated antibodies. Upper panels: western blot of co-immunoprecipitated proteins. Lower panels: control direct western blot proteins.
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Ectopic expression of DN-ICSBP inhibits the growth of J558L and U937 cells, but not the growth of NIH 3T3 cells
Both PU.1 and IRF-4 are crucial factors for Ig production in general, and IgL
in particular. We wanted to further characterize the role of such proteinprotein interaction in the expression of IgL
in the plasmacytoma cell line J558L. In addition, IRF-8\ICSBP was recently shown to be essential for the maturation and the activity of macrophages. For that purpose, we used the promyelocytic U937 cells that can be further induced to differentiate to mature macrophages in vitro. DN-ICSBP was designed as a molecular tool to test the role of IRF proteinprotein interaction in these processes. To achieve these goals, we attempted to stably express both DN-ICSBP and DN-L331P-ICSBP in these cells following retroviral transduction and G418 selection. J558L and U937 cells resistant to the selective antibiotic and expressing DN-L331P-ICSBP were easily isolated. To our surprise, we could not isolate resistant cells expressing DN-ICSBP in both cell lines during the first 8 days following retroviral transduction. To further characterize this inhibitory effect of the transduced DN-ICSBP, the growth rate of the cultured cells was monitored. At 48 h after transduction, antibiotic selection was employed and the number of living cells was monitored daily. J558L cells transduced with DN-L331P-ICSBP proliferated with a replication time of 22 h, which was similar to that of untransduced cells (Fig. 3A and data not shown). On the other hand, cells transduced with DN-ICSBP did not proliferate during the first 45 days following antibiotic selection and then replicated with a doubling time of
48 h (Fig. 3A). Only after
10 days from the onset of the antibiotic selection did resistant cells start to appear with a replication time that was similar to that of the control cells. Similar results were also observed for U937 cells transduced with the same vectors (Fig. 3B). On the other hand, no inhibitory effect on growth rate was noted with NIH 3T3 cells transfected with DN-ICSBP as compared to cells transduced with DN-L331P-ICSBP (Fig. 3C).

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Fig. 3. DN-ICSBP affects the growth rate of J558L and U937 cells, but not NIH 3T3 cells. J558L (A), U937 (B) and NIH 3T3 (C) cells were infected with retroviruses encoding GAL4, DN-ICSBP and DN-L331P-ICSBP. At 48 h post-infection, expressing cells were selected with 200 (J558L), 800 (U937) or 450 (NIH 3T3) µg/ml G418. During the selection period viable cells were counted using a hemocytometer following Trypan blue staining every 24 h from the first day of selection for 200300 h. Triangles: DN-ICSBP; circles: DN-L331P-ICSBP. The data represent the average of four independent experiments performed in duplicate.
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The data suggested that cells of hematopoietic origin were either inhibited or could not survive the enforced expression of DN-ICSBP. However, we could not detect expression of DN-ICSBP in these cells, while expression of DN-L331P-ICSBP was easily recorded by western blot analysis (data not shown). The inability of the stable clones to express DN-ICSBP is not due to inefficient transduction since these cells expressed comparable levels of EGFP as the DN-L331P-ICSBP- or GAL-4-transduced cells as determined by FACS analysis (data not shown). Further, integration of these bicistronic RVV was also verified by genomic PCR. As seen in Fig. 4, all the transduced cells incorporated both the EGFP and DN-ICSBP genes, indicating that the whole bicistronic cassette was integrated in their genomes.

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Fig. 4. Genomic PCR analysis for the integration of DN-ICSBP and EGFP in retrovirus-infected cells. Genomic DNA was extracted from stable clones of J558L, U937 and NIH 3T3 cell lines, transduced with RVV as illustrated (A). PCR was preformed on 100 ng of genomic DNA with the indicated primers (B). Integration of DN-ICSBP was determined with H3/H6 primers and EGFP with R3/R4 primers. The various lanes represent genomic PCR products from the indicated cells transduced with RRV-GAL4 or RRV-DN-ICSBP. DNA fragments were separated on 1.5% agarose gel and stained with ethidium bromide.
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Ectopic expression of DN-ICSBP in two hematopoietic cell lines leads to enhanced apoptosis that is not observed in fibroblastoid cells
Altogether, our results suggest that selection for a high level of expression of DN-ICSBP in either J558L or U937 cells was inhibitory or lethal to the expressing cells. Cells that were rescued following the selection process, which led to stable integration of the gene, did not express detectable levels of DN-ICSBP. This phenomenon was not observed with NIH 3T3 cells or in the human embryonic kidney 293 cell line (data not shown), suggesting that overexpression of DN-ICSBP leads to enhanced apoptosis of hematopoietic cells. To test this possibility, cells were transduced with either DN-ICSBP or DN-L331P-ICSBP and 48 h later the cells were stained with Annexin-V to determine their apoptotic state. These cells were gated by FACS analysis for EGFP expression, which serves as a reporter gene for viral integration, and the level of Annexin-V staining was analyzed. During these experimental conditions no antibiotic selection was employed (for details, see Methods). As seen in Fig. 5, both J558L cells and U937 cells transduced with DN-ICSBP were significantly more apoptotic than cells transduced with DN-L331P-ICSBP. Cells transduced with DN-L331P-ICSBP had the same apoptotic index as cells transduced with just GAL4 or untransduced cells (data not shown). Unlike the results with J558L or U937 cells, no increase in apoptosis was observed in NIH 3T3 cells transduced with DN-ICSBP when compared with cells transduced with DN-L331P-ICSBP (Fig. 5, lower panel). These results are in agreement with the data presented in Fig. 3 demonstrating that DN-ICSBP elicited significant growth inhibition only on cells of hematopoietic origin.

