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J. Biol. Chem., Vol. 282, Issue 9, 6629-6643, March 2, 2007
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1
From the
The Feinberg School of Medicine and The Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois 60611 and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois 60612
Received for publication, August 14, 2006 , and in revised form, December 26, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, we found that NF1 transcription and Nf1 expression increase during cytokine-induced differentiation of myeloid leukemia cell lines or murine myeloid progenitor cells (4). We also found that cytokine-induced NF1 transcription requires the interferon consensus sequence-binding protein (ICSBP or IRF8) (4). ICSBP/IRF8 is expressed exclusively in myeloid and B-cells (10), and acquired ICSBP/IRF8 deficiency is found in bone marrow cells from subjects with chronic myeloid leukemia, AML, and MDS (11, 12). Interestingly, ICSBP/IRF8 deficiency induces myeloproliferation in mice, and myeloid cells from these mice are hypersensitive to the same cytokines as Nf1-deficient cells (4, 13, 14). Consistent with a role in NF1 transcription, proliferative abnormalities in ICSBP/IRF8-deficient myeloid progenitor cells can be rescued by expression of the Nf1GAP-related domain (4).
In previous studies, we also found that activation of NF1 transcription requires cytokine-induced phosphorylation of a specific ICSBP/IRF8 tyrosine residue (Tyr-95) (15). This residue is in the conserved IRF domain that is thought to be involved in DNA-binding or protein/protein interactions of IRF proteins. ICSBP/IRF8 is a substrate for SHP1 and SHP2 protein-tyrosine phosphatases in undifferentiated myeloid cells (15, 16). However, a constitutively activated SHP2 mutant, described in human subjects with MDS, AML, and juvenile myelomonocytic leukemia, dephosphorylates ICSBP/IRF8 in differentiated and undifferentiated myeloid cells (15, 17). Such activated SHP2 mutants also induce cytokine hypersensitivity in myeloid progenitors (15, 18).
IRF proteins regulate target gene transcription by interacting with several different DNA-binding site consensus sequences. ICSBP/IRF8 represses cis elements with PRDI consensus sequences (5'-TCACTT-3') by interacting directly with DNA. In contrast, tyrosine-phosphorylated ICSBP/IRF8 can activate PRDI-cis elements by interaction with DNA-bound IRF1 (19). ICSBP/IRF8 also binds to interferon-stimulated response elements (5'-GAAANNGAAA-3'). Interferon-stimulated response elements can be repressed by an interaction between ICSBP/IRF8, the ets protein Tel, and histone deacetylase 3 (20).
ICSBP/IRF8 activates gene transcription by interacting with composite ets/IRF-cis elements (also referred to as EICE sequences). Such ets/IRF sequences have a loose consensus (5'-GGAA(A/G)TGNNA-3') and are found in a number of genes expressed in mature phagocytes and involved in the immune response. Examples include genes encoding the respiratory burst oxidase proteins gp91PHOX and p67PHOX and the gene encoding the Toll-like receptor 4 (the CYBB, NCF2, and TLR4 genes, respectively) (11, 21, 22). DNA-bound PU.1 interacts with IRF1, and this DNA-bound heterodimer recruits ICSBP/IRF8 and the CREB-binding protein (11, 21, 22). These interactions require post-translational modification of the proteins in differentiating myeloid cells. Specifically, interaction of ICSBP/IRF8 with the DNA-bound PU.1-IRF1 heterodimer requires phosphorylation of PU.1 Ser-148 and ICSBP/IRF8 Tyr-95 (16, 21).
The ICSBP/IRF8-binding cis element in the proximal NF1 promoter has homology to composite ets/IRF consensus sequences found in myeloid-specific genes. This suggests possible involvement of PU.1 in NF1 transcriptional regulation and perhaps another IRF protein. The goal of these investigations is to determine the mechanism of cytokine-induced NF1 transcription in differentiating myeloid cells. This will be approached by identifying the components of the NF1 transcriptional activation complex.
Although composite ets/IRF consensus sequences have been identified in a number of genes involved in the inflammatory response, no such cis elements have previously been identified in target genes regulating proliferation. Identification of homologous cis elements that interact with common trans-factors in genes that regulate both differentiation and proliferation would suggest a common mechanism of cytokine activation of different types of genes. This could have implications for understanding the inter-relationship between differentiation-progression and proliferation-arrest during myelopoiesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mammalian expression vector. These cDNAs were also subcloned into the pGEX1 vector (Amersham Biosciences) for expression in Escherichia coli as glutathione S-transferase (GST) fusion proteins. Reporter Constructs—An artificial promoter construct with four copies of the ICSBP/IRF8-binding cis element from the NF1 promoter (bp -320 to -336) linked to a minimal promoter and a CAT reporter (the p-TATACAT vector) was previously described (15). This construct is referred to as nf1TATACAT.
Oligonucleotides
Oligonucleotides were custom-synthesized by MWG Biotec (Piedmont, NC). Double-stranded oligonucleotides used in EMSA and DNA affinity co-immunoprecipitation assays represent the -320- to -336-bp NF1 promoter sequence (NF1-320 to -336; 5'-ggatcccacttccggtggc-3'). The underlined sequence has homology to composite ets/IRF-cis elements from other ICSBP target genes. A double-stranded oligonucleotide with mutation of the ICSBP/IRF8-binding site (previously described (4)) was also used in these assays (mutNF1 -320 to -336; 5'-ggatcccacaaccggtggc-3'). For in vitro binding competition assays, an oligonucleotide with the composite ets/IRF sequence for the CYBB gene was used 5'-gttttcatttcctcattgg-3') (21). Subcloning these oligonucleotides into a plasmid vector to generate probes has been described (4).
