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Originally published In Press as doi:10.1074/jbc.M706710200 on January 14, 2008

J. Biol. Chem., Vol. 283, Issue 12, 7921-7935, March 21, 2008
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The Interferon Consensus Sequence-binding Protein (ICSBP/IRF8) Represses PTPN13 Gene Transcription in Differentiating Myeloid Cells*

Weiqi Huang{ddagger}, Chunliu Zhu{ddagger}, Hao Wang{ddagger}, Elizabeth Horvath{ddagger}§, and Elizabeth A. Eklund{ddagger}§1

From the {ddagger}The Feinberg School of Medicine and The Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois 60611 and §Jesse Brown Veterans Health Administration Medical Center, Chicago, Illinois 60612

Received for publication, August 13, 2007 , and in revised form, January 11, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The interferon consensus sequence-binding protein (ICSBP/IRF8) is an interferon regulatory factor that is expressed in myeloid and B-cells. ICSBP-deficient mice develop a myeloproliferative disorder characterized by cytokine hypersensitivity and apoptosis resistance. To identify ICSBP target genes involved in these effects, we screened a CpG island microarray with chromatin that co-immunoprecipitated with ICSBP from myeloid cells. Using this technique, we identified PTPN13 as an ICSBP target gene. PTPN13 encodes Fas-associated phosphatase 1 (Fap-1), a ubiquitously expressed protein-tyrosine phosphatase. This was of interest because interaction of Fap-1 with Fas results in Fas dephosphorylation and inhibition of Fas-induced apoptosis. In this study, we found that ICSBP influenced Fas-induced apoptosis in a Fap-1-dependent manner. We also found that ICSBP interacted with a cis element in the proximal PTPN13 promoter and repressed transcription. This interaction increased during myeloid differentiation and was regulated by phosphorylation of conserved tyrosine residues in the interferon regulatory factor domain of ICSBP. ICSBP deficiency was present in human myeloid malignancies, including chronic myeloid leukemia. Therefore, these studies identified a mechanism for increased survival of mature myeloid cells in the ICSBP-deficient murine model and in human myeloid malignancies with decreased ICSBP expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The interferon consensus sequence-binding protein (ICSBP2 or IRF8) is an interferon regulatory factor that regulates multiple aspects of myelopoiesis. ICSBP was cloned by homology to interferon regulatory factors 1 and 2 (IRF1 and IRF2) (1). However, unlike these ubiquitous proteins, ICSBP is expressed exclusively in myeloid and B-cells. Similar to other IRF proteins, ICSBP interacts with cis elements containing ISRE, PRDI, or composite ets/IRF consensus sequences (2). ICSBP either activates or represses target gene transcription, depending upon the sequence of the cis element and the cellular context (3).

The first identified ICSBP target genes encoded proteins involved in the inflammatory response. For example, ICSBP activates transcription of genes encoding the NADPH oxidase proteins gp91PHOX and p67PHOX, the Toll-like receptor 4, IL18, and IL12 in differentiating myeloid cells (4-8). Transcriptional activation involves binding of ICSBP to the cis elements of these genes via DNA-bound PU.1 and/or another IRF. This interaction requires phosphorylation of conserved tyrosine residues in the IRF domain of ICSBP (9). Because ICSBP is tyrosine-phosphorylated during myelopoiesis, this provides a mechanism for differentiation stage-specific transcription of such genes (9, 10). ICSBP also represses transcription of the gene encoding 3',5'-oligoadenylate synthase (OAS) during macrophage differentiation (11). Therefore, ICSBP may down-regulate some phagocyte functions during differentiation of monocytes to macrophages, a process necessary to prevent tissue damage.

To better understand the role of ICSBP in myelopoiesis, an ICSBP-deficient murine model was generated (12). These mice exhibit specific defects in immune function as anticipated. In addition, 100% of homozygous ICSBP knock-out mice develop a myeloproliferative disorder. This MPD is characterized by leukocytosis with mature appearing neutrophils that accumulate in the bone marrow and tissues (12). Acute myeloid leukemia (AML) develops in 80% of ICSBP-/- mice by 6 months (12). Further studies indicate that myeloid cells from ICSBP-/- mice are resistant to apoptosis and hypersensitive to various cytokines, including GM-CSF, M-CSF, and stem cell factor (13, 14). These results imply the existence of a set of ICSBP target genes that regulate proliferation and apoptosis. ICSBP expression was also investigated in human myeloid malignancies. A decrease in ICSBP mRNA is found in bone marrow cells from subjects with chronic myeloid leukemia (CML) in comparison with normal (15, 16). ICSBP-expression is also decreased in CD34+ bone marrow cells from a poor prognosis subset of subjects with therapy-related myelodysplasia and AML (17).

To identify target genes involved in the leukemia-suppressor function of ICSBP, we screened a CpG island microarray with chromatin that co-immunoprecipitated with ICSBP from myeloid cells (14). As reported previously, we identified the gene encoding neurofibromin 1 (Nf1) as an ICSBP target (10, 14, 18). Because Nf1 is a Ras-GAP, these results provide a mechanism for cytokine hypersensitivity in ICSBP-deficient cells.

In this study, we identified PTPN13 as an ICSBP target gene. PTPN13 encodes Fas-associated phosphatase-1 (Fap-1), a protein-tyrosine phosphatase that antagonizes Fas-induced apoptosis (19, 20). Because Fas is important for apoptosis of mature phagocytic cells, this result had potential significance for ICSBP-deficient myelopoiesis (21). Previous studies demonstrated that Fap-1 inhibits Fas-induced apoptosis by dephosphorylating Fas and by inhibiting cell membrane trafficking (22). If ICSBP represses PTPN13 transcription, Fap-1 expression would increase in ICSBP-deficient cells. The resultant Fas resistance might provide a mechanism for accumulation of phagocytes in the ICSBP-deficient murine model and in human diseases such as CML.

Fas resistance correlates with increased Fap-1 expression in some leukemia cell lines (23). Fas expression and/or function is associated with treatment resistance and prognosis in AML (24). Also, Fas resistance may result in persistence of malignant clones during treatment of CML (25). Therefore, identification of PTPN13 as an ICSBP target gene would have implications for understanding the phenotype of ICSBP-deficient myeloid malignancies and might suggest therapeutic approaches to such diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids
Protein Expression Vectors—The ICSBP cDNA was obtained from Dr. Ben Zion-Levi (Technion, Haifa, Israel); the full-length cDNA was generated by PCR and subcloned into the mammalian expression vector pcDNA and the pMSCVpuro retroviral vector (Stratagene, La Jolla, CA) (14). A mutant form of ICSBP with conserved IRF domain tyrosine residues changed to phenylalanine has been described previously (Y92F/Y95F ICSBP) (9).

shRNA Expression Vectors—ICSBP-specific shRNA and scrambled control sequences were designed with the assistance of the Promega website. Double-stranded oligonucleotides representing the complementary sequences separated by a hairpin loop were subcloned into the pLKO.1puro vector (a gift from Dr. Kathy Rundell, Northwestern University, Chicago). Several sequences were tested, and the most efficient for ICSBP suppression (and scrambled negative control) was used in the experiments.

PTPN13 Reporter Constructs—Various fragments of the PTPN13 5' flank were obtained from U937 chromatin by genomic PCR. The fragments were sequenced on both strands to ensure identity with the published sequence and subcloned into the pCATE reporter vector (Promega, Madison, WI).

