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PU.1 and Multiple IFN Regulatory Factor Proteins Synergize to Mediate Transcriptional Activation of the Human IL-1 Gene¹

Pulmonary Center and Department of Pathology, Boston University School of Medicine, Boston MA 02118

	Abstract

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Both lymphoid and myeloid cells express two related members of the IFN regulatory factor (IRF) family of transcription factors, specifically IRF-4 and IFN consensus binding protein (ICSBP or IRF-8). We previously reported that macrophages express IRF-4 and in combination with the ETS-like protein PU.1 can synergistically activate a human IL-1 reporter gene. Here we report that this synergy is mediated by a composite PU.1/IRF element located within an upstream enhancer known to confer cytokine- and LPS-inducible expression. In macrophages, synergistic activation of IL-1 reporter gene expression was preferentially mediated by IRF-4, whereas IRF-4 and ICSBP were equally capable of synergizing with PU.1 when coexpressed in fibroblasts. Furthermore, coexpression of IRF-1 and IRF-2 dramatically increased the capacity of both PU.1/IRF-4 and PU.1/ICSBP to induce IL-1 reporter gene expression in fibroblasts. The additional synergy observed with IRF-1 and IRF-2 coexpression is mediated by a region of DNA distinct from either the IL-1 enhancer or promoter. We also assessed the capacity of these transcription factors to activate endogenous IL-1 gene when overexpressed in human embryonic kidney 293 cells. Although ectopic expression of PU.1 alone was sufficient to activate modest levels of IL-1 transcripts, endogenous IL-1 expression was markedly increased following coexpression of additional IRF proteins. Thus, maximal expression of both a human IL-1 reporter gene and the endogenous IL-1 gene was observed in cells that coexpressed PU.1, IRF-4 (or ICSBP), IRF1, and IRF2. Together, our observations suggest that these factors may function together as an enhanceosome.

	Introduction

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Responses of immune cells to IFN- are mediated by a variety of transcription factors, including STAT proteins and members of the IFN regulatory factor (IRF)³ family. The former bind largely to genes containing IFN--activated sequence (GAS) elements, whereas the latter bind largely to IFN sequence response elements (ISRE). The IRF family is comprised of 10 mammalian members and several viral homologues (reviewed in Refs. 1, 2, 3). Although most IRF proteins are ubiquitously expressed, IRF-4 and IFN consensus sequence binding protein (ICSBP, also known as IRF-8) are primarily expressed in lymphoid and myeloid cells (4, 5, 6, 7, 8). ICSBP was initially reported to function solely as a transcriptional repressor, but was subsequently shown to also function as a transcriptional activator (4, 9, 10, 11). IRF-4 functions as a transcriptional repressor of ISRE-containing genes, presumably when interacting with other IRF proteins. In contrast, IRF-4 can serve as a transcriptional activator when interacting with the ETS-like PU.1 (also known as Spi-1). Thus, all IRF proteins may be capable of serving dual roles in transcriptional regulation, with their ultimate function being defined by interaction with additional factors. Potential IRF interaction partners include ETS proteins, NF-B family members, as well as the transcriptional coactivators CBP (CREB binding protein) and PCAF (a phorbol-ester-inducible coactivator of the IRF family) (9, 12, 13, 14).

PU.1 is a critical regulator of myelopoiesis and is predominately expressed in cells of the hemopoietic lineage (reviewed in Refs. 15, 16, 17). PU.1 can bind alone to a core motif (A/GGAA) that is shared by most ETS proteins, although additional flanking sequences define the ultimate binding specificity (18). Genes regulated by PU.1 include CD11b, the macrophage mannose receptor, the type A scavenger receptor, the M-CSF receptor, the IL-1 receptor antagonist, and others (17, 19, 20, 21, 22). PU.1 can also regulate gene expression through a composite PU.1/IRF DNA motif, a sequence element containing adjacent PU.1 and ISRE motifs. Of all the IRF family members, only IRF-4 and ICSBP have been reported to physically interact with PU.1 and bind to composite elements (23, 24, 25, 26, 27). Several genes contain composite PU.1/IRF elements, including the Ig and light chains, gp91^phox, CD20, IL-18, and Toll-like receptor-4 (9, 10, 28, 29, 30, 31) (Table I). PU.1 binds to DNA and then recruits either IRF-4 or ICSBP to the PU.1/IRF composite element through protein-protein interaction that is mediated (in part) by the proline-glutamate-serine-threonine-rich (PEST) domain of PU.1 (26). This interaction is significantly enhanced by phosphorylation of serine 148 on PU.1, a residue located within the PEST domain (28).

Our previous finding that an IL-1 reporter plasmid could be synergistically activated by PU.1 and IRF-4 prompted us to identify the region within the gene that mediated this synergy. Here we present evidence that the upstream IL-1 enhancer contains a functional PU.1/IRF composite element, and that this synergistic activation can be further augmented by coexpression of IRF-1 and IRF-2. Interestingly, this IRF-1- and IRF-2-dependent synergy is mediated by a region of DNA not contained in either the upstream enhancer or the promoter regions. Lastly, ectopic coexpression of PU.1 and these IRF proteins was found to activate the endogenous IL-1 gene, further suggesting that these factors may regulate IL-1 production in vivo.

	Materials and Methods

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Reagents and Abs

Trypsin, proteinase K, antipain, aprotinin, chymostatin, and leupeptin were purchased from Sigma (St. Louis, MO). Polyclonal rabbit antisera raised against human PU.1 as well as polyclonal goat antisera raised against IRF-4 or ICSBP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Normal preimmune goat and rabbit sera were purchased from Pierce (Rockford, IL). These antisera were not cross-reactive, as determined by the manufacturer and confirmed in the laboratory (data not shown).