Myeloid Cell Line Culture
The human myelomonocytic cell line U937 (24) was obtained from Andrew Kraft (Hollings Cancer Center, Medical University of South Carolina, Charleston, SC). Cells were maintained and differentiated as described (21). For differentiation experiments, U937 cells were treated for 24 or 48 h with 500 units/ml human recombinant IFN
(Roche Applied Science) (21).
Transfections and Reporter Gene Assays
U937 cells were cultured and transfected as described previously (4, 15, 21, 22). Cells (32 x 106 per sample) were transfected with a vector to express the following: wild type ICSBP/IRF8, Y95F ICSBP/IRF8, or empty vector control; wild type IRF2, Y109F IRF2, or empty vector control; wild type PU.1, S148A PU.1, S132A/S133A PU.1, or empty vector control; the minimal promoter/reporter vector pTATACAT with four copies of the -320- to -336-bp NF1 sequence (nf1TATACAT) or empty vector control (pTATACAT); and p-CMV
gal (to control for transfection efficiency). Transfectants were harvested 48 h after transfection, with or without incubation with recombinant human IFN (500 units/ml). Lysates were analyzed for CAT and -galactosidase activity, as described (21).
Isolation of Nuclear Proteins and Electrophoretic Mobility Shift Assays
Nuclear extract proteins were isolated from U937 cells by the method of Dignam et al. (25) (with the addition of protease inhibitors but not phosphatase inhibitors, as described). In some experiments, U937 cells were differentiated with 500 units/ml of IFN before nuclear protein isolation. Oligonucleotides probes were prepared, and EMSA and antibody supershift assays were performed, as described (21). Antibodies to phosphotyrosine, ICSBP/IRF8, IRF1, IRF2, PU.1, and irrelevant, control GST antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Immunoprecipitation and Western Blots
Western Blots of U937 Lysates Proteins—U937 cells were lysed by boiling in 2x SDS sample buffer. Lysate proteins (30 µg) were separated by SDS-PAGE (12% acrylamide) and transferred to nitrocellulose, according to standard techniques. Western blots were serially probed with antibodies to Nf1, ICSBP/IRF8, IRF2, PU.1, and GAPDH (to control for loading). In other studies, nuclear proteins were isolated from U937 cells (with or without IFN treatment). 30 µg of protein were separated by SDS-PAGE (20% acrylamide gel) and transferred to nitrocellulose. Western blots were serially probed with antibodies to PU.1, IRF2, pERK2 (as a control for IFN treatment), total ERK2 (as a loading control), or GAPDH (as a loading control).
Immunoprecipitation and Western Blots—Nuclear proteins were isolated from U937 cells with or without IFN treatment and immunoprecipitated under denaturing conditions with antibody to PU.1, IRF2, phosphotyrosine (Tyr(P)), or irrelevant control antibody (anti-GST), as described previously (14, 15). Precipitated proteins were collected with staphylococcus protein A-Sepharose, separated by SDS-PAGE, and transferred to nitrocellulose, as above. Western blots were serially probed with an anti-phosphotyrosine antibody (clone 4G10, Upstate) and antibodies to IRF2 or PU.1.
DNA Affinity Co-immunoprecipitation—Nuclear proteins were isolated from U937 cells with or without IFN treatment. Proteins (300 µg) were incubated with a biotin-labeled double-stranded oligonucleotide probe representing the -320- to -336-bp NF1 promoter sequence or a specific mutant, non-ICSBP/IRF8-binding sequence. Probes were immunoprecipitated with anti-biotin antibody or irrelevant control antibody under nondenaturing conditions and recovered with staphylococcus protein A-Sepharose beads. Immunoprecipitates were separated by SDS-PAGE (10% acrylamide) and proteins transferred to nitrocellulose. Western blots were serially probed with antibodies to ICSBP/IRF8, IRF2, and PU.1.
In other experiments, 35S-labeled, in vitro translated proteins were generated using rabbit reticulocyte lysate from in vitro transcribed RNA, as described previously (16). These proteins were also used in DNA affinity co-immunoprecipitation experiments with biotin-labeled probes, as described above. For these experiments, DNA/protein interaction was identified by autoradiography of SDS-PAGE.
Metabolic Labeling and Immunoprecipitation—U937 cells treated with IFN- for 0, 24, and 48 h were incubated for 4 h at 37 °C with [32P]orthophosphate. Cells were lysed under denaturing conditions, and lysate proteins (100 µg) were immunoprecipitated with antibody to PU.1 or irrelevant control antibody. Immunoprecipitates were collected with staphylococcus protein A-Sepharose beads and separated by SDS-PAGE (20% acrylamide). Phosphorylated PU.1 protein was identified by autoradiography of the fixed and dried gel.