Oligonucleotides
Oligonucleotides were custom-synthesized by MWG Biotec (Piedmont, NC). Double-stranded oligonucleotides used in EMSA and/or DNA affinity purification experiments represented the -587- to -627-bp sequence of the PTPN13 promoter (5'-CTCCCGGAGTCTGTTTCTAATTTCTGCAAATGATTGTGG-3', the ISRE-like sequence is underlined), an ISRE consensus sequence (5'-GGATCCAGAAAGCGAAAGTGGTCTGTAAATCCCTCGAG-3'), a mutant form of the PRDI consensus that does not bind ICSBP (5'-TGTCTTTGTCTTTGTCTT-3'), and an irrelevant oligonucleotide with a CCAAT box from the β-globin gene (5'-CCTGGTAAGGGCCAATCTGCTCAC-3'). Oligonucleotides used to amplify PTPN13 sequences by quantitative real time PCR were -627 to -613 (5'-CTCCCGGAGTCTG-3') and -602 to -587 (5'-TGCAAATGATTGTGG-3').

Myeloid Cell Line Culture
The human myelomonocytic leukemia cell line U937 (26) was obtained from Andrew Kraft (Hollings Cancer Center, Medical University of South Carolina, Charleston, SC). The human myeloblastic cell line KG1 was obtained from Dr. Leon Platanias (Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago). Cells were maintained as described (14). U937 or KG1 cells were treated for 48 h with 500 units per ml of human recombinant IFN{gamma} for differentiation (Roche Applied Science).

Murine Bone Marrow Culture
Animal studies were performed according to a protocol approved by the Animal Care and Use Committees of North-western University and Jesse Brown Veterans Affairs Medical Center. Bone marrow mononuclear cells were obtained from the femurs of WT or ICSBP-/- C57/BL6 mice. Sca1+ cells were separated using the Miltenyi magnetic bead system (Miltenyi Biotechnology, Auburn, CA). Bi-potential myeloid progenitor cells were cultured (at a concentration of 2 x 105 cells per ml) for 48 h in Dulbecco's modified Eagle's media supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 10 ng/ml murine GM-CSF (R & D Systems Inc., Minneapolis, MN), and 10 ng/ml murine recombinant IL3 (R & D Systems). Some ICSBP-/- myeloid progenitors were transduced with a retroviral vector to express ICSBP or Y92F/Y95F ICSBP or empty vector control, as described (10). Cells were either maintained in GM-CSF + IL3 for 48 h, or were differentiated over 72 h in 10 ng/ml of G-CSF (granulocyte) or 10 ng/ml of murine M-CSF (monocyte) ± 500 units/ml of murine IFN{gamma} for the last 24 h.

Chromatin Co-immunoprecipitation and CpG Island Microarray Screening
U937 cells were cultured with or without IFN{gamma} for 48 h (14). Cells were incubated briefly in media supplemented with formaldehyde, and cell lysates were sonicated to generate chromatin fragments with an average size of 2.0 kb (27). Lysates were immunoprecipitated with either ICSBP antiserum or preimmune serum (10). Antibody to ICSBP (and control preimmune serum) was a kind gift of Dr. Stephanie Vogel (University of Maryland, Baltimore, MD). Chromatin was PCR-amplified, as described (27). Several batches of immunoprecipitated, amplified chromatin were combined for each experiment.

Aliquots of ICSBP-specific and preimmune serum control precipitated and amplified chromatin were labeled with Cy3 or Cy5 by the random primer method (with dye swapping to control for differences in incorporation efficiency). Labeled DNA was used to probe a CpG island microarray, as described (27). Microarrays were obtained from the Microarray Center, University Health Network (Ontario Cancer Institute, Ontario, Canada). "Spots" with 3-fold enhancement in ICSBP specific versus control serum precipitated chromatin in three independent hybridization experiments were further considered. Arrays were scanned using an Agilent microarray scanner (G2565BA, Wilmington, DE), and feature intensity statistics were extracted using GenePix (Molecular Devices, Union City, CA). The GenBankTM accession number from the array was used to search the NCBI human genome data base for adjacent genes.

Some co-precipitated chromatin was analyzed by PCR for co-precipitation of the PTPN13 gene. For these experiments, total input chromatin (not precipitated) was a positive control, and chromatin precipitated by preimmune serum was a negative control. PCR products were analyzed by acrylamide gel electrophoresis or real time PCR.

Quantitative Real Time PCR
For Determining mRNA Expression—RNA was isolated using the TRIzol reagent (Invitrogen) and tested for integrity by denaturing gel electrophoresis. Primers were designed with Applied Biosystems software, and real time PCR was performed using SYBR green according to the "standard curve" method. Result were normalized to 18 S.

For Quantifying Chromatin Immunoprecipitation—Chromatin which co-precipitated with ICSBP antibody or preimmune serum was amplified with primers flanking the ICSBP-binding cis element in the PTPN13 promoter (-669 to -646, 5'-GTCGTGCTTGCACAGCTCCGCTCT-3', and -512 to -489, 5'-ACCTTGCATCAGACAGTGTCTCTC-3'). Results were normalized to PCRs with total nonprecipitated chromatin to control for differences in DNA abundance between samples.

Myeloid Cell Line Transfections and Reporter Gene Assays
Stable Transfectant Cell Lines—KG1 cells were transfected by electroporation with equal amounts of an ICSBP expression vector or empty vector control (ICSBP/pcDNAamp or pcDNAamp) plus a vector with a neomycin phosphotransferase cassette (pSR{alpha}) (30 µg each). Stable pools of cells were selected in G418 (0.5 mg/ml), and aliquots of cells were tested for ICSBP expression by Western blot. Other KG1 cells were transfected by electroporation with a construct to express an ICSBP-specific shRNA or scrambled control shRNA using the pLKO.1puro vector. Stable pools of transfected cells were selected in puromycin (1.2 µg/ml) and tested for ICSBP expression by Western blot.

Transient Transfections for Reporter Gene Assays—U937 cells (32 x 106/ml) were transfected with a vector to express ICSBP or vector control (50 µg) and reporter constructs with 2.0 kb, 1.3 kb, 670 bp, or 500 bp of PTPN13 3' flank in the pCATE reporter vector or empty vector control (70 µg). Cells were co-transfected with CMV/β-gal reporter to control for transfection efficiency. Transfectants were assayed for CAT and β-galactosidase expression after 48 h, as described (10, 14).

Apoptosis Assays
Apoptosis assays were performed using annexin V/propidium iodide double staining. The cells were washed with culture medium and adjusted to a concentration of 1 x 106 cells/ml, incubated with annexin-V/fluorescein isothiocyanate solution (2.5 µg/ml) and propidium iodide (12.5 µg/ml) on ice for 15 min, and analyzed on a FACScan flow cytometry (BD Biosciences).

U937 and KG1 Cells—Cells were cultured for 48 h with or without IFN{gamma} (500 units/ml). Cells were incubated with Fas-agonist antibody CH11 or control, as described (28). In some experiments, cells were incubated for the last 12 h with SLV peptide (or control VLS peptide) to disrupt Fas-Fap-1 interaction (29).

Murine Bone Marrow Myeloid Cells—Murine bone marrow myeloid progenitors from WT or ICSBP-/- cells were incubated in GM-CSF or differentiated with M-CSF or G-CSF as described above ±500 units/ml of IFN{gamma} for the last 24 h. Cells were incubated with Fas-agonist antibody, as above. In some experiments, Fas antibody-treated cells were incubated with SLV peptide or VLS peptide.

Immunoprecipitation and Western Blots
Western Blots of Lysates Proteins—U937, KG1, or murine bone marrow cells were lysed by boiling in 2x SDS sample buffer. Lysate proteins (50 µg) were separated by SDS-PAGE (8% acrylamide) and transferred to nitrocellulose. Western blots were serially probed with antibodies to ICSBP and GAPDH or tubulin (to control for loading). Each experiment was repeated at least three times with different batches of lysate proteins. A representative blot is shown.