Cell lines and tissue culture conditions

RAW264.7 murine macrophage and NIH-3T3 murine fibroblast cell lines were purchased from American Type Culture Collection (Manassas, VA). Human embryonic kidney (HEK) 293 fibroblasts were a gift from Douglas Golenbock (Boston Medical Center, Boston, MA). RAW264.7 cells were maintained in RPMI culture medium (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT), 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (BioWhittaker). NIH-3T3 and HEK 293 cells were cultured in DMEM (BioWhittaker) supplemented as described above. All tissue culture reagents contained <10 pg/ml endotoxin contamination as determined by the manufacturer.

EMSA analysis

Nuclear extracts were prepared as described previously (4). Protein concentrations of the extracts were determined using a commercial kit (Bio-Rad, Hercules, CA). All nuclear extracts were stored at -70°C, and multiple freeze-thawing cycles were avoided. A double-stranded oligonucleotide corresponding to a single copy of the human IL-1 enhancer composite element was used as a probe in EMSA experiments (5'-GA CAT AAG AGG TTT CAC TTC CTG AGA TGG ATG GA-3', the single composite PU.1/IRF binding site is underlined). An unlabeled competitor double-stranded oligonucleotide containing a single NF-B site from the IL-2R -chain promoter (5'-GGGGAATTCC-3') was also used. DNA probes were radiolabeled with [-³²P]dATP (DuPont-NEN, Boston, MA) using Escherichia coli DNA polymerase Klenow fragment (Promega, Madison, WI) as recommended by the manufacturer. Unincorporated nucleotides were removed using Sephadex G-25 columns (5 Prime3 Prime, Boulder, CO). Nuclear extracts (typically 5 µg) were incubated with radiolabeled probe DNA (0.1 ng, typically 10,000 cpm) in the presence of 2 µg of poly(dI-dC)·(dI-dC) (Pharmacia, Piscataway, NJ), 1.0 mM EDTA, 10 mM Tris-HCl (pH 7.9), 25 mM glycerol, and 0.5 mM DTT as previously described (36, 38). Binding reactions were incubated at room temperature for 30 min. In competition experiments, unlabeled oligonucleotide DNA was added to the nuclear extracts at a 100 fold-molar excess before addition of the labeled binding reaction, where indicated. For supershift experiments, 2 µg of antiserum was added to the nuclear extracts before addition of the labeled binding reaction as indicated. Following incubation, a portion of each binding reaction was electrophoresed through a 7% nondenaturing low ionic strength polyacrylamide gel in an electrophoresis buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM borate, and 0.5 mM EDTA. The gels were dried without fixation and visualized by autoradiography.

RT-PCR

Total RNA from transiently transfected HEK 293 fibroblasts was extracted using Qiashredder and RNA Easy RNA purification columns exactly as recommended by the manufacturer (Qiagen, Valencia, CA). Briefly, 600 µl of cell lysis buffer was added per well of a six-well tissue culture dish (Corning-Costar, Cambridge, MA) containing 0.6–2 x 10⁶ transiently transfected cells. Following passage through a Qiashredder spin column (Qiagen), the flow-through was mixed 1/1 (v/v) with isopropanol and passed over an RNA-Easy spin column. RNA was quantified by spectrophotometry using a Beckman DU-65 spectrophotometer (Columbia, MD).

Reverse transcriptase reactions to generate cDNA were performed using avian myeloblastosis virus reverse transcriptase (Promega). Briefly, 10 µg of total RNA was reverse transcribed in a 60-µl reaction containing a final concentration of 5 mM MgCl₂, 1x RT buffer, 1 mM dNTP, 0.5 µg of oligo(dT)/µg of RNA, 1 U of RNase inhibitor/µl of reaction, and 15 U of avian myeloblastosis virus RT/µg of RNA. PCR were performed using between 100 ng and 2 µg of cDNA, 1 mM oligonucleotide primers (each), 1.5 mM MgCl₂, 150 µM dNTPs, and 2.5 U of Taq in a final reaction volume of 75 µl for 30 cycles (95°C denaturation, 30 s; 55°C annealing, 1 min; 72°C extension, 1.5 min). The intron-spanning PCR primers for human IL-1 and -actin used in this study are listed below. Following amplification, a portion of the PCR was electrophoresed through a 1.2% agarose gel and the 599-bp IL-1 and 402-bp -actin products were visualized using ethidium bromide: 5' human IL-1 primer, 5'-TTC TTC GAC ACA TGG GAT AAC GA-3'; 3' human IL-1 primer, 5'-GGA AAG TCC AGG CTA TAG CCG TAC-3'; 5' human -actin primer, 5'-TGG TGG GCA TGG GTC AGA AG-3'; and 3' human -actin primer, 5'-GTC CCG GCC CAG CCA GGT CCA G-3'

Plasmids

An expression plasmid encoding wild-type PU.1 was provided by Michael L. Atchison (University of Pennsylvania, Philadelphia, PA) and was previously described (23). An expression plasmid encoding the full-length murine ICSBP protein was provided by Keiko Ozato (National Institutes of Health, Bethesda, MD) and was previously described (39). Expression plasmids encoding C-terminal deletion mutants of ICSBP (m363 and m373) were provided by Ben-Zion Levi (University of Haiifa, Haiifa, Israel) and were previously described (40). Expression plasmids encoding the wild-type murine IRF-4 protein, a mutant IRF-4 protein lacking an -helical domain spanning aa 396–413 (IH), and a mutant IRF-4 protein containing a single amino acid substitution at position 399 (K399A) were provided by Harinder Singh (University of Chicago, Chicago, IL) and were previously described (27). Full-length murine IRF-1 and IRF-2 expression plasmids were provided by Stephanie Vogel (Uniformed Services University of the Health Sciences, Bethesda, MD) and were previously described (41). The control vector pcDNA3.1 was purchased from Invitrogen (Carlsbad, CA) and was used to maintain a constant amount of total plasmid DNA in each transfection.