In Vitro Protein Translation and GST Co-affinity Purification Assay
In vitro transcribed IRF2, Y109F IRF2, ICSBP/IRF8, Y95F ICSBP/IRF8, PU.1, S148A PU.1, or S132A/S133A PU.1 mRNAs were generated from linearized template DNA using the riboprobe system, according to manufacturer's instructions (Promega). In vitro translated proteins were generated in rabbit reticulocyte lysate, according to the manufacturer's instructions (Promega). Control (unprogrammed) lysates were generated in similar reactions in the absence of input RNA, and proteins were radiolabeled by including [35S]methionine in the translation reaction. JM109 E. coli transformed with PU.1, IRF2, or ICSBP/IRF8 in the pGEX vector (or empty control vector) were grown to log phase, supplemented to 0.1 mM isopropyl 1-thio--D-galactopyranoside, and incubated for 3 h at 37 °C with shaking. The cells were harvested and resuspended in HN buffer (20 mM HEPES (pH 7.4), 0.1 M NaCl, 2 mM MgCl2, 0.1 mM EDTA, 0.5% Nonidet P-40, 0.1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 5 mM NaF) and sonicated on ice (22). Debris was removed by centrifugation, and the lysate was incubated with glutathione-agarose beads (Sigma) and washed extensively. The beads were preincubated with control rabbit reticulocyte lysate to induce serine/threonine phosphorylation of PU.1/GST and tyrosine phosphorylation of ICSBP/GST or IRF2/GST as described (16, 26). GST proteins were then incubated with [35S]methionine-labeled in vitro translated protein. Proteins were eluted with SDS-PAGE sample buffer, separated on 15% SDS-PAGE, and identified by autoradiography of the fixed and dried gel.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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treatment of U937 cells increases NF1 promoter activity and ICSBP/IRF8 binding to a positive cis element in the NF1 promoter (4, 15). To identify other proteins that interact with this cis element, we used in vitro DNA binding assays. EMSAs were performed with a radiolabeled, double-stranded oligonucleotide representing the NF1-cis element and nuclear proteins from U937 cells. We found that IFN differentiation of U937 cells increases in vitro protein binding to this probe, consistent with our previous results (Fig. 1A) (4, 15).
We noted that the NF1-cis element has homology with the ets/IRF consensus sequences from the CYBB or NCF2 promoters. Therefore, we used double-stranded oligonucleotide competitors representing these cis element to investigate binding specificity of the NF1-protein complex. These competitors were compared with homologous oligonucleotide (ds NF1) or an oligonucleotide with mutation which abolishes protein binding to the NF1-cis element (ds mutNF1) (described (4)). In EMSA with the NF1 probe and nuclear proteins from IFN-differentiated U937 cells, we found that the CYBB and NCF2 oligonucleotides compete for protein complex binding (Fig. 1B). This result suggests that the NF1-cis element interacts with PU.1 and one or more IRF proteins.
Therefore, we performed EMSA with the NF1 probe, nuclear proteins from IFN-treated U937 cells, and antibodies to various transcription factors (Fig. 1C). In these studies, we found that the shifted complex is cross-immunoreactive with PU.1, ICSBP/IRF8, and IRF2 but not IRF1. Because the NF1-cis element appears to bind a multiprotein complex, we tested the ability of various combinations of PU.1, IRF2, and ICSBP/IRF8 antibodies to disrupt the complex (Fig. 1C). We found that the complex is completely disrupted by combining all three antibodies.
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In this assay, we also find that IFN treatment increases interaction of PU.1, IRF2, and ICSBP/IRF8 with the NF1-cis element probe. In contrast, we found previously that PU.1 and IRF1 bind the CYBB- and NCF2-cis elements in undifferentiated myeloid cells. For those cis elements, differentiation increases ICSBP/IRF8 interaction with these proteins but does not increase DNA binding of PU.1 and IRF1. Our current results suggest differences in the activation of various ets/IRF-cis elements, even in the same lineage.
Identification of Protein/Protein/DNA Interactions Required for Assembly of the NF1-cis Element-binding Complex—Previous studies suggest that PU.1 binding to ets/IRF consensus sequences is required to provide an anchor for IRF proteins. Therefore, we investigated the role of PU.1 in mediating IRF2 and ICSBP/IRF8 binding to the NF1-cis element. For these studies, we used in vitro translated, 35S-labeled PU.1, IRF2, and ICSBP/IRF8 (Fig. 2A). Because of differences in size, the three proteins can be detected simultaneously by SDS-PAGE. These in vitro translated proteins were tested for binding to biotin-labeled ds NF1 or ds mutNF1 probes in DNA-affinity purification assays (DAPA), as above. In these experiments the total amount of reticulocyte lysate in the binding assays was kept constant by inclusion of control (no RNA) lysate.
In initial assays, we tested each of these proteins individually for binding to the ds NF1 probe (Fig. 2B). We found that only PU.1 is able to interact independently with this probe, and none of the proteins interact with the ds mutNF1 probe. Previous studies indicate that phosphorylation of serine residues in the PU.1 PEST domain is important for PU.1 protein/protein/DNA interactions. For example, PU.1 serine 148 is necessary for recruitment of ICSBP/IRF8 to the CYBB promoter (21). Similarly, serines 132 and 133 in PU.1 are essential for PU.1 activation of macrophage-specific genes (27). Because these residues are phosphorylated in reticulocyte lysate (28, 29), we used DAPA to investigate the role of PU.1 phosphorylation in the assembly of the NF1-binding protein complex. We found that in vitro translated wild type PU.1 and PU.1 with mutation of serine 148 or 132/133 to alanine (S148A PU.1 and S132A/S133A PU.1, respectively) have similar binding to the NF1-cis element probe (Fig. 2C). This result is important for interpretation of transfection experiments as shown in the sections below.