Immunoprecipitation and Western Blots—Lysate proteins were isolated from U937, KG1, or murine bone marrow cells, with or without differentiation, and immunoprecipitated under denaturing conditions with antibody to Fap-1 or Fas (Santa Cruz Biotechnology, Santa Cruz CA). Precipitated proteins were collected with Staph protein A-Sepharose, separated by SDS-PAGE, and transferred to nitrocellulose. Western blots of Fap-1 antibody-precipitated proteins were probed with Fap-1 antibody. Western blots of Fas antibody-precipitated proteins were probed with an anti-phosphotyrosine antibody (clone 4G10, Upstate Biotechnology, Charlottesville, VA). In other experiments, lysates proteins were immunoprecipitated with anti-Fas antibody under nondenaturing conditions. Precipitated proteins were analyzed by Western blots serially probed with antibodies to Fas, Fap-1, and phosphotyrosine. Each experiment was repeated at least three times with different batches of lysate proteins. A representative blot is shown.

Isolation of Nuclear Proteins, Nuclear Protein Extraction—Nuclear proteins were extracted from U937 or KG1 cells by the method of Dignam (30) with protease inhibitors, as described (10, 14).

In Vitro DNA Binding Assays
DNA Affinity Purification Assays—Nuclear proteins (300 µg) were incubated with biotin-labeled double-stranded oligonucleotide probe representing the -587- to -627-bp PTPN13 promoter sequence or a non-ICSBP-binding PRDI mutant sequence overnight in DAPA buffer (25 mM HEPES (pH 7.6), 60 mM KCl, 5 mM MgCl2, 7.5% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, and 0.25% Triton X-100). The DNA-protein complexes were precipitated with 50 µl of a 50% slurry of neutravidin-coated agarose beads (Pierce). Proteins bound to the beads were eluted, separated by SDS-PAGE (10% acrylamide), and transferred to nitrocellulose. Western blots were probed with an antibody to ICSBP obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Electrophoretic Mobility Assays—Oligonucleotides probes were prepared, and EMSA and antibody supershift assays were performed as described (10, 14). Each assay was repeated with at least three different batches of nuclear proteins, and representative experiments are shown. In some experiments, binding assays were preincubated with unlabeled double-stranded oligonucleotide competitors. In other experiments, binding reactions were preincubated with an ICSBP antibody or an irrelevant antibody to the DNA binding domain of the yeast Gal4 transcription factor (Santa Cruz Biotechnology, Santa Cruz, CA). Each experiment was repeated several times with at least three different batches of nuclear proteins. Equal loading for nuclear proteins was determined in control EMSA with a probe representing a classical CCAAT box from the {alpha}-globin gene.

Genomic Sequence Analysis
Conserved genomic sequences and consensus sequences for IRF protein DNA binding were identified using the VISTA software (Genomics Division of the Lawrence Berkley National Laboratory (Berkeley, CA) (31-33).

Statistical Analysis
Statistical significance was determined by Student's t test and analysis of variance methods using the SigmaPlot and Sigma-Stat software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of PTPN13 as an ICSBP Target Gene—To identify target genes involved in the leukemia-suppressor function of ICSBP, we screened a CpG island microarray with ICSBP co-immunoprecipitating chromatin. We used the U937 myeloid leukemia cell line in these studies. These cells undergo differentiation in response to various cytokines, including IFN{gamma} (26). Over a 24-h period, differentiating U937 cells acquire characteristics of mature phagocytes, including respiratory burst activity and phagocytosis (4, 5). Differentiation of U937 cells is also characterized by cell cycle arrest by 24 h and programmed cell death over 48-96 h. U937 cells express a number of interferon regulatory factors, including ICSBP. Previous studies demonstrated that IRF1, IRF2, and ICSBP are tyrosine-phosphorylated during differentiation of either U937 cells or primary myeloid progenitor cells (9, 10, 18). Therefore, U937 cells represented a reasonable model to study events that are influenced by ICSBP during myelopoiesis.

To identify differentiation stage-specific interactions of ICSBP with potential target genes, human CpG island microarrays were screened with chromatin that co-immunoprecipitated with ICSBP from U937 cells with or without 48 h of IFN{gamma} differentiation. Prior to immunoprecipitation, lysates were sonicated to generate chromatin fragments of 2.0 kb or less (27). Using this technique, we identified a number of putative ICSBP target genes involved in the inflammatory response (Table 1). This was consistent with the known role of ICSBP in regulating phagocyte and B-cell effector genes. We also identified a CpG island in the 5' flank of the PTPN13 gene in experiments with co-precipitating chromatin from differentiated cells. This gene encodes Fas-associated phosphatase 1 (Fap-1), a protein that antagonizes Fas-induced apoptosis. This was of interest because ICSBP deficiency is associated with apoptosis resistance.


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  		TABLE 1
ICSBP target genes

 
The identified CpG island was between -875 and -2230 bp from the PTPN13 transcription start site (TSS), thereby identifying a potential ICSBP-binding site within the proximal 4.0 kb of the 5' flank (Fig. 1A). To assist in identifying functionally significant cis elements, we analyzed the PTPN13 gene for conserved sequences. This analysis determined that the human and murine PTPN13 sequences were highly conserved throughout the coding region and within the proximal 2.0 kb of PTPN13 5' flank (Fig. 1B). Sequence homology between the two species decreased rapidly beyond 3.0 kb 5' of the TSS. We searched the proximal 4.0 kb of PTPN13 5' flank for IRF protein DNA-binding site consensus sequences. We identified eight potential binding sites, all located within the proximal 700 bp.

To verify these results, independent chromatin immunoprecipitation experiments were performed. For these studies, chromatin was co-precipitated from lysates of differentiated U937 cells with ICSBP antibody or control preimmune serum. Precipitated chromatin was analyzed by PCR with primers representing various sequences in the PTPN13 5' flank. PCR products were sized by acrylamide gel electrophoresis. We found that 2.0 or 1.0 kb of the proximal PTPN13 5' flank specifically co-precipitated with ICSBP, but the proximal 500 bp did not (Fig. 1C). Additional PCR primers were designed to further localize the binding site. ICSBP co-precipitating chromatin was amplified by primers for the proximal 670 bp, but not 600 bp, of PTPN13 5' flank (Fig. 1C). This region included one of the IRF-binding consensus sequences identified by data base search. This sequence was homologous to ISRE or composite ets/IRF consensus sequences (-602 to -612, 5'-GAAATTAGAAA-3', compared with ISRE, 5'-GAAANNGAAA-3', or ets/IRF, 5'-GAA(A/G)TGNNA-3').

We also investigated more distal sequences. We found that ICSBP-co-precipitating chromatin was not amplified by primers flanking the 670-bp to 2.0-kb sequence (Fig. 1C) nor the sequence between 2.0 and 4.0 kb (not shown). These results suggested that ICSBP interacts with an IRF-binding consensus sequence in the proximal PTPN13 promoter. However, a role for ICSBP in Fap-1-expression had not been described previously. Therefore, we investigated whether Fap-1 expression was ICSBP-dependent.