A wild-type IL-1 luciferase reporter construct containing the entire human IL-1 promoter (positions -3757 to +11) was previously described (4). Mutant IL-1 reporter constructs containing mutations of either the PU.1 or IRF binding sites were generated using the Quick-Change PCR-plasmid mutagenesis kit exactly as recommended by the manufacturer (Stratagene, La Jolla, CA). The mPU.1 reporter contains a mutation at the PU.1 binding site within the putative PU.1/IRF composite element (GGAA to TTCA), while the mIRF reporter contains a mutation of the IRF binding site (GAAACC to GAATCA). These changes are also shown in Fig. 1A. A mutant IL-1 chloramphenicol acetyltransferase (CAT) reporter construct containing a deletion of the PU.1 site located within the cap site-proximal promoter at position -40 (PU.1) was generated by PCR mutagenesis and was previously described (35). A luciferase reporter containing four tandem copies of the IL-1 PU.1/IRF composite element under control of the IL-1 promoter was generated by ligating an annealed oligonucleotide containing four copies of the IL-1 PU.1/IRF composite element (each identical in sequence to the EMSA probe) into the BglII site of the pGL3 basic luciferase reporter (Promega). The promoter fragment from the wild-type reporter was blunt end ligated into the XhoII site of the pGL3. Endotoxin levels in plasmid preparations were <10 pg/ml final concentration as measured by the Limulus amebocyte lysate kit (BioWhittaker). All plasmid preparations were passed through a 0.2-µm pore size syringe filter before use (Fisher Scientific, Pittsburgh, PA).

View larger version (30K): [in this window] [in a new window]   FIGURE 1. PU.1/IRF synergy requires DNA binding sites. A, Schematic diagram representing the human IL-1 reporter constructs used. Sequences of mutations introduced into the PU.1/IRF composite element (mIRF and mPU.1) are included as well as the cap site-proximal PU.1 binding site. B, Transient transfections of RAW264.7 macrophages were performed using the wild-type or mutated IL-1 reporter constructs (mIRF or mPU.1) and expression plasmids encoding either IRF-4 or ICSBP, as indicated. Although basal activities of all reporters were normalized to 1, the mPU.1 and mIRF reporters exhibited lower (2- to 4-fold) raw luciferase values than the wild-type reporter. C, Transient transfections of NIH-3T3 fibroblasts with the IL-1 reporter constructs used in A and expression plasmids encoding PU.1, IRF-4, or ICSBP as indicated. Data are representative of at least three separate experiments performed in triplicate. All data presented are the mean luciferase reporter activity expressed as fold activity over reporter alone ± SD, where the basal level of reporter activity is normalized to a value of 1.

Transient transfections were performed using SuperFect reagent (Qiagen) according to the manufacturer’s instructions. Briefly, cells were plated in six-well dishes 1–2 days before transfection, and transfections were performed when cells reached 80% confluence. Plasmid DNA was added to 100 µl of Opti-Mem reduced serum medium (Life Technologies, Gaithersburg, MD). RAW264.7 transfections used a total of 3 µg of plasmid DNA, while NIH-3T3 and HEK 293 transfections were performed using a total of 4 µg of plasmid DNA. A constant quantity of transfected plasmid DNA was maintained by inclusion of the empty vector pcDNA3.1. Ten microliters (RAW264.7, NIH-3T3), or 12 µl (HEK 293) of SuperFect was added to the DNA-medium mixture, incubated for 10 min at ambient temperature, diluted with 600 µl of serum-containing medium, and added to individual wells. Each mixture was prepared individually and each transfection condition was performed in triplicate. Mixtures were incubated with cells for 2–3 h, except in HEK 293 transfections, where the mixtures were incubated overnight. Cells were then washed, cultured in complete medium, and harvested 18–24 h post-transfection.

Luciferase activities were measured using the Luciferase Assay System (Promega) according to the manufacturer’s instructions and were performed as previously described (4). Thirty micrograms of total protein from each lysate was assayed for luciferase activity using a Monolight 3010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PU.1/IRF synergy requires DNA contact at the upstream IL-1 enhancer

We previously reported that PU.1 synergized with either IRF-4 or ICSBP in NIH-3T3 fibroblasts to activate a reporter plasmid under control of the full-length human IL-1 promoter and enhancer (4). The cis-acting DNA element that mediated this synergy was not identified. After careful screening of the promoter and enhancer regions, a consensus PU.1/IRF composite element (^A/_GGAAGTGAAAN^T/_C) was identified within the upstream IL-1 enhancer at position -2846 (Fig. 1A). Sequence comparison of known PU.1/IRF composite elements and the putative composite element contained within the IL-1 enhancer revealed strong sequence similarity (Table I), suggesting that this element may be a functional PU.1/IRF composite element.