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We previously identified a conserved tyrosine residue in the ICSBP/IRF8 IRF domain that is necessary for interaction with a PU.1-IRF1 heterodimer bound to the CYBB- or NCF2-cis element. Because IRF domain tyrosine residues are phosphorylated in reticulocyte lysate (16), we used DAPA to determine the role of the IRF2 IRF domain tyrosine (Tyr-109) in interaction with PU.1. We incubated PU.1 and wild type or Y109F IRF2 (tyrosine 109 mutated to phenylalanine) with the biotin-labeled ds NF1 or ds mutNF1 probe (Fig. 2D). We found that Y109F IRF2 interacts less efficiently with DNA-bound PU.1 than wild type IRF2.
We next investigated whether ICSBP/IRF8 can interact with the DNA-bound PU.1-IRF2 heterodimer. We found that in vitro translated ICSBP/IRF8 interacts with the ds NF1 probe (but not the ds mutNF1 probe) in binding assays with PU.1 and IRF2 (Fig. 2E). Because the conserved IRF domain tyrosine in ICSBP/IRF8 (Tyr-95) is required for activation of the NF1-cis element, we performed binding assays to determine the role of this residue in interaction with DNA-bound PU.1 + IRF2 (Fig. 2E). We found that Y95F ICSBP/IRF8 interacts less efficiently with the ds NF1 probe + PU.1 + IRF2.
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These studies identify the order of binding of the activation complex proteins to the NF1-cis element. We also investigated interaction between these proteins using an in vitro assay system that does not require DNA binding of component proteins. For these studies, we expressed IRF2 in E. coli as a fusion protein with glutathione S-transferase. The IRF2/GST fusion protein was phosphorylated by incubation with reticulocyte lysate, as described previously (16). We investigated interaction with in vitro translated wild type versus S132A/S133A PU.1 versus S148A PU.1 by "pulldown" assay. Previously, we found that mutation of Ser-148 impairs the ability of PU.1 to interact with ICSBP/IRF8 in such assays (16). In this study, less in vitro translated S132A/S133A PU.1 co-purifies with IRF2/GST in comparison with the wild type PU.1 (Fig. 3A). In contrast, S148A PU.1 and wild type PU.1 copurify equivalently with IRF2/GST (Fig. 3B).
We also determined the role of IRF2 IRF domain phosphorylation for interaction with non-DNA-bound PU.1. For these studies, PU.1 was expressed in E. coli as a GST fusion protein and phosphorylated by incubation with reticulocyte lysate, as described (29). Wild type and Y109F IRF2 were translated in vitro in reticulocyte lysate. We found that wild type IRF2 co-affinity purifies with PU.1/GST more efficiently than Y109F IRF2 (Fig. 3C). We also performed similar experiments to pursue the hypothesis that ICSBP/IRF8 tyrosine phosphorylation is necessary for interaction with IRF2. For these studies, IRF2 was expressed as a GST fusion protein, as described above, and incubated with in vitro translated wild type or Y95F ICSBP/IRF8. We found that the wild type protein co-precipitates more efficiently with IRF2/GST than does Y95F ICSBP/IRF8 (Fig. 3D).
None of these in vitro translated proteins co-precipitate with control GST. Phosphorylation of GST fusion proteins and in vitro translated proteins is discussed under "Materials and Methods."
PU.1, IRF, and ICSBP/IRF8 Cooperate to Activate NF1 Transcription—We next investigated the functional significance of PU.1 and IRF2 for NF1 transcription. In previous studies, we determine that overexpressed ICSBP/IRF8 activates transcription from an artificial promoter construct with multiple copies of the NF1-cis element in U937 transfectants (15). For this study, we used the same reporter system to determine the impact of overexpressed PU.1 and IRF2 on this NF1-cis element. Expression of IRF2 is ubiquitous, and PU.1 and ICSBP/IRF8 are expressed in all myeloid cell lines. Therefore, overexpressed ICSBP/IRF8 may have activated the NF1-cis element in our previous studies by interacting with endogenous PU.1 and IRF2. In this case, overexpression of various combinations PU.1, IRF2, and ICSBP/IRF8 might be expected to result in more reporter gene expression than overexpression of any protein individually.
In initial experiments, U937 cells were transfected with a reporter construct containing a minimal promoter with or without four copies of the NF1-cis element (nf1TATACAT and pTATACAT control, respectively). The cells were co-transfected with mammalian expression vectors to overexpress ICSBP/IRF8, PU.1, or IRF2 or control expression vector (Fig. 4A). Because of the impact of differentiation on the NF1-cis element, these transfectants were analyzed with and without IFN treatment (4, 15).
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-differentiated transfectants (p < 0.0001, n = 5). PU.1 overexpression induces a smaller but statistically significant increase in reporter expression from the NF1-cis element in undifferentiated transfectants (p 
0.04, n = 12) and a larger increase in differentiated transfectants (p < 0.0001, n = 12).