ICSBP Expression Inversely Correlated with Fap-1 Expression in Myeloid Cells—In previous studies, Fas sensitivity and Fap-1 expression were determined in various leukemia cell lines (23). U937 cells were found to be relatively Fas-resistant with abundant Fap-1 expression. In contrast, KG1 myeloid cells were relatively Fas-sensitive with lower levels of Fap-1 (23). Because ICSBP-deficient cells are resistant to apoptosis (21), we investigated whether expression of ICSBP and Fap-1 were inversely correlated in U937 cells versus KG1 cells. We found that ICSBP protein was relatively more abundant and Fap-1 protein relatively less abundant in KG1 cells in comparison with U937 cells (Fig. 2A). We also found that Fap-1 expression decreased in both lines during differentiation. These studies were repeated at least three times, and representative results are shown. As in our previous studies, ICSBP protein abundance was not altered by differentiation of U937 or KG1 cells (9, 10). Therefore, ICSBP expression levels did not explain decreased Fap-1 expression during differentiation. This is addressed further below.


Figure 1
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  		FIGURE 1.
ICSBP interacts with the proximal PTPN13 5' flank. A, sequence of the human and murine PTPN13 5' flanks. PTPN13 was identified as a potential ICSBP target gene by screening a CpG island microarray with ICSBP-co-precipitating chromatin. The human sequence (black) was aligned with the murine sequence (blue). The human gene sequence is shown from -3.0 kb to the TSS. Conserved sequences between the human and murine 5' flanks are indicated in gray. The 5' and 3' ends of the CpG island are indicated by arrows. Conserved IRF-DNA-binding site consensus sequences are indicated by a blue line. The -587 to -627 bp sequence is identified by brackets. B, conserved sequences in the human PTPN13 gene. Homology between the human and murine PTPN13 locus is indicated in the upper panel. Homology in the 5' flank rapidly declines beyond -2.0 kb from the TSS. Sequence homology between human, mouse, and dog for the proximal 2.0 kb of 5' flank are shown in the lower panel. Arrows indicate conserved IRF-DNA binding consensus sequences. C, ICSBP binds the PTPN13 5' flank between -670 and -500 bp from the TSS in vitro. Chromatin immunoprecipitation was performed using lysates from IFN{gamma}-differentiated U937 cells and antibody to ICSBP or control preimmune serum. Co-precipitating chromatin was amplified by PCR with primer sets representing the proximal 2.0, 1.0, 0.67, 0.60, and 0.50 kb of 5' flank, and products were sized on acrylamide gels. PCR was also performed with a primer set to amplify the sequence between 670 bp and 2.0 kb from the PTPN13 TSS.

 


Figure 2
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  		FIGURE 2.
ICSBP expression was inversely correlated with Fap-1 expression in myeloid cells. A, comparison of ICSBP and Fap-1 expression in U937 and KG1 myeloid leukemia cells. Total lysates were isolated from U937 or KG1 cells, with or without IFN{gamma} differentiation. Cell lysates were separated by SDS-PAGE, and Western blots (WB) were serially probed with antibodies to ICSBP and GAPDH (to control for loading). An aliquot of lysate was immunoprecipitated with Fap-1 antibody (or irrelevant antibody), and immunoprecipitates were separated by SDS-PAGE, and Western blots were probed with Fap-1 antibody. No Fap-1 cross-immunoreactive protein co-precipitated from these lysates with irrelevant antibody (not shown). B, altering ICSBP expression altered Fap-1 expression and Fas phosphorylation in KG1 cells. Stable transfectants of KG1 cells were generated with a vector to overexpress ICSBP (or empty vector control) or a vector to express an ICSBP-specific shRNA (or scrambled control shRNA). Cell lysates were separated by SDS-PAGE, and Western blots were serially probed with antibodies to ICSBP and tubulin (to control for loading). Aliquots of lysate proteins were immunoprecipitated with antibody to Fap-1 or Fas (or irrelevant antibody) and separated by SDS-PAGE. Western blots of Fap-1 immunoprecipitates were probed with Fap-1 antibody, and Western blots of Fas immunoprecipitates were probed with antibody to phosphotyrosine. No Fap-1 or phosphotyrosine cross-immunoreactive protein co-precipitated with irrelevant antibody (not shown). C, expression of Fap-1 mRNA was sustained during ex vivo differentiation of ICSBP-deficient murine myeloid progenitors. Bone marrow-derived WT and ICSBP-/- murine myeloid progenitors were cultured in GM-CSF, IL3, and SCF. Some cells were differentiated to granulocytes with G-CSF or to monocytes with M-CSF (±IFN{gamma}). mRNA expression was determined by quantitative real time PCR. Statistically significant differences in Fap-1 mRNA expression (p ≤ 0.005, n = 6) are indicated by *, **, or ***. Statistically significant differences in gp91phox mRNA expression (p ≤ 0.001, n = 6) are indicated by #, ##, or ###. D, expression of Fap-1 protein was sustained during ex vivo differentiation of ICSBP-deficient murine myeloid progenitors. Lysate proteins from cultured, primary WT, and ICSBP-/- cells, described above, were immunoprecipitated with Fap-1 antibody (or irrelevant control antibody), separated by SDS-PAGE, and Western blots were probed with anti-Fap-1 antibody. Aliquots of lysate were also separated by SDS-PAGE, and Western blots were probed with antibody to GAPDH to control for protein content in the lysates. No Fap-1 cross-immunoreactive protein co-precipitated with irrelevant antibody (not shown).

 
U937 and KG1 are leukemia cell lines that might be anticipated to have abnormalities in a number of signaling pathways. To directly determine the impact of ICSBP on Fap-1 expression, stable KG1 transfectants were generated in which ICSBP expression was specifically manipulated. In initial studies, KG1 cells were transfected with a vector to overexpress ICSBP (or empty expression vector). We found that ICSBP overexpression decreased Fap-1 expression and increased Fas-tyrosine phosphorylation in these cells (Fig. 2B). In the converse experiment, stable KG1 transfectant pools were generated with a vector to express an ICSBP-specific shRNA (or a scrambled control shRNA) (Fig. 2B). We found that decreased ICSBP expression correlated with increased Fap-1 expression and decreased Fas-tyrosine phosphorylation. Three independent transfectant pools were generated for each of these constructs, and blots were repeated several times for each pool. A representative blot from a single experiment is shown. Therefore, perturbation of ICSBP expression altered Fap-1 expression and Fas phosphorylation in myeloid cell lines.

We also investigated the association between expression of ICSBP and Fap-1 in a nontransformed model of myeloid differentiation. For these studies, Sca1+ cells were isolated from the bone marrow of ICSBP-/- mice or WT control littermates, and myeloid progenitors were cultured in GM-CSF, IL3, and SCF. Some cells were differentiated to granulocytes with G-CSF or to monocytes with M-CSF. IFN{gamma} was added to some cultures to induce terminal differentiation. Both WT and ICSBP-/- cells cultured in GM-CSF, IL3, and SCF were Sca1+CD14dimCD20-CD4-. Ex vivo differentiated cells were Sca1-CD14+Gr1+ or Sca1-CD14+Mac1+, respectively, consistent with published results (12, 14). These results indicated a block in terminal differentiation in ICSBP-/- cells, as anticipated based on known target genes (4-8).

Gene expression was studied in these cells using quantitative real time PCR. Each study was performed three times in duplicate. We found slightly more Fap-1 mRNA in ICSBP-/- myeloid progenitors in comparison with WT (p = 0.24, n = 6) (Fig. 2C). The amount of Fap-1 mRNA decreased significantly in WT progenitor cells upon differentiation with G-CSF or M-CSF (p < 0.005, n = 6). In contrast, Fap-1 expression was not significantly altered by differentiation of ICSBP-/- progenitor cells with these cytokines (p = 0.8, F = 0.33, n = 6). Therefore, Fap-1 expression was significantly greater in ICSBP-/- cells that were differentiated with G-CSF or M-CSF + IFN{gamma} in comparison with similarly treated WT cells (p < 0.001, n = 6). ICSBP was not expressed in ICSBP-/- cells, as expected, and differentiation did not alter ICSBP expression in WT cells, consistent with previous results (14, 18). Expression of gp91PHOX, an ICSBP activation target, was a positive control. There was significantly less gp91PHOX mRNA in differentiated ICSBP-/- cells in comparison with WT (p < 0.001, n = 6).