To determine whether this putative PU.1/IRF composite element was functional, we introduced mutations into the PU.1 and IRF binding motifs within the context of a large genomic fragment (positions -3757 to +12). These mutations (Fig. 1A) were identical with those previously demonstrated to decrease binding of these transcription factors to the Ig enhancer composite element (28). Transient transfection studies using wild-type and mutant IL-1 reporter plasmids were performed using RAW264.7 macrophages and NIH-3T3 fibroblasts. As shown in Fig. 1B, mutation of the PU.1 binding site (mPU.1) decreased IRF-4-dependent IL-1 reporter activity in macrophages by 40%, whereas mutation of the IRF binding site (mIRF) essentially abolished IRF-4-dependent reporter gene expression. In contrast to IRF-4, overexpression of ICSBP had little effect on IL-1 reporter activity in RAW264.7 macrophages (Fig. 1B). Macrophages constitutively express PU.1, IRF-4, and ICSBP, making it difficult to assess the individual contribution of each transcription factor. Therefore, these experiments were repeated using NIH-3T3 fibroblasts, cells that do not express these factors. Transient cotransfection of the wild-type, mPU.1, or mIRF reporters along with the PU.1, IRF-4, and ICSBP expression plasmids revealed that IL-1 reporter activity was similarly affected by these mutations in transfected NIH-3T3 fibroblasts (Fig. 1C). Cells expressing either PU.1/IRF-4 or PU.1/ICSBP could only activate mPU.1 reporter activity to 40–50% of that observed with the wild-type IL-1 reporter, whereas no PU.1/IRF synergy was observed using the mIRF reporter. Furthermore, when a reporter plasmid lacking the PU.1/IRF composite element was transfected into NIH-3T3 fibroblasts, no PU.1/IRF synergy was detectable (data not shown). These observations suggest that the upstream IL-1 enhancer contains a PU.1/IRF composite element capable of supporting PU.1/IRF synergy.

PU.1/IRF synergy requires protein-protein interaction

Several studies have demonstrated a direct physical association of PU.1 with either IRF-4 or ICSBP (9, 25, 26, 27, 28, 29, 42). In the case of the Ig and light chain enhancers, direct protein-protein interaction between PU.1 and IRF-4 (in addition to DNA binding) must occur to activate these enhancers. The protein-protein interaction domains (protein-protein IADs) of IRF-4 and ICSBP have been localized to their carboxyl-terminal regions by several groups (27, 40, 42, 43). We sought to determine whether protein-protein interaction was necessary for synergistic activation of the IL-1 composite element by PU.1 and IRF proteins. The capacities of wild-type and mutant IRF-4 and ICSBP proteins to synergize with PU.1 were examined in macrophages and NIH-3T3 fibroblasts. These IRF mutants were selected based on their reported inability to functionally interact with PU.1. IRF-4 IH encodes a protein lacking an -helical region (aa 396–413) that mediates interaction with PU.1, while the K399A mutant IRF-4 contains a single amino acid substitution within this helix (K399A) (27, 43). We also examined the ICSBP mutants m363 and m377, which encode proteins lacking 57 or 47 C-terminal amino acids (respectively), truncating ICSBP at or near the protein IAD (40). These ICSBP mutants were selected because they correspond to the -helical domain of IRF-4 involved in interaction with PU.1. These IRF-4 and ICSBP mutations are represented in Fig. 2A.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Protein-protein interaction domains present in IRF-4 and ICSBP are required for synergy with PU.1. A, Schematic diagram representing IRF-4 and ICSBP, drawn to scale. The locations of the IRF-4 mutations (IH, K399A) and ICSBP mutations (m363, m377) are denoted by arrows. B, Mutated IRF-4 and ICSBP do not maximally activate the IL-1 reporter in macrophages. RAW264.7 macrophages were transiently transfected with the wild-type IL-1 reporter and either wild-type or mutated IRF-4 (wt, IH, or K399A) or ICSBP (wt, m363, or m377). C, Mutated IRF-4 and ICSBP fail to maximally synergize with PU.1 compared with the wild-type proteins in fibroblasts. NIH-3T3 fibroblasts were transiently cotransfected with the IL-1 reporter, wild-type or mutated IRF proteins, and/or wild-type PU.1 as indicated. Data are representative of at least three separate experiments performed in triplicate and are presented as the mean fold luciferase activity of triplicate transfections ± SD over reporter alone, which is normalized to 1.

IRF-4 and ICSBP bind to the IL-1 PU.1/IRF composite element in a PU.1-dependent manner

We subsequently sought to determine whether PU.1, IRF-4, and ICSBP directly bind to the composite element in vitro using EMSA analysis. Nuclear extracts were prepared from RAW264.7 macrophages and incubated with a radiolabeled oligonucleotide probe containing a single copy of the PU.1/IRF IL-1 composite element. As shown in Fig. 3, we observed four specific DNA-protein complexes (designated I, II, III, and IV). Complex I contained only PU.1, as judged by Ab supershifting (lane 5), while complex III contained both PU.1 and either IRF-4 or ICSBP (lanes 3 and 4). Longer exposures of the EMSA revealed detectable complex III even after supershifting with either IRF-4 or ICSBP (data not shown). Thus, complex III is not a trimolecular complex containing PU.1, IRF-4, and ICSBP. As previously shown for other composite elements (28, 29), DNA binding by IRF proteins was not observed in the absence of PU.1 binding, presumably because PU.1 is necessary to recruit IRF-4 and ICSBP to the ISRE half-site. Lastly, Abs specific for STAT-1, Ets-1, IRF-1, IRF-2, and C/EBP yielded no detectable supershifts (Fig. 3, lane 6, and data not shown). Thus, the identities of the proteins present in complexes II and IV remain to be determined. Preimmune rabbit and goat antisera yielded no detectable supershifted complexes (data not shown). The specificity of DNA binding was demonstrated by competition with excess unlabeled oligonucleotide containing a single copy of the PU.1/IRF composite element (lane 7) and an unlabeled oligonucleotide containing four copies of the PU.1/IRF composite element (lane 8), but not with an unlabeled oligonucleotide containing a single NF-B motif (lane 9). In summary, our data reveal that the human IL-1 enhancer contains a PU.1/IRF composite element capable of binding PU.1 and either IRF-4 or ICSBP in vitro; combined with the transfection data, this supports the hypothesis that the upstream IL-1 contains a functional PU.1/IRF element.