Based on these results, we tested the effect of overexpressing combinations of these proteins (Fig. 4A). We found that co-overexpressing PU.1 with either IRF2 or ICSBP/IRF8 results in a statistically significant increase in activity of the NF1-cis element in comparison with overexpressing either IRF protein alone (p 0.002, n = 4 for all combinations, with and without IFN). We also tested the effect of co-overexpressing all three proteins on NF1-cis element activity. We were interested in determining whether the effect of overexpressing PU.1 and both IRF proteins was different from overexpressing PU.1 and either IRF protein alone. To determine this, the total amount of IRF expression vector was kept constant when comparing PU.1 + IRF2 versus PU.1 + ICSBP versus PU.1 + IRF2 + ICSBP.
We found that reporter expression from the NF1-cis element-containing construct is significantly greater in transfectants with PU.1 + ICSBP + IRF2 in comparison with PU.1 + ICSBP or PU.1 + IRF2 (p 0.002, n = 7; with and without IFN) (Fig. 4A). These results suggest that IRF2 and ICSBP are not functionally redundant for their impact on NF1 transcription. In control experiments, none of these proteins significantly influence reporter expression from the empty pTATACAT vector, with or without IFN treatment of the transfectants.
Because these studies involve expressing multiple proteins from multiple vectors, we performed a control experiment to verify that these proteins could be simultaneously overexpressed. U937 cells were co-transfected with vectors to overexpress PU.1, IRF2, and ICSBP/IRF8 or with an equivalent amount of control vector. Transfectants were incubated with IFN, as in the reporter gene assays above. Lysate proteins were separated by SDS-PAGE and analyzed for protein expression by Western blot (Fig. 4B). We found that these proteins are indeed co-overexpressed in U937 cells under these conditions. We also find that this co-overexpression increases expression of endogenous Nf1, consistent with the reporter assays.
PU.1 Is Essential for Activation of the NF1-cis Element in U937 Myeloid Cells—We hypothesize that PU.1 binding to the NF1-cis element is essential for binding of IRF2, which is essential for binding of ICSBP/IRF8. If this hypothesis is correct, decreasing endogenous PU.1 in U937 cells should decrease activity of the NF1-cis element. To investigate this hypothesis, U937 cells were co-transfected with a vector to express a PU.1-specific short hairpin RNA (shRNA) or scrambled control shRNA. We found that expression of a PU.1-specific shRNA induces a small but significant decrease in reporter expression from the NF1-cis element in U937 transfectants with and without IFN differentiation (p < 0.002, n = 3) (Fig. 5A).
Therefore, we next determined the impact of co-overexpressing PU.1-specific shRNA with IRF2, ICSBP/IRF8, or IRF2 + ICSBP/IRF8. This experiment would test our hypothesis that activation of the NF1-cis element by overexpressed IRF2 or ICSBP/IRF8 requires the presence of either endogenous or overexpressed PU.1. We found that expression of PU.1-specific shRNA (but not control scrambled shRNA) blocks activation of the nf1TATACAT reporter construct by overexpressed IRF2, ICSBP/IRF8, or IRF2 + ICSBP/IRF8 (Fig. 5A). Indeed, we found that activity of the NF1-cis element containing construct is not significantly influenced by overexpression of these IRF proteins in the presence of PU.1 shRNA expression (p = 0.61, F = 0.54, n = 3). Expression of PU.1-specific shRNA does not impact reporter expression from pTATACAT control vector.
Based on these results, we also investigated the effect of PU.1 knockdown on expression of endogenous Nf1. For these experiments, U937 transfectants with PU.1-specific or scrambled control shRNA were IFN-treated, and cell lysates were analyzed by Western blot (Fig. 5B). We found that decreased PU.1 expression in U937 cells is associated with decreased Nf1 expression. This is consistent with the reporter gene assays.
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-differentiation (p 0.002, n = 6). Indeed, reporter activity from the nf1TATACAT construct is not significantly different in transfectants with S132A/S133A PU.1 than in transfectants with control expression vector (p = 0.122, n = 6 for undifferentiated transfectants, and p = 0.54, n = 6 for IFN-treated transfectants). We hypothesize S132A/S133A PU.1 can bind the NF1-cis element but not recruit the IRF proteins necessary for transcriptional activation.
Therefore, we tested the effect of S132A/S133A PU.1 on cooperation with IRF2 for NF1-cis element activation (Fig. 6A). We found that overexpression of S132A/S133A PU.1 + IRF2 induces significantly less reporter expression from the NF1-cis element-containing construct than PU.1 + IRF2, with and without IFN differentiation (p < 0.0001, n = 4). Indeed, reporter activity from the NF1-cis element containing construct is less in transfectants overexpressing S132A/S133A PU.1 + IRF2 than in transfectants overexpressing only IRF2 (p = 0.001, n = 4 for undifferentiated and IFN-treated transfectants). These results support the hypothesis that S132A/S133A PU.1 prevents endogenous PU.1 from binding to the NF1-cis element, therefore preventing interaction with overexpressed IRF2.
Our DNA binding assays indicate that a DNA-bound PU.1-IRF2 heterodimer is essential for interaction of ICSBP/IRF8 with the NF1-cis element. This suggests activation of the NF1-cis element by overexpressed PU.1 and ICSBP/IRF8 requires endogenous IRF2. Because IRF2 does not interact with DNA-bound S132A/S133A PU.1, one would anticipate that overexpressed S132A/S133A PU.1 + ICSBP will also not activate the NF1-cis element. Consistent with this, we found that S132A/S133A PU.1 + ICSBP induces significantly less reporter expression from the nf1TATACAT construct than PU.1 + ICSBP in undifferentiated and IFN-treated transfectants (p 0.0001, n = 4).