We determined whether differences in Fap-1 mRNA in ICSBP-/- versus WT cells correlated with protein abundance. Lysates proteins from WT or ICSBP-/- bone marrow-derived cells were analyzed for Fap-1 expression by Western blot. Each of these studies was repeated at least three times, and a representative blot is shown. We found that Fap-1 protein decreased in WT murine myeloid progenitors during granulocyte or monocyte differentiation. In contrast, differentiation did not alter Fap-1 protein expression in ICSBP-deficient progenitors (Fig. 2D). However, this correlation did not provide a functional connection between ICSBP, Fap-1, and susceptibility to Fas-induced apoptosis. Therefore, additional studies were performed.

ICSBP Inversely Correlated with Inhibition of Fas-induced Apoptosis by Fap-1 in Myeloid Cells—To investigate the role of Fap-1 in resistance to Fas-induced apoptosis in U937 cells, we employed a previously described peptide that specifically disrupts the Fas-Fap-1 interaction (the SLV peptide) (29). For these studies, U937 cells were cultured for 48 h with or without IFN{gamma}. Cells were analyzed for apoptosis with or without treatment with a Fas-agonist antibody and with SLV or control VLS peptide (28, 29). We found that treatment of U937 cells with the Fas-agonist antibody did not significantly alter the percent of apoptotic cells, with or without differentiation (p > 0.1, n = 3) (Fig. 3A). However, Fas antibody induced a significant amount of apoptosis in SLV peptide-treated, IFN{gamma}-differentiated U937 cells (p = 0.005, n = 3) (Fig. 3A). These results suggested that Fas resistance of U937 cells was Fap-1-dependent.

To verify that SLV peptide was interfering with Fas-Fap-1 interaction, lysates from SLV or VLS peptide-treated, IFN{gamma}-differentiated U937 cells were immunoprecipitated with an anti-Fas antibody (or control antibody). Immunoprecipitates were separated by SDS-PAGE, and interaction between Fas and Fap-1 was determined by Western blot (Fig. 3B). This experiment was repeated twice, and a representative blot is shown. SLV peptide treatment decreased co-immunoprecipitation of Fap-1 and Fas and increased Fas phosphorylation.

We next determined whether manipulation of ICSBP expression in KG1 cells altered Fas-induced apoptosis, using the stable transfectant pools described above. Each experiment was performed with several different pools. We found that Fas-agonist antibody induced significant apoptosis in IFN{gamma}-treated control or ICSBP-overexpressing KG1 cells (p = 0.01, n = 3) (Fig. 3C). Differentiation significantly increased Fas-induced apoptosis in ICSBP-overexpressing KG1 cells (p = 0.03, n = 3). Conversely, ICSBP knockdown significantly decreased Fas-induced apoptosis in differentiated KG1 transfectants in comparison with control (p < 0.001, n = 6) (Fig. 3D). This difference was abolished by treatment with SLV peptide (p = 0.13, n = 6). Although SLV peptide significantly increased apoptosis in differentiated KG1 transfectants with ICSBP knockdown (p < 0.001, n = 6), this effect was not seen in control transfectants (p = 0.44, n = 6). These studies suggested that resistance to Fas-induced apoptosis in cells with relative ICSBP deficiency was Fap-1-dependent.

We extended these studies to ex vivo differentiated murine myeloid progenitors. Sca1+ cells were isolated from WT or ICSBP-/- murine bone marrow. Common granulocyte-monocyte progenitors were cultured in GM-CSF, IL3, and SCF, and some cells were differentiated with G-CSF or M-CSF with or without IFN{gamma} for the last 24 h. We found that apoptosis was not significantly different in WT versus ICSBP-/- myeloid progenitor cells, with or without Fas-antibody (p = 0.14, F = 2.2, n = 4) (Fig. 4A). In contrast, Fas-agonist antibody induced significant apoptosis in G-CSF or M-CSF differentiated WT cells, and this was increased by IFN{gamma} treatment (p < 0.02, n = 4). In contrast, Fas-agonist antibody did not induce significant apoptosis in ICSBP-/- cells under any of these cytokine conditions (Fig. 4A) (p = 0.3, n = 4). We noted that base-line apoptosis (i.e. without Fas antibody) was significantly less in ex vivo differentiated ICSBP-/- versus WT murine myeloid cells (p < 0.001, n = 4).

We found that treatment with SLV peptide significantly increased Fas-induced apoptosis in ICSBP-/- cells that had been differentiated with either G-CSF + IFN{gamma} or M-CSF + IFN{gamma} (p < 0.001, n = 3) (Fig. 4B). In these studies, Fas-induced apoptosis in SLV peptide-treated, G-CSF + IFN{gamma}-differentiated ICSBP-/- cells was not significantly different from similarly treated WT cells (p = 0.4, n = 3). Interestingly, Fas antibody induced more apoptosis in SLV peptide-treated, M-CSF + IFN{gamma}-differentiated ICSBP-/- cells than in similarly treated WT cells (p = 0.001, n = 3).

These results suggested that increased Fap-1 expression contributed to Fas resistance in ICSBP-/- myeloid cells. However, these studies had not demonstrated a direct impact of ICSBP on PTPN13 transcription. Therefore, this issue was specifically addressed.

ICSBP Repressed PTPN13 Transcription via a Cis Element in the Proximal Promoter—Based on chromatin immunoprecipitation, we hypothesized that there was an ICSBP-binding cis element between 600 and 670 bp in the proximal PTPN13 5' flank. To investigate this, we generated a series of reporter constructs with 2.0 kb, 1.3 kb, 670 bp, and 500 bp of the PTPN13 5' flank. These PTPN13/reporter constructs were co-transfected into U937 cells with a vector to overexpress ICSBP or empty control vector. U937 cells were chosen for these experiments based on their relatively high level of Fap-1 expression. Because our results suggested that Fap-1 expression was functionally relevant to Fas resistance in IFN{gamma}-treated U937 cells, transfectants were assayed with or without differentiation.

We found that the construct with 2.0 kb of PTPN13 5' flank had significantly more reporter activity than the 1.3-kb construct (Fig. 5A) (p < 0.001, n = 3), indicating the presence of a positive cis element between 2.0 and 1.3 kb (region A in Fig. 5B). Differentiation of the transfectants abolished the difference in reporter activity between the 2.0-and 1.3-kb constructs. This suggested the positive cis element in region A was not active in differentiated cells. Activity of both of the 2.0 -and 1.3-kb constructs was significantly decreased by overexpression of ICSBP (p < 0.001, n = 3). This implied an ICSBP-binding negative cis element in the common proximal region.