View larger version (63K): [in this window] [in a new window]   FIGURE 3. Detection of IRF and PU.1 binding to the IL-1 enhancer in vitro. EMSA analysis was performed using nuclear extracts prepared from RAW264.7 macrophages and a radiolabeled oligonucleotide PU.1/IRF composite element probe. Four complexes were resolved, labeled I, II, III, and IV. These nuclear extracts were also incubated with Abs specific for IRF-4 (lane 3), ICSBP (lane 4), PU.1 (lane 5), and STAT-1 (lane 6) as indicated. Nuclear extracts were also incubated with unlabeled IL-1 probe (cold 1X), an unlabeled probe containing four copies of the PU.1/IRF composite element (cold 4X), or an unlabeled NF-B probe (cold NF-B). The location of supershifted PU.1, IRF-4, and ICSBP DNA complexes are noted with asterisks, and the migrations of the protein-DNA complexes are denoted by arrows and numbered I, II, III, and IV. The migration of free probe is shown in lane 1, and that of nuclear extract without supershifting Ab is shown in lane 2.

Coexpression of IRF-1 and IRF-2 with PU.1 and either IRF-4 or ICSBP further enhances synergy

Recently, Eklund et al. showed that IRF-1 could further enhance composite element-dependent transcription induced by PU.1 and ICSBP (9). This suggested that other IRF proteins, such as IRF-1 and IRF-2, could further augment synergistic activation of the IL-1 promoter and enhancer. To address this possibility, we cotransfected IRF-1 and IRF-2 with PU.1 and IRF-4 or with PU.1 and ICSBP into NIH-3T3 fibroblasts. As presented in Fig. 4A, a striking synergy was observed when IRF-1 was coexpressed with PU.1/IRF-4 or PU.1/ICSBP. Coexpression of IRF-1 with PU.1 plus IRF-4 (or ICSBP) enhanced IL-1 reporter expression 10-fold over the synergy observed using PU.1/IRF-4 or PU.1/ICSBP alone. In contrast, coexpression of IRF-2 with PU.1/IRF-4 or PU.1/ICSBP had a minimal effect on synergy induced by PU.1/IRF-4 (4.5-fold enhancement) or PU.1/ICSBP (1.2-fold enhancement). Together, these studies show that IRF-1 alone has the ability to substantially enhance IL-1 expression activated by PU.1 and IRF-4 (or ICSBP), whereas IRF-2 alone lacks this ability.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Coexpression of IRF-1 and IRF-2 dramatically enhances PU.1/IRF synergy. A, NIH-3T3 fibroblasts were transiently cotransfected with the wild-type IL-1 reporter and expression plasmids encoding PU.1, IRF-1, IRF-2, IRF-4, and/or ICSBP as indicated. B, IRF-1 and IRF-2 coexpression activates the IL-1 reporter to a level similar to PU.1/IRF-4 and PU.1/ICSBP. NIH-3T3 fibroblasts were transfected with PU.1 and/or various IRF proteins, as indicated. Data are the mean fold luciferase activity over the basal activity of the reporter ± SD and are representative of at least three separate experiments performed in triplicate.

The cap site-proximal PU.1 is required for but does not mediate PU.1/IRF synergy

In addition to the PU.1/IRF composite element located within the enhancer, the IL-1 promoter contains a functional PU.1 binding site, centered at position -40 (35, 45). As shown above, the wild-type and mutated IL-1 reporters could be activated 3- to 10-fold in NIH-3T3 fibroblasts transfected with a PU.1 expression plasmid (Figs. 1C and 2C). To determine whether the promoter-proximal PU.1 site mediated this activity and whether this site was involved in PU.1/IRF synergy, a mutated IL-1 reporter lacking this cap site-proximal PU.1 site (PU.1) was used. As shown in Fig. 5, overall reporter activity was greatly diminished in the absence of the promoter-proximal PU.1 site (compare Fig. 4A with Fig. 5), consistent with previous reports (35, 45). Nonetheless, synergistic activation by PU.1 and these IRF proteins was still observed in transient transfections using the PU.1 IL-1 reporter, although the overall reporter activity was diminished. Furthermore, reporter activation induced by PU.1 alone, which was observed with the wild-type and mutant IL-1 reporters in fibroblasts (Fig. 1C), was absent in cells transfected with the PU.1 IL-1 reporter (Fig. 5). Thus, reporter activation induced by PU.1 expression alone appears to be mediated through the cap site-proximal promoter and not the PU.1/IRF composite element. Moreover, the absence of the promoter-proximal PU.1 site resulted in a dampening of the overall activity elicited by PU.1 and IRF proteins. One possibility is that this promoter-proximal PU.1 site is critical for overall gene expression, and deletion of this site globally decreases reporter activity. An alternate hypothesis is that while this PU.1 site may not be directly involved in PU.1/IRF synergy, it may potentially serve as a signal integrator, transmitting activation signals from the enhancer to the basal transcriptional machinery.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. The cap site-proximal PU.1 binding site is required for but does not mediate PU.1/IRF synergy. NIH-3T3 fibroblasts were transiently transfected with a mutated IL-1 reporter lacking a promoter-proximal PU.1 binding site (PU.1) and various combinations of expression plasmids encoding PU.1, IRF-1, IRF-2, IRF-4, and ICSBP, as indicated. Data are the mean of triplicate transfections ± SD and are presented as fold luciferase activity over that with reporter alone, which is normalized to 1. The data presented are representative of at least three separate experiments performed in triplicate.