We also tested the impact of mutating PU.1 serine residues 132 and 133 on activation of the NF1-cis element by overexpressed IRF2 + ICSBP (Fig. 6A). We found significantly less reporter expression from nf1TATACAT in transfectants with S132A/S133A PU.1 + IRF2 + ICSBP than in transfectants with wild type PU.1 + these two IRF proteins. This was true in transfectants both with and without IFN-differentiation (p 0.0003, n = 6). Indeed, activity of the NF1-cis element is not significantly different in transfectants overexpressing S132A/S133A PU.1 + IRF2 + ICSBP than in transfectants with empty control expression vector (p 
0.7, n = 4).
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(p 0.01, n = 8). These results suggest that activation of this cis element by overexpressed PU.1 involves interaction with both endogenous IRF2 and ICSBP.
Therefore, we investigated the impact of S148A PU.1 on IRF2 activation of the NF1-cis element (Fig. 6B). We found that S148A PU.1 + IRF2 is significantly less efficient than wild type PU.1 + IRF2 in activation of reporter expression from the NF1-cis element in untreated and IFN-differentiated U937 transfectants (p 0.04, n = 4). These results suggest that this PU.1 mutation impairs the ability of the PU.1-IRF2 heterodimer to interact with endogenous ICSBP/IRF8. Therefore, we assayed for nf1TATACAT reporter expression in transfectants co-overexpressing ICSBP/IRF8 and either wild type or S148A PU.1. We found significantly less reporter expression in transfectants with S148A PU.1 + ICSBP/IRF8 than in transfectants with wild type PU.1 + ICSBP/IRF8 both without IFN treatment (p 0.0001, n = 4).
We also tested the ability of S148A PU.1 to induce NF1-cis element activation in cooperation with both overexpressed IRF2 and ICSBP/IRF8. We found that nf1TATACAT expression in transfectants with S148A PU.1 + the two IRF proteins is less than half that in transfectants with wild type PU.1 + IRF2 + ICSBP/IRF8 (p = 0.001, n = 4). This effect is even greater in IFN-treated transfectants (p < 0.00002, n = 6).
We found that there is no significant difference in activation of nf1TATACAT reporter activity by S148A PU.1 in comparison with S132A/S133A PU.1 (p = 0.61, n = 8 and p = 0.98, n = 8, respectively). Similarly, we found that there is no significant difference in NF1-cis element activation in transfectants with S148A PU.1 + ICSBP/IRF8 + IRF2 versus S132A/S133A PU.1 + ICSBP/IRF8 + IRF2, with and without IFN treatment (p = 0.1, n = 8 and p = 0.99, n = 8, respectively). These results suggest that impairing ICSBP/IRF8 interaction with the NF1-cis element has the same effect, whether it is because of impairing interaction between PU.1 and IRF2 (by mutating Ser-132 and Ser-133) or by impairing interaction of ICSBP/IRF8 with the PU.1-IRF2 heterodimer.
Expression from the empty reporter vector pTATACAT is not significantly altered by expression of any of these proteins. Previous studies and our own preliminary data indicate that expression of wild type PU.1 and various serine mutants is equivalent in such transfection experiments (not shown in this study) (26).
Mutation of a Conserved IRF Domain Tyrosine Prevents IRF2 from Activating NF1 Transcription—We found that the conserved IRF domain tyrosine in IRF2 increases interaction with DNA-bound PU.1. Therefore, we investigated the role of this residue (Tyr-109 IRF2) in activation of the NF1-cis element. We performed U937 transfection experiments with the NF1-cis element-containing minimal promoter/reporter construct or pTATACAT control vector and vectors to overexpress various combinations of wild type or Y109F IRF2, ICSBP, and PU.1 (Fig. 7A). The reporter gene assays in this section were performed simultaneously with the experiments in Fig. 4A and Fig. 6, A and B, but are presented in a separate graph for convenience of interpretation.
We initially tested the impact of Y109F IRF2 overexpression alone on the NF1-cis element in the nf1TATACAT reporter construct. In these studies, we found significantly less reporter expression in transfectants with Y109F IRF2 in comparison with wild type IRF2 with and without differentiation (p 0.002, n = 6). We hypothesize that this is because of the inability of overexpressed Y109F IRF2 to interact with endogenous, DNA-bound PU.1. This results in an inability to recruit ICSBP/IRF8. Therefore, we next tested the impact of mutating the conserved IRF2 IRF domain tyrosine on cooperation with overexpressed PU.1. We found that Y109F IRF2 + PU.1 induces significantly less reporter expression from the NF1-cis element-containing construct than wild type IRF2 + PU.1 both in untreated and IFN-treated transfectants (p < 0.002, n = 6). Consistent with the hypothesis that overexpressed Y109F IRF2 is unable to interact with overexpressed PU.1, there is no difference in reporter activity in transfectants with PU.1 + Y109F IRF2 and with PU.1 alone (p = 0.64, n = 3 for undifferentiated transfectants and p = 0.25, n = 3inIFN-treated transfectants).