Figure 3
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  		FIGURE 3.
ICSBP deficiency impaired Fas-induced apoptosis in a Fap-1-dependent manner in myeloid cell lines undergoing differentiation. A, blocking the Fap-1-Fas interaction with SLV peptide increased Fas-induced apoptosis in U937 cells. U937 cells were cultured for 48 h, with or without IFN{gamma}-differentiation, and treated with Fas-agonist antibody (or irrelevant control). Some cells were also treated with SLV peptide (or control) to block interaction of Fap-1 with Fas. Statistically significant differences in apoptosis are indicated by *. B, SLV peptide blocked Fap-1 co-precipitation with Fas in U937 cells. The U937 cells described above were also analyzed for interaction between Fas and Fap-1. Cell lysates were immunoprecipitated (IP) with antibody to Fas (or irrelevant antibody) and separated by SDS-PAGE. Western blots were serially probed with antibody to Fap-1 or phosphotyrosine. C, ICSBP overexpression increased Fas-induced apoptosis in KG1 cells. Stable KG1 transfectant pools were generated with a vector to overexpress ICSBP or with empty control vector, as above. Cells were analyzed for apoptosis, with or without IFN{gamma} differentiation, with or without Fas agonist antibody (Ab). Statistically significant differences in apoptosis are indicated by *, ** and ***. D, ICSBP knockdown inhibited Fas-induced apoptosis in KG1 cells in a Fap-1-dependent manner. Stable KG1 transfectant pools, described above, were generated with a vector to express an ICSBP-specific shRNA or scrambled shRNA control. Cells were analyzed for apoptosis, with or without IFN{gamma} differentiation, with or without Fas-agonist antibody. Statistically significant difference in Fas-induced apoptosis are indicated by * or **.

 


Figure 4
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  		FIGURE 4.
ICSBP deficiency impaired Fas-induced apoptosis in a Fap-1-dependent manner in primary murine myeloid progenitors during differentiation. A, Fas-induced apoptosis was impaired in ex vivo differentiated ICSBP-/- murine myeloid cells. Bone marrow-derived WT or ICSBP-/- murine myeloid progenitors were cultured in GM-CSF, IL3, and SCF. Some cells were differentiated to granulocytes with G-CSF (±IFN{gamma}) or to monocytes with M-CSF (±IFN{gamma}). Cells were analyzed for apoptosis with or without Fas-agonist antibody (Ab). Statistically significant differences in Fas-agonist antibody induced apoptosis are indicated by *, **, #, ##. B, impaired Fas-induced apoptosis in ex vivo differentiated ICSBP-/- murine myeloid cells was Fap-1-dependent. Bone marrow-derived WT or ICSBP-/- murine myeloid progenitors were differentiated to granulocytes with G-CSF + IFN{gamma} or to monocytes with M-CSF + IFN{gamma}. Cells were treated with SLV blocking peptide (or control) and analyzed for Fas antibody-induced apoptosis. Statistically significant differences in Fas-induced apoptosis are indicated by * or **.

 
Truncation from 1.3 kb to 670 bp resulted in further loss of promoter activity in undifferentiated transfectants (p < 0.0001, n = 3). This suggested a second positive cis element within this region (region B in Fig. 5B). This conclusion was supported by the observation that repression of the 670-bp construct by overexpressed ICSBP was relatively greater than repression of the 1.3-kb construct. Indeed, overexpressed ICSBP abolished activity of the 670-bp construct in differentiated transfectants. This would be consistent with more efficient ICSBP repression of the 670-bp construct because there was less transcriptional activation to overcome in comparison with the 1.3-kb construct. This result also suggested that there was an ICSBP-binding negative cis element within the proximal 670-bp PTPN13 5' flank that was not effectively opposed by positive cis elements within this region (the proximal 670 bp is region C and D in Fig. 5B).

Activity of the 500-bp construct was significantly greater than activity of the 670-bp construct in undifferentiated and differentiated transfectants (p ≤ 0.001, n = 3). Overexpressed ICSBP did not significantly alter reporter expression from the 500-bp construct (p = 0.1, n = 3), in contrast to the profound repression of the 670-bp construct. This suggested that the ICSBP-binding negative cis element was eliminated by truncation of the sequence between 500 and 670 bp (i.e. region C in Fig. 4B).

Activity of the 500-bp construct decreased with IFN{gamma} (p ≤ 0.01, n = 3). This implied that positive cis elements in the proximal 500 bp were more active in undifferentiated transfectants or that there was a negative cis element that was not repressed by ICSBP within this sequence. The observation that the 670-bp construct was significantly less active than the 500-bp construct suggested that positive cis elements in the proximal 500 bp of PTPN13 5' flank (region D in Fig. 5B) were over-whelmed by the ICSBP-binding, negative cis element between 500 and 670 bp (region C). Neither IFN{gamma} differentiation nor ICSBP overexpression altered activity of the empty, control reporter vector, which was consistently low (<200 cpm) and was subtracted as background.

These results suggested that the -500- to -670-bp region of the PTPN13 5' flank included an ICSBP-binding repressor element. The most highly conserved sequence within this region was 100% identical in human, mouse, and dog and included an IRF-binding consensus (-627 and -587 bp) (Fig. 5C). Therefore, we hypothesized that the ICSBP-binding repressor cis element was -627 to -527 bp of the PTPN13 5' flank. However, we had not specifically demonstrated ICSBP binding to this sequence. Therefore, further studies were performed.

ICSBP Bound to the Proximal PTPN13 Promoter in a Differentiation Stage-specific Manner—We investigated in vivo ICSBP binding to the putative PTPN13 cis element using chromatin immunoprecipitation from U937 cells. Co-precipitated chromatin was quantitated by real time PCR with a primer set designed to amplify this region of the promoter (-639 to -567 bp). We found that differentiation significantly increased in vivo binding of endogenous ICSBP to this region of the PTPN13 promoter (p < 0.0001, n = 6) (Fig. 6A). This promoter sequence did not co-precipitate from U937 lysates with preimmune serum.


Figure 5
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  		FIGURE 5.
ICSBP repressed a specific cis element in the PTPN13 promoter. A, ICSBP repressed PTPN13 transcription via a cis element between -500 and -670 bp in the promoter. U937 cells were co-transfected with a series of PTPN13 promoter/reporter constructs (2.0 kb, 1.3 kb, 670 bp, and 500 bp, or empty control vector) and a vector to overexpress ICSBP (or empty control vector). Transfectants were analyzed after 48 h, with or without IFN{gamma} differentiation. Statistically significant differences in reporter expression are indicated by *, **, ***, #, ##, ###, & or &&. Neither ICSBP nor IFN{gamma} overexpression significantly altered expression from empty control reporter vector that was subtracted as background. B, schematic representation of the PTPN13 proximal 5' flank. A schematic representation of the proximal 2.33 kb of PTPN13 5' flank is shown. The CpG island is indicated as is the IRF DNA-binding consensus sequence between -587 and -627 bp. The various constructs used in transfection experiments are graphically designated below with identification of the sequences sequentially deleted to generate these constructs (A-D). The table indicates the location of positive cis elements and ICSBP-repressed negative cis elements as inferred by the results of the reporter gene assays. C, sequence conservation of the -670- to -500-bp sequence of the PTPN13 promoter. Sequences from the proximal PTPN13 promoters of human, mouse, and dog in the region of the putative ICSBP-repression cis element were aligned. Conserved sequences are underlined. The IRF-DNA binding consensus sequence in this region of the promoter is in red.

 
We also used two different in vitro assays to investigate ICSBP binding to the PTPN13 promoter. We first investigated this interaction using a DNA-co-affinity purification assay. For these studies, nuclear proteins were isolated from untreated or IFN{gamma}-differentiated U937 cells. Proteins were incubated with a biotin-labeled, double-stranded oligonucleotide probe representing the -587- to -627-bp sequence from the PTPN13 promoter. In control experiments, proteins were incubated with a biotin-labeled, double-stranded oligonucleotide containing a mutant PRDI sequence, known not to bind ICSBP (14). The double-stranded oligonucleotides were precipitated and co-precipitating proteins separated by SDS-PAGE. Western blots were probed with an antibody to ICSBP to identify co-precipitating proteins. Nonprecipitated nuclear proteins were a control for protein equivalence between the preparations in these experiments.