The data reported above demonstrate that PU.1 and IRF-4 (or ICSBP in some cases) can synergistically activate IL-1 reporter activity, and that this synergy can be further enhanced by coexpression of IRF-1 and IRF-2. We next determined whether IRF-1 and IRF-2 act directly on the IL-1 composite element or at a distinct region within the IL-1 gene. Reporter plasmids were constructed containing four copies of the IL-1 PU.1/IRF element (identical with the probe used in EMSA analysis) ligated upstream of either the heterologous SV-40 promoter, or the cap site-proximal IL-1 promoter (positions -131 to +11). These plasmids were designated 4X-SV and 4X-IL, respectively.

When the 4X-SV reporter was cotransfected with expression plasmids encoding PU.1, IRF-1, IRF-2, and IRF-4 (ICSBP) into NIH-3T3 fibroblasts, the reporter failed to be activated (Fig. 6). These data suggested that either the identified PU.1/IRF composite element was not functional in isolation or this enhancer could not activate the SV-40 promoter. We subsequently examined 4X-IL, which contains the IL-1 promoter. When this reporter was transiently transfected into NIH-3T3 fibroblasts with PU.1 and either IRF-4 or ICSBP, a 5- to 7-fold activation of the reporter was observed (Fig. 6). IRF-1 and IRF-2 failed to activate this reporter alone and also failed to further synergize with PU.1/IRF-4 or PU.1/ICSBP when coexpressed in NIH-3T3 fibroblasts (Fig. 6). Together, these findings suggest that the IL-1 PU.1/IRF composite element alone is sufficient to mediate PU.1/IRF-4 and PU.1/ICSBP synergy in a promoter-specific manner, but is insufficient to support further synergy mediated by IRF-1 and IRF-2.

View larger version (51K): [in this window] [in a new window]   FIGURE 6. A multicopy PU.1/IRF composite element is synergistically activated by PU.1/IRF-4 and PU.1/ICSBP when the human IL-1 promoter is present. NIH-3T3 fibroblasts were transiently transfected with a reporter containing four copies of the PU.1/IRF composite element contained in the IL-1 enhancer ligated to either the SV-40 promoter () or the human IL-1 promoter (). All data represent the mean of triplicate transfections ± SD and are presented as the fold luciferase activity over reporter alone, which is normalized to 1. Data are representative of at least three separate experiments performed in triplicate.

View larger version (37K): [in this window] [in a new window]   FIGURE 7. IRF-1 and IRF-2 synergy is mediated by cis elements located between the 5' IL-1 enhancer and promoter. NIH-3T3 fibroblasts were transiently transfected with X2-HT CAT and various combinations of PU.1, IRF-1, IRF-2, IRF-4, and ICSBP as indicated. All data are the mean of triplicate transfections ± SD and are presented as the fold CAT activity over reporter alone, which is normalized to 1. Data are representative of at least three separate experiments performed in triplicate.

Endogenous IL-1 gene expression can be synergistically induced by PU.1 and IRF proteins

Our data demonstrated that PU.1 and multiple IRF family members could synergize to activate an IL-1 reporter gene in transiently transfected cells. One drawback of using reporter constructs is that they are not always regulated in the same manner as the endogenous gene locus. To determine whether PU.1 and IRF proteins can regulate IL-1 gene expression from the endogenous locus, HEK 293 cells were transfected with expression plasmids encoding PU.1 and IRF proteins, and the induction of IL-1 mRNA expression was determined by RT-PCR. As shown in Fig. 8, HEK 293 cells did not constitutively express IL-1 mRNA (Fig. 8), and transfection with the control vector did not induce IL-1 expression (Fig. 8A, lane 1). Expression of PU.1 alone was able to induce a detectable quantity of IL-1 message (Fig. 8A, lane 2). Expression of either IRF-4 or ICSBP alone did not induce IL-1 gene expression (lanes 3 and 4). However, transcription was synergistically activated upon coexpression of PU.1 and either IRF-4 or ICSBP (lanes 5 and 6). These observations are qualitatively consistent with those obtained in transient transfections of NIH-3T3 cells (Fig. 1C) and HEK 293 cells (data not shown), where the reporter is under the control of 3.7 kb of the human IL-1 promoter and enhancer.

View larger version (39K): [in this window] [in a new window]   FIGURE 8. Endogenous IL-1 transcription can be synergistically induced by coexpression of PU.1 and IRF proteins in HEK 293 cells. A, Expression of PU.1 is sufficient to induce IL-1 transcription in HEK 293 fibroblasts. HEK 293 fibroblasts were transiently transfected with empty vector (pCDNA3.1) or expression plasmids encoding PU.1, IRF-4, and ICSBP as indicated. RT-PCR was performed using 2000 ng of total cDNA to detect IL-1 message and 200 ng of total cDNA to detect -actin transcripts. Arrows indicate the 599-bp IL-1 and 402-bp -actin transcripts. The data presented are representative of at least three separate experiments. B, Coexpression of PU.1, IRF-1, IRF-2, and either IRF-4 or ICSBP can synergistically induce transcription of the IL-1 gene. HEK 293 fibroblasts were transiently transfected with empty vector or with expression plasmids encoding PU.1, IRF-1, IRF-2, IRF-4, or ICSBP as indicated. RT-PCR analysis was performed exactly as described in A, and PCR products are indicated by arrows. The photograph in A is overexposed relative to that in B to allow visualization of weak bands.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We previously reported that PU.1 could synergize with either IRF-4 or ICSBP in NIH-3T3 fibroblasts and augment IL-1 reporter activity (4). Here we have identified and characterized the regulatory element within the human IL-1 gene responsible for this PU.1/IRF synergy. A functional PU.1/IRF composite element located 2.8 kb 5' of the transcriptional start site of the IL-1 gene was identified. IRF-4 and ICSBP could bind to this DNA sequence in vitro in a PU.1-dependent manner (Fig. 3), as has been observed with other composite elements (28, 29). Similarly, transcriptional synergy required physical interaction between PU.1 and either IRF-4 or ICSBP (Fig. 2). Furthermore, coexpression of IRF-1 and IRF-2 with PU.1/IRF-4 or PU.1/ICSBP resulted in additional and dramatic synergy (Fig. 4). This IRF-1/2 responsiveness required a region of the IL-1 gene located between the PU.1/IRF composite element and the promoter (Figs. 6 and 7). Lastly, we have shown that transcription of the endogenous IL-1 gene can be activated by ectopic expression of PU.1 and the IRF proteins in a manner virtually identical with that observed in transiently transfected cells (Fig. 8). Together these studies have identified a new regulatory motif within the human IL-1 gene and have extended our understanding of the functional interactions between PU.1 and IRF proteins.