If ICSBP/IRF8 interaction with the NF1-cis element requires pre-binding of PU.1 and IRF2, overexpressed Y109F IRF2 would not be anticipated to increase activation of the NF1-cis element by overexpressed ICSBP/IRF8. Consistent with this, we found that activation of the NF1-cis element in transfectants overexpressing Y109F IRF2 + ICSBP/IRF8 is not significantly different from reporter activity in transfectants overexpressing ICSBP/IRF8 alone (p = 0.83, n = 3 for undifferentiated transfectants and p = 0.69, n = 3 for IFN-treated transfectants). This results suggests ICSBP/IRF8 is interacting with only endogenous IRF2 in transfectants overexpressing this IRF2 mutant.
We next investigated the effect on activity of the NF1-cis element of combining Y109F IRF2 with both PU.1 and ICSBP/IRF8. We found significantly less reporter expression in transfections with Y109F IRF2 + PU.1 + ICSBP in comparison with wild type IRF2 and these other two proteins (p 0.03, n = 6, with and without IFN). These results suggest that Tyr-109 in IRF2 is necessary for the overexpressed protein to interact with PU.1. This heterodimer recruits ICSBP/IRF8 and activates transcription.
In control experiments, overexpression of these proteins did not significantly alter reporter expression from the empty minimal promoter control vector. In additional control experiments, we demonstrate that expression of wild type and Y109F IRF2 is equivalent in U937 transfections (Fig. 7B).
Mutation of a Conserved IRF Domain Tyrosine Prevents ICSBP from Activating NF1 Transcription—In our studies above, we found that Tyr-95 in ICSBP/IRF8 is necessary for interaction with the DNA-bound PU.1-IRF2 heterodimer. Also, we previously found that phosphorylation of ICSBP/IRF8 Tyr-95 is essential for activation of NF1-cis element in U937 transfections (15). Therefore, we investigated the role of ICSBP/IRF8-tyrosine phosphorylation in functional interaction with PU.1, IRF2, and the NF1-cis element. These reporter gene assays were performed simultaneously with the experiments in Fig. 4A, Fig. 6, A and B, and Fig. 7A but are presented in a separate graph for convenience of interpretation.
For these studies, U937 cells were co-transfected with the NF1-cis element-containing reporter construct (nf1TATACAT) or control pTATACAT and various combinations of vectors to overexpress wild type or Y95F ICSBP/IRF8, IRF2, and PU.1 (or empty expression vector control) (Fig. 7C). In initial studies, we confirm that Y95F ICSBP/IRF8 induces significantly less reporter activity from the NF1-cis elementcontaining construct than wild type ICSBP, which is consistent with our previous results (15) (p 0.01, n = 6 for transfectants with and without IFN).
Therefore, we co-overexpressed PU.1 and wild type versus Y95F ICSBP/IRF8. We found that overexpressed PU.1 cooperates significantly less efficiently with overexpressed Y95F ICSBP/IRF8 than with wild type ICSBP/IRF8 in either untreated or IFN-differentiated transfectants (p 0.01, n = 4). We hypothesize that the interaction of overexpressed PU.1 + ICSBP/IRF8 with the NF1-cis element involves interaction with endogenous IRF2. Because Tyr-95 ICSBP/IRF8 is required for interaction with PU.1 + IRF2, we anticipate that nf1TATACAT reporter activity would be the same with overexpressed PU.1 as with PU.1 + Y95F ICSBP/IRF8. Indeed, there is no difference in reporter activity in these transfectants (p = 0.16, n = 3 for undifferentiated and p = 0.46, n = 3 for differentiated transfectants).
Therefore, we next tested the impact of ICSBP/IRF8 Tyr-95 phosphorylation on interaction with IRF2. We found that reporter expression from the NF1-cis element-containing constructs is not significantly different in transfectants overexpressing IRF2 + Y95F ICSBP/IRF8 in comparison with transfectants overexpressing IRF2 only (p 0.1, n = 3 for both undifferentiated and differentiated U937 transfectants). Based on these results, we tested the impact of overexpressed Y95F ICSBP/IRF8 on cooperation with overexpressed PU.1 + IRF2. We found expression of Y95F ICSBP/IRF8 + PU.1 + IRF2 induces significantly less expression from the NF1-cis element-containing construct than ICSBP/IRF8 + PU.1 + IRF2 in both untreated and differentiated transfectants (p 0.01, n = 4). Indeed, activity of the NF1-cis element is not significantly different in transfectants with PU.1 + IRF2 + Y95F ICSBP/IRF8 than transfectants with PU.1 alone (untreated transfectants p = 0.43, n = 3; IFN-treated transfectants p = 0.46, n = 3).
In control experiments, none of these combinations of overexpressed proteins influenced expression of the empty minimal promoter/reporter vector (pTATACAT). In previous control experiments, we demonstrated that wild type and Y95F ICSBP is equivalently overexpressed in U937 cells using this vector (15, 16).
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Differentiation of U937 Cells—If PU.1 is serine-phosphorylated in response to cytokine stimulation, this would provide a mechanism for increased NF1 transcription during myeloid differentiation. Initially, we determined the impact of IFN differentiation on PU.1 abundance in U937 cells (Fig. 8A). We found that PU.1 protein increases with IFN treatment, and there is a relative increase in a higher molecular weight immunoreactive band. This higher molecular weight band, which is readily detected on 20% acrylamide gels (but not lower % gels), might represent phosphorylated PU.1. The blot was stripped and re-probed for total ERK protein as a loading control and phospho-ERK as a control for IFN signaling.