Figure 6
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  		FIGURE 6.
ICSBP interacted with a specific cis element in the PTPN13 promoter. A, ICSBP interacted in vivo with a sequence between -490 and -670 bp in the PTPN13 promoter. Chromatin immunoprecipitation was performed with an ICSBP antibody (or control preimmune serum) and lysates from U937 cells. Co-precipitating chromatin was amplified by quantitative real time PCR with primers flanking the putative ICSBP-binding cis element in the PTPN13 promoter. Results from undifferentiated and IFN{gamma}-differentiated cells were compared. Statistically significant increase in ICSBP binding is indicated by *. B, ICSBP co-purified with a double-stranded oligonucleotide representing the -587- to -627-bp sequence from the PTPN13 promoter. DNA-affinity purification was performed with nuclear proteins from U937 cells and a double-stranded, biotin-labeled probe representing the -587- to -627-bp sequence from the PTPN13 promoter, or a non-ICSBP-binding mutant PRDI consensus sequence. Oligonucleotides were precipitated with avidin-conjugated agarose, and precipitates were separated by SDS-PAGE. Western blots were probed with an antibody to ICSBP. A 1/10 volume of input (nonprecipitated) nuclear proteins was a loading control in this experiment. C, ICSBP specifically interacted with the -587- to -627-bp sequence from the PTPN13 promoter by electrophoretic mobility shift assay. EMSAs were performed with U937 cell nuclear proteins and a double-stranded (ds), radiolabeled probe representing the -587- to -627-bp sequence from the PTPN13 promoter. Nuclear proteins from IFN{gamma}-differentiated cells were compared with untreated cells. Some binding assays were preincubated with excess unlabeled double-stranded oligonucleotide competitor representing the homologous sequence, a consensus ISRE sequence, or an irrelevant sequence, as indicated. Other binding assays were preincubated with ICSBP antibody or preimmune serum. Equal protein loading in assays with nuclear proteins from untreated versus IFN{gamma}-differentiated U937 cells was determined in control EMSA with a CCAAT box probe.

 
We found specific interaction of ICSBP with the PTPN13 cis element probe (Fig. 6B). Consistent with our in vivo results, co-precipitation of ICSBP with the PTPN13 cis element was more efficient with nuclear proteins from differentiated U937 cells. This experiment was repeated with two different batches of nuclear proteins, and a representative blot is shown.

We also investigated in vitro ICSBP binding to the PTPN13 cis element by EMSA. For these studies, U937 nuclear proteins were incubated with a radiolabeled, double-stranded oligonucleotide probe representing the PTPN13 -587- to -627-bp sequence. Some binding assays were preincubated with unlabeled, double-stranded oligonucleotide competitors representing homologous sequence, an ISRE consensus, or an irrelevant oligonucleotide (Fig. 6C). A specific protein complex bound to this PTPN13 sequence and exhibited cross-competitive binding specificity with the ISRE consensus. This complex was relatively more abundant in assays with nuclear proteins from differentiated U937 cells in comparison with undifferentiated cells. Equivalence in protein loading between nuclear protein preparations was determined in control EMSA with a CCAAT box-binding oligonucleotide (Fig. 6C). These studies were performed with three different batches of nuclear proteins, and representative experiments are shown.

To determine whether the complex that bound the PTPN13 probe included ICSBP, nuclear proteins were preincubated with either ICSBP antibody or irrelevant control antibody. We found that ICSBP antibody disrupted the protein complex that bound to the -587- to -627-bp probe (Fig. 6C). Therefore, these studies suggested that ICSBP interacted with this ISRE-like sequence in the PTPN13 promoter.

ICSBP Interacted with the PTPN13 Cis Element in a Tyrosine Phosphorylation-dependent Manner—These studies indicated that interaction of ICSBP with the PTPN13 cis element increased during differentiation. The mechanism for this was not obvious, since differentiation of U937 or KG1 cells did not increase ICSBP expression. We previously found that interaction of ICSBP with the CYBB, NCF2, and NF1 genes increased during differentiation (5, 9, 14). For these genes, ICSBP binding was regulated by phosphorylation of conserved tyrosine residues in the ICSBP IRF domain (9, 10). Because ICSBP-tyrosine phosphorylation occurs during myelopoiesis (3, 9, 10), this provided a mechanism for differentiation stage-specific regulation of these target genes.

Therefore, we performed transfection experiments to determine the role of the IRF domain tyrosines in ICSBP-induced PTPN13 repression. For these studies, U937 cells were co-transfected with the 2.0-kb PTPN13 reporter construct (or control vector) and vectors to overexpress ICSBP, a form of ICSBP with the conserved IRF domain tyrosine residues mutated to phenylalanine (Y92F/Y95F ICSBP), or empty control vector. Reporter activity was determined with or without IFN{gamma}-induced differentiation. We found that overexpressed WT ICSBP repressed PTPN13 promoter activity significantly more efficiently than Y92F/Y95F ICSBP, with or without differentiation (p ≤ 0.0002, n = 6) (Fig. 7A).

Overexpression of these proteins did not alter activity of the control, empty reporter vector, which was subtracted as background. In previous studies, we demonstrated that these two forms of ICSBP were equivalently expressed in U937 cells and equivalently stable under the conditions of these assays (9, 10, 18). In those studies, we also determined that overexpressed ICSBP is somewhat tyrosine-phosphorylated in U937 transfectants, and tyrosine phosphorylation increased with IFN{gamma}-induced differentiation (9, 10).

We were also interested in the role of IRF domain tyrosine residues on ICSBP-induced repression of endogenous Fap-1 expression. Previously, we found that expression of WT ICSBP in ICSBP-/- myeloid cells rescued expression of gp91PHOX and Nf1 but expression of Y92F/Y95F ICSBP did not (9, 10). Similar to those studies, ICSBP-/- myeloid progenitors were cultured in GM-CSF, IL3, and SCF, transduced with a retroviral vector to express ICSBP, Y92F/Y95F ICSBP or with empty control vector, and differentiated with M-CSF + IFN{gamma} (as in Ref. 10). Fap-1 expression was determined by real time PCR (Fig. 7B).

We found that Fap-1 expression was not significantly different in ICSBP-/- cells that had been transduced with empty control vector in comparison with nontransduced ICSBP-/- cells (p < 0.02, n = 3). Fap-1 expression was also not significantly different in cells that were transduced with a Y92F/Y95F ICSBP expression vector in comparison with empty vector control (p = 0.3, n = 3). In contrast, ICSBP-/- cells that were transduced with a vector to express WT ICSBP had significantly less Fap-1 expression in comparison with these other groups (p = 0.007, n = 3). Expression of Fap-1 in these cells was not significantly different from WT cells (p = 0.02, n = 3).

In control experiments, we verified that WT and Y92F/Y95F ICSBP were equivalently expressed in transduced ICSBP-/- cells. For these studies, we performed real time PCR with a set of primers designed to amplify the product of either the WT or mutant ICSBP transgene (see Ref. 38). We found equivalent expression of WT and Y92F/Y95F ICSBP in ICSBP-/- cells (p = 0.9, n = 3) (Fig. 7B). As anticipated, no transgene was detected in control cells that were transduced with empty retroviral vector. These results were consistent with protein expression results in our previous studies (10).