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Fig. 5. DN-ICSBP induces apoptosis in J558L and U937 cells, but not NIH 3T3 cells. J558L, U937 and NIH 3T3 cells were infected with the different retroviruses. At 72 h post-infection, the cells were reacted with biotinylated Annexin-V and labeled with streptavidinTriColor conjugate (for details, see Methods) and analyzed by FACS. Cells expressing EGFP were gated using the FL-1 channel and co-analyzed for the levels of Annexin-V by the FL-3 channel. White histogram: DN-ICSBP; black histogram: DN-L331P-ICSBP. Numbers represent the mean fluorescence value. Each set of transfection experiments was repeated at least 3 times generating similar results.
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Short-term expression of DN-ICSBP in J558L cells inhibits IgL
expression
Finally, we wanted to test whether DN-ICSBP can inhibit the expression of endogenous genes such as IgL
in the plasmacytoma J558L cells. For that purpose, the cells were transduced with RVV harboring DN-ICSBP or DN-L331P-ICSBP or empty vector containing only EGFP as the second cistron. After 48 h, the cells were stained for cytoplasmic IgL
expression and gated by FACS for EGFP expression, and the level of IgL
expression was determined in these cells. As seen in Fig. 6, in cells transduced with DN-ICSBP, the level of IgL
expression was half of the level detected in the control cells transduced with DN-L331P-ICSBP, GAL4 or empty vector. These results suggest that shortly after transduction and prior to its apoptotic effect in J558L cells, DN-ICSBP was able to compete for the endogenous transcription factor(s), probably IRF-4, for the synergistic interaction with PU.1. This interaction subsequently led to the formation of a DN-ICSBP/PU.1 heterocomplex, which is incapable of binding to the proper enhancer element of the IgL
gene.
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Discussion
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IRF-8\ICSBP exerts its transcriptional activity through association with other transcription factors. Association with IRF members like IRF-1 or IRF-2 leads mainly to transcriptional repression of ISRE-containing promoters. Conversely, IRF-8\ICSBP also associates with non-IRF members such as PU.1 or E47 on DNA composite elements, which leads to transcriptional synergy. These latter associations were originally identified for IRF-4, which is the closest homologue to IRF-8\ICSBP. Accumulating data suggests that IRF-8\ICSBP is engaged in numerous associations with additional transcription factors [for review, see (2)]. To dissect the role of the various proteinprotein interactions in immune cells, a DN-ICSBP was constructed. As shown here, this DN-ICSBP, which is truncated at the DBD and cloned in a bicistronic RVV, was able to negate the association of either IRF-8\ICSBP or IRF-4 with PU.1 (Fig. 1) and with E47 (data not shown). As expected, DN-ICSBP interacted with its partners, but the heterocomplex formed could not bind target DNA since intact DBD of both interacting partners are critical (17). Surprisingly, attempts to stably express DN-ICSBP in cells of hematopoietic origin resulted in clones in which the expression of DN-ICSBP was silent, although EGFP from the same bicistronic transcript was expressed. This was not due to non-integration of the bicistronic cassette since correct size integration of DN-ICSBP was verified by genomic PCR (Fig. 4). During the process of selecting these stable clones a significant inhibition in the growth rate and increased apoptosis of the transfected cells was noted (Figs 3 and 4 respectively). This inhibitory effect of DN-ICSBP was observed only in hematopoietic cells and was not observed in the murine fibroblast NIH 3T3 cells. The immune cells restricted selection against overexpression of DN-ICSBP, and the inhibitory effect on cell growth and enhanced apoptosis is attributed to the ability of DN-ICSBP to interact with other factor(s) and block their transcriptional capacity. This is due to the fact that a similar construct, in which Leu331 was mutated to Pro, was readily overexpressed in all tested cells (hematopoietic and non-hematopoietic) and demonstrated no inhibitory effect on cell growth. We have shown that this L331A mutation within the IAD of IRF-8\ICSBP ablated its ability to interact with all factors. Further, mutation of the corresponding Leu in the IAD of IRF-4 or IRF-9 produced similar effects, e.g. mutant IRF-4 did not interact with PU.1 or E47 (11) and mutant IRF-9 did not interact with Stat1 and Stat2 (D. E. Levy, pers. commun.).