Therefore, we determined the impact of IFN treatment on PU.1 phosphorylation in U937 cells. U937 cells were incubated for 48 h with or without IFN, followed by metabolic labeling with [32P]orthophosphate. Cell lysates were immunoprecipitated under denaturing conditions with an antibody to PU.1 (or control antibody), separated by SDS-PAGE, and visualized by autoradiography. We found that IFN differentiation of U937 cells increases PU.1 phosphorylation (Fig. 8B). This study shows multiple phospho-bands, suggesting multiple phosphorylated forms of PU.1 in these cells.
To determine whether these phospho-residues are serine/threonine versus tyrosine, nuclear proteins from U937 cells were immunoprecipitated under denaturing conditions with an antibody to PU.1 (or control irrelevant antibody). Immunoprecipitates were separated by SDS-PAGE (7.5% acrylamide), and Western blots were serially probed with antibody to phosphotyrosine and PU.1 (Fig. 8C). We were not able to detect tyrosine-phosphorylated PU.1 by using this approach, consistent with previous results (28). PU.1 appears as a single band in this study because of the percent of acrylamide in the SDS-PAGE.
IRF2 Is Tyrosine-phosphorylated during IFN Differentiation of U937 Cells—In previous studies, we found that IRF1 and ICSBP/IRF8 become tyrosine-phosphorylated during myeloid differentiation. If IRF2 is similarly modified, this might provide an additional mechanism for cytokine-induced Nf1 expression in differentiating myeloid cells. Therefore, we determined the tyrosine phosphorylation state of IRF2 in IFN-treated U937 cells. We initially investigated nuclear abundance of IRF2 in differentiating U937 cells (Fig. 8A). We found that total IRF2 protein is not significantly altered by IFN treatment.
To determine the tyrosine phosphorylation state of IRF2 in these cells, U937 nuclear proteins were immunoprecipitated under denaturing conditions with an antibody to IRF2 (or control antibody). Immunoprecipitates were separated by SDS-PAGE and Western blots serially probed with antibodies to phosphotyrosine and IRF2 (Fig. 8D). We found that IRF2-tyrosine phosphorylation increases during IFN differentiation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we note that the NF1-cis element is homologous to composite ets/IRF elements in the CYBB and NCF2 genes. However, we found that there are differences in protein binding to the NF1-cis element in comparison with the homologous cis elements in these oxidase genes. The CYBB- and NCF2-cis elements bind PU.1 and IRF1 in undifferentiated myeloid cells (16, 21, 22). This interaction provides basal transcription at early stages of differentiation (21). During terminal differentiation, the PU.1-IRF1 heterodimer recruits ICSBP/IRF8 and the CREB-binding protein to the CYBB and NCF2 promoters (22). Assembly of this complex requires phosphorylation of PU.1 Ser-148 and of the conserved tyrosine residue (Tyr-95) in the ICSBP/IRF8 IRF domain (16).
In contrast, we found no equivalent activation complex binding the NF1-cis element in undifferentiated myeloid cells. In response to differentiating cytokines, an activation complex assembles on the NF1-cis element. Similar to the CYBB- and NCF2-cis elements, assembly of this complex requires initial binding of PU.1. We found that PU.1 recruits IRF2 to the NF1 promoter in a manner that requires phosphorylation of PU.1 serine residues 132 and 133. This interaction also requires the conserved IRF domain tyrosine in IRF2 (Tyr-109). Recruitment of ICSBP/IRF8 to the NF1-cis element by the PU.1/IRF2 heterodimer requires phosphorylation of the conserved tyrosine residue in the ICSBP IRF domain. This provides a specific molecular mechanism for our previous observations of the functional significance of this Tyr residue (15). We also find that recruitment of ICSBP to the NF1-cis element by the PU.1/IRF2 heterodimer requires phosphorylation of serine 148 in PU.1.
These results suggest different residues in PU.1 are responsible for interaction with different IRF proteins. If these residues were substrates for different kinases (or phosphatases), differential signal-dependent events might regulate the interaction of PU.1 with various protein partners. This could specify PU.1 target gene activation patterns at various stages of differentiation or during the inflammatory response. Additionally, these results suggest there are differences in protein/protein/DNA interactions between different ets/IRF-cis elements.
Previous studies found that PU.1 and IRF4 bind to a composite ets/IRF sequence in the immunoglobulin K gene (referred to as the K3'E) (29). This interaction requires PU.1 Ser-148 and activates transcription in differentiating B-cells. Similar to the CYBB-, NCF2-, and NF1-cis elements, recruitment of the IRF protein to the K3'E requires PU.1 binding. For this cis element, the minimal PU.1-consensus is 3'-GGAA-5', consistent with our present results (30). Crystallography studies have been performed to obtain additional information regarding protein interaction with composite ets/IRF sequences. In these studies, the PU.1 ets domain and the IRF4 IRF domain were co-crystallized with the K3'E. These studies indicate that the PU.1 PEST domain is sterically available for protein/protein interactions (31). These studies also indicate that the conserved IRF domain Tyr residue in IRF4 is positioned to interact with the PU.1 ets domain. Th