Our data also demonstrate that IRF-4 and ICSBP differ in their capacities to activate the IL-1 reporter plasmid in macrophages. Specifically, IRF-4, but not ICSBP, possessed the capacity to activate this reporter plasmid in transfected macrophages (Fig. 1B). However, interpretation of these data is difficult because macrophages constitutively express PU.1, IRF-4, and ICSBP (4). For this reason, experiments were also performed in NIH-3T3 fibroblasts, cells naturally deficient in the expression of PU.1, IRF-4, and ICSBP. We found that both PU.1/IRF-4 and PU.1/ICSBP synergistically activated the wild-type IL-1 reporter in fibroblasts by 40- to 50-fold (Fig. 1C). PU.1/IRF synergy decreased by 50% when the mPU.1 reporter was used, compared with synergy with the wild-type reporter. Furthermore, the mIRF reporter could not be synergistically activated by IRF-4 or ICSBP in the presence of PU.1. These data confirm that IRF-4 and ICSBP must contact DNA for transcriptional synergy to occur. It is intriguing that the mPU.1 mutation only decreased reporter activity, while the mIRF mutation essentially abolished reporter activity. A reporter plasmid lacking the entire PU.1/IRF composite element could not be synergistically activated by PU.1/IRF-4 or PU.1/ICSBP (data not shown), suggesting that the partial effect with mPU.1 is due to residual PU.1 binding to this mutated site. Indeed, EMSA analysis revealed that some PU.1 DNA binding could be detected using EMSA probes containing the mPU.1 mutation (data not shown). Nevertheless, these combined observations clearly demonstrate that a functional PU.1/IRF composite element is located in the 5' enhancer of the human IL-1 gene.

EMSA analysis of RAW264.7 macrophage nuclear extracts revealed DNA binding by PU.1, IRF-4, and ICSBP to the IL-1 composite element in vitro (Fig. 3). Similar to other genes containing PU.1/IRF composite elements, IRF-4 and ICSBP DNA binding was undetectable in the absence of association with PU.1 (lanes 3 and 4). Of the four DNA-protein complexes resolved, only the proteins present in complexes I and III could be identified by Ab supershifting experiments, namely PU.1, IRF-4, and ICSBP. Complexes II and IV specifically bound to the probe, although their identities and potential contributions to IL-1 gene regulation remain to be determined. The PU.1/IRF composite element lies within a domain previously reported to bind recombinant IRF-1 in vitro (37), although an anti-IRF-1 Ab did not supershift any of the observed complexes. Furthermore, a STAT-like molecule (termed LIL-STAT) present in extracts prepared from cells stimulated with LPS or the cytokines IL-1 and IL-6 was also reported to bind to this site (37). However, Abs against these molecules as well as against IRF-2, C/EBP, Ets-1, and Ets-2 all failed to yield any supershifted complexes. It is possible that LIL-STAT binding could not be detected in our studies because the nuclear extracts used were prepared from unstimulated cells. Likewise, all transient transfection experiments were performed under unstimulated conditions, making it unlikely that an inducible LIL-STAT protein participated in the synergistic responses reported here. Nonetheless, PU.1, IRF-4, and ICSBP DNA binding can be detected with the IL-1 enhancer, supporting the hypothesis that this gene contains a functional PU.1/IRF element.

Eklund et al. reported that PU.1, IRF-1, and ICSBP could synergize to activate the expression of a reporter containing multiple copies of the gp91^phox PU.1/IRF composite element (9). Despite the fact that we could not detect IRF-1 binding to the IL-1 composite element, the gp91^phox observation prompted us to examine whether coexpression of IRF-1 could affect PU.1/IRF-4 or PU.1/ICSBP synergy. We observed that coexpression of IRF-1 with either PU.1/IRF-4 or PU.1/ICSBP dramatically enhanced IL-1 reporter activity in NIH-3T3 fibroblasts, and the inclusion of IRF-2 further enhanced synergy (Fig. 4). Although coexpression of PU.1/IRF-4, PU.1/ICSBP, or IRF-1/IRF-2 could activate the IL-1 reporter 50-fold (Fig. 4B), coexpression of IRF-1 and IRF-2 with either PU.1/IRF-4 or PU.1/ICSBP resulted in reporter activation of >1000-fold over basal levels. Thus, each of these factors is required for maximal IL-1 reporter activity.