Previously, the DBD of IRF-8\ICSBP was used as DN, which inhibited both type I and type II IFN-stimulated genes, and in addition inhibited human HIV type 1 and vaccinia virus infection in monocytic cells (18,19). However, since all IRF bind to similar DNA motifs, it is possible that this DBD of IRF-8\ICSBP was competing for the binding of other IRF members such as IRF-3 and IRF-7, thus acting as an IRF-specific DN rather than IRF-8\ICSBP specific. To avoid such non-specific inhibition, Brass et al. have fused the DBD of IRF-4 and the DBD of PU.1, and this chimeric protein was capable of inhibiting promoters harboring EtsIRF composite elements to which these interacting factors can bind (16). We generated a DN-ICSBP that takes part in proteinprotein interaction, but is incapable of binding to the target DNA sequence since the DBD of both interacting partners are essential (11,20). Similar to that reported for the IRF-4-PU.1 DBD fusion construct, our DN-ICSBP construct was also able to inhibit the expression of IgL
in the plasmacytoma cell line J558L [(16) and Fig. 6 respectively]. Native forms of DN IRF, truncated at their DBD, were recently reportedIRF-3a, a splice variant of IRF-3, and viral-encoded IRF-like factors (21,22). It is thought that these native DN factors affect the activity of IRF through proteinprotein interaction, thus interfering with the ability of the normal partner to bind to the DNA.
Similar to the DN-ICSBP described here, other DN IRF defective in their DBD were recently reported. The DN construct of IRF-3 was used to show that the factor is essential for the IFN-
response against viral myocarditis in mice (23) and for Sendai virus-induced apoptosis (24). The ability of IRF-7 to form homodimers or heterodimers with IRF-3 and its role in virus-activated transcription of IFN-
genes was also demonstrated using DN IRF-7 truncated at its DBD (25).
The IAD of IRF show structural similarities with the MH2 domain of the Smad family of transcription factors, which facilitate proteinprotein interaction (3,7). Overall, this is a conserved structural element that confers proteinprotein interaction. However, the interaction specificity is dictated by non-conserved amino acid residues. Based on this, we reasoned that our DN-ICSBP will compete for the interaction of endogenous IRF-8\ICSBP and for some of the interactions of IRF-4, since both IRF members associate with PU.1 and E47. Here, we show that DN-ICSBP can inhibit both IRF-4- and ICSBP-mediated interaction with PU.1 using a reporter gene assay and co-immunoprecipitation studies in overexpressing cells. We were not able to generate stable clones overexpressing DN-ICSBP in the plasmacytoma or the pro-monocytic cell lines, while such clones were readily achieved with NIH 3T3 cells. The fact that DN-ICSBP harboring L331A had no inhibitory effect on any cell line tested supports our assumption that DN-ICSBP exerts its inhibitory effect via proteinprotein interaction only in cells of hematopoietic origin. Although ICSBP was shown to be engaged in numerous proteinprotein interactions, this inhibitory effect is most probably through interaction with hematopoietic-specific factors. We speculate that PU.1, which is an essential factor for myeloid/lymphoid cell differentiation and maturation, may be the target gene for DN-ICSBP. Supportive data comes from a recent study by Sevilla et al. (26) demonstrating that Ets2 and PU.1 are also essential factors for macrophage survival. These factors function together to inhibit apoptosis by transcriptionally activating the expression of the Bcl-xL gene. Any interference in the expression level of either factor by the DN construct led to a massive increase of apoptosis. It is most probable that our failure to overexpress DN-ICSBP only in hematopoietic cells is due to its interaction with PU.1. This aberrant heterocomplex neutralizes PU.1 from synergizing with Ets2, leading to reduced expression of Bcl-xL and consequently to apoptosis. In addition, it was recently shown that overexpression of ICSBP resulted in decreased expression of Bcl-xL (27). It is most probable that excess expression of ICSBP led to efficient formation of heterocomplexes with PU.1 and, like DN-ICSBP, it served as a sink preventing PU.1 from synergizing with Ets2 to keep essential levels of Bcl-xL. Our results suggest that PU.1 together with Ets2 also serve as essential survival factors for B cells, and this is in line with the fact that PU.1 gene disruption studies showed that PU.1-deficient mice lack mature B cells, neutrophils and macrophages (28,29)
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Acknowledgements
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Financial support from The Niedersachsen-Israel Research Cooperation Program and from the fund for the promotion of research at the Technion is gratefully acknowledged.
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Abbreviations
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DBDDNA-binding domain
DNdominant negative
EGFPenhanced green fluorescent protein
IADIRF association domain
ICSBPIFN consensus sequence-binding protein
IRESinternal ribosome entry site
IRFIFN-regulatory factor
ISREIFN simulated response element
RV-SNretrovirus-containing supernatant
RVVretroviral vector
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