Transient transfections using the PU.1 mutant IL-1 reporter demonstrated that the promoter-proximal PU.1 site (centered at position -40) does not directly mediate synergy among PU.1, IRF-1, IRF-2, IRF-4, and ICSBP (Fig. 5). These factors were still able to synergize in the absence of the -40 PU.1 site, albeit to a much lesser degree. Unlike in the wild-type IL-1 reporter, where PU.1/IRF-4, PU.1/ICSBP, or IRF-1/IRF-2 expression activated IL-1 reporter expression 50-fold (Fig. 4B), PU.1/IRF-4 and PU.1/ICSBP could only activate the PU.1 reporter 3- to 5-fold and IRF-1/IRF-2 coexpression activated this reporter 10- to 15-fold (compare Figs. 1B, 4, and 5). It is intriguing to speculate that the function of the PU.1/IRF composite element is somehow dependent on the promoter-proximal PU.1 site. It is possible that this promoter site is involved in conveying enhancer activity to the basal transcriptional machinery. EMSA analysis revealed that the promoter-proximal PU.1 site does not bind IRF-4 or ICSBP (data not shown), yet the transfection data demonstrate that its presence is critical for maximal synergy between PU.1 and these IRF proteins. Elements within the proximal promoter are necessary for functional PU.1/IRF synergy as well as the additional synergy conferred by IRF-1 and IRF-2. For example, the 4X-SV reporter plasmid could not be activated by PU.1 and/or IRF proteins (Fig. 6). In contrast, the 4X-IL reporter plasmid could be synergistically activated by PU.1 and IRF-4/ICSBP, but this synergy could not be further augmented by IRF-1 or IRF-2. Together, these data suggest that the cap site-proximal IL-1 promoter contains a functional PU.1 site that is required for maximal transcriptional activation, but is not necessary for composite element synergy. Moreover, the upstream PU.1/IRF composite element requires distinct promoter elements (in addition to the downstream PU.1 site) to exert an effect on transcription.

It appears that at least three separate regions of the human IL-1 gene are required for maximal synergy between PU.1 and these IRF proteins, although the exact locations of IRF-1 and IRF-2 binding remain to be determined. IRF-1 and IRF-2 synergy is lost when a portion of the IL-1 gene between -2700 and -140 is removed, a region that presumably contains the IRF-1 and IRF-2 DNA binding sites. Nevertheless, it is clear that maximal transcriptional synergy requires that all proteins contact DNA. Furthermore, overall synergy was decreased when the promoter-proximal PU.1 binding site was deleted and was even further reduced if this deletion was combined with the mPU.1 or mIRF mutation (data not shown). Interestingly, synergy mediated by IRF-1 and IRF-2 was also decreased, even though they bound to a site unaffected by these mutations. This observation suggests that the cap site-proximal PU.1 site is also required for maximal IRF-1 and IRF-2 synergy. Together, these combined observations suggest that the human IL-1 gene may be regulated by an enhanceosome that involves the upstream composite element, the IRF-1 and IRF-2 binding site, and the promoter-proximal PU.1 site. It is intriguing to speculate that the PEST domains of PU.1, IRF-1, and IRF-2 are used to bring these discreet functional elements together. Rationale for this possibility comes from the finding that PU.1, IRF-1, and IRF-2 can interact with IRF-4 and ICSBP via their PEST domains (23, 29, 42).

Lastly, IL-1 transcripts could be induced in human HEK 293 fibroblasts following ectopic expression of PU.1 and these IRF proteins. The pattern of expression was qualitatively similar to that observed using transiently transfected reporters in both NIH-3T3 (Fig. 8) and HEK 293 fibroblasts (data not shown). The capacities of these factors to activate transcription of the endogenous IL-1 gene in HEK 293 fibroblasts suggest that these factors may regulate IL-1 transcription in vivo. Further studies are required to determine precisely how these transcription factors interact in vivo and how this molecular mechanism might be implemented in activated macrophages. It should be noted that HEK 293 cells, rather than murine NIH-3T3 cells, were used for these experiments because the murine IL-1 enhancer differs markedly from the human enhancer and lacks a PU.1/IRF composite element (46). Nevertheless, transiently transfected human IL-1 reporter plasmids cotransfected with PU.1 and IRF expression plasmids were essentially identically regulated in both cell lines. One remaining question is how the IL-1 composite element is regulated in cells stimulated with LPS and/or IFN-. Although IRF-4 protein levels are not altered in macrophages following IFN- treatment (4), it is tempting to speculate that IFN--induced expression of ICSBP, IRF-1, and IRF-2 might play a role in the enhancement of IL-1 expression that is observed in LPS-stimulated macrophages that have been pretreated (primed) with IFN-. Moreover, LPS and IFN- stimulation may post-translationally modify IRF-4 or the other factors in a manner that would not be apparent in ourexperiments. LPS stimulation of macrophages results in phosphorylation of PU.1 (36) and thus enhances interaction with IRF-4. Future studies will seek to determine whether IFN- priming acts via the recruitment and/or activation of the IRF proteins that mediate activation of the IL-1 enhancer.

	Acknowledgments

 
We thank Drs. Barbara Nikolajczyk for useful discussions, and GregViglianti for critical review of the manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant GM57053 (to M.J.F.).

² Address correspondence and reprint requests to Dr. Matthew J. Fenton, Pulmonary Center, R-220, Boston University School of Medicine, Boston, MA 02118-2394. E-mail address: mfenton{at}bu.edu

³ Abbreviations used in this paper: IRF, IFN regulatory factor; ICSBP, IFN consensus sequence binding protein; HEK, human embryonic kidney; ISRE, IFN sequence response element; LIL-STAT, LPS- and IL-1-inducible STAT; LIL-RE, LPS- and IL-1-responsive element; IAD, interaction domain; PEST, proline-glutamate-serine-threonine-rich; CAT, chloramphenicol acetyltransferase.

Received for publication October 4, 2000. Accepted for publication March 16, 2001.

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