We were also interested in determining the role of the conserved IRF domain tyrosine residues in ICSBP binding to the PTPN13 promoter in vivo. To do this, aliquots of these transduced ICSBP-/- cells were used for chromatin immunoprecipitation studies. Chromatin that co-precipitated with ICSBP antibody (or irrelevant control antibody) was analyzed by real time PCR with primers flanking the ICSBP-binding site in the PTPN13 promoter. We found that significantly more WT ICSBP bound to the PTPN13 cis element in comparison with Y92F/Y95F ICSBP (p < 0.0001, n = 3). No ICSBP binding to the PTPN13 cis element was detected in control ICSBP-/- cells that had been transduced with empty retroviral vector, as anticipated. In contrast, the PTPN13 cis element did not co-precipitate with control, preimmune serum.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although ICSBP-deficient myeloid cells were known to be apoptosis-resistant (21), direct target genes involved in this effect had not been previously identified. In this study, we used high throughput screening to identify the gene encoding Fap-1 as an ICSBP target gene. This was of interest, because Fas dephosphorylation by Fap-1 impairs Fas-induced apoptosis. We found that ICSBP interacted with the PTPN13 promoter in a tyrosine phosphorylation-dependent manner and repressed transcription. We also found that decreased ICSBP expression increased Fap-1 expression and Fap-1-dependent resistance to Fas-induced apoptosis. Therefore, the current studies identified a pathway involved in apoptosis resistance in myeloid malignancies that are characterized by decreased ICSBP expression.

Most previously identified ICSBP target genes encoded proteins involved in the phagocyte or B-cell immune response. Our high throughput screening identified a number of additional genes consistent with this well described function of ICSBP. However, the goal of these studies was to better understand mechanisms for the ICSBP-leukemia-suppressor effect. Therefore, we were especially interested to identify a CpG island -875 bp from PTPN13 exon 1. Using additional chromatin immunoprecipitation studies, in vitro binding assays, and transfection experiments, we identified an ICSBP-binding cis element between -627 and -587 bp in the PTPN13 5' flank. This region of the promoter included a highly conserved sequence with homology to both ISRE and composite ets/IRF sequences for IRF-protein DNA binding.

However, these results did not exclude the possibility that ICSBP also interacted with other cis elements, more remote from the TSS. Because our goal was to identify ICSBP target genes functionally involved in leukemia suppression, we did not exhaustively investigate the PTPN13 locus for additional binding sites. Also, we found that conservation of the PTPN13 5' flank between human, mouse and dog declined rapidly at beyond -2.0 kb from the TSS. This decreased the possibility of additional, functionally significant ICSBP-binding cis elements further 5'.

We found that in vitro and in vivo binding of endogenous ICSBP to the PTPN13 cis element increased during myeloid differentiation. Consistent with this, the impact of ICSBP deficiency on Fap-1 expression and apoptosis inhibition was greatest in differentiated primary myeloid cells or cell lines. Therefore, these studies identified a mechanism for differentiation stage inappropriate resistance to Fas-induced apoptosis in ICSBP-deficient cells. There are several mechanisms that might account for increased interaction of endogenous ICSBP with the PTPN13 cis element during myelopoiesis. One possibility was that increased ICSBP expression during differentiation increased PTPN13 binding. This would be consistent with our transfection experiments in which overexpressed ICSBP repressed PTPN13 transcription. However, ICSBP expression is not significantly altered by differentiation of myeloid leukemia cell lines or primary murine myeloid progenitors (5, 10).


Figure 7
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  		FIGURE 7.
ICSBP interacted with the PTPN13 cis element in a tyrosine phosphorylation-dependent manner. A, conserved IRF domain tyrosine residues are involved in ICSBP-induced repression of the PTPN13 promoter. U937 cells were co-transfected with the 2.0-kb PTPN13 promoter/reporter construct (or empty control vector) and a vector to overexpress WT or Y92F/Y95F ICSBP or empty control vector. Transfectants were analyzed after 48 h with or without IFN{gamma} differentiation. Statistically significant differences in reporter expression are indicated by * or **. B, Fap-1 expression in ex vivo differentiating primary myeloid progenitors required the conserved IRF domain tyrosine residues in ICSBP. ICSBP-/- myeloid progenitors were transduced with retroviral vectors to express ICSBP, Y92F/Y95F ICSBP, or empty murine stem cell retrovirus vector control. Cells were ex vivo differentiated with M-CSF + IFN{gamma} and Fap-1 expression was determined by real time PCR. ICSBP-transgene expression was also determined as a control, and results were normalized to 18 S RNA. C, in vivo interaction of ICSBP with the PTPN13 cis element required the conserved IRF domain tyrosines. Some of the ICSBP-/- transduced cells, described above, were used for chromatin immunoprecipitation (IP) experiments. Chromatin was co-precipitated with antibody to ICSBP or preimmune control serum. Real time PCR was used to amplify chromatin fragments representing the ICSBP-binding cis element in the PTPN13 promoter. Results were normalized to total input chromatin.

 
Another possible mechanism by which differentiation might increase ICSBP interaction with the PTPN13 promoter is post-translational modification. Previous studies found that endogenous ICSBP is tyrosine-phosphorylated during differentiation of various myeloid cell lines or murine myeloid progenitors (9, 10). We previously found that phosphorylation of conserved tyrosine residues in the IRF domain of ICSBP was required for assembly of multiprotein complexes that activate the CYBB, NCF2, and NF1 genes (9, 18). In this study we found that the same IRF domain tyrosines were involved in differentiation stage-specific ICSBP binding to and repression of the PTPN13 promoter. Therefore, these studies represent identification of the first target gene for which ICSBP-tyrosine phosphorylation regulates repression activity in differentiating myeloid cells.

Although IRF domain-tyrosine mutant ICSBP was less efficient at repressing the PTPN13 promoter than WT ICSBP, repression activity was not abolished. One possible explanation was that phosphorylation of these residues increases ICSBP affinity for the PTPN13 cis element, but it is not an absolute requirement. In this case, the overexpressed mutant protein might have decreased, but not absent, interaction with the cis element. This would be consistent with our studies of ICSBP interaction with positive cis elements in other target genes (9, 10, 18). Another possibility would be that other ICSBP phosphoresidues, in addition to the IRF-domain tyrosines, are involved in PTPN13 regulation. Identification of ICSBP partner proteins will facilitate investigation of these possibilities.

PTPN13 may share a common, bi-directional promoter with the gene that encodes Jnk3 (MAPK10) (34). These two genes are immediately adjacent in the chromosome with PTPN13 transcription proceeding in the telomeric direction and MAPK10 transcription proceeding in the centromeric direction. Jnk3 is a stress-activated protein kinase with neural cell expression (35). We found low levels of Jnk3 protein in myeloid leukemia cell lines, with or without differentiation (not shown). These results suggested that the choice between transcription of PTPN13 versus MAPK10 may be dictated in a tissue-specific manner. It will be of interest to investigate this hypothesis further.

We found little Fas-induced apoptosis in U937 cells. However, treatment of U937 cells with the SLV peptide induced Fas sensitivity which was comparable with KG1 cells. ICSBP over- expression increased and knockdown inhibited Fas-induced apoptosis in KG1 cells. Also, there was no significant difference in Fas-induced apoptosis in IFN{gamma}-treated KG1 cells with ICSBP knockdown + SLV peptide versus IFN{gamma}-treated control KG1 cells. Therefore, ICSBP influenced Fas-induced apoptosis by dose-dependent alteration of Fap-1 expression.

Although ex vivo differentiated WT primary murine myeloid cells exhibited Fas-induced apoptosis, there was also significant apoptosis in these cells without Fas antibody (i.e."endogenous" apoptosis). Ex vivo differentiated ICSBP-/- myeloid cells exhibited less Fas-induced and endogenous apoptosis than WT cells. These results suggested that ICSBP also influences transcription of target genes involved in endogenous apoptosis. This would be consistent with the function of several putative ICSBP target genes we identified using this screening technique. The significance of ICSBP-regulated transcription of these genes is currently under investigation and will be the subject of future reports.

Treatment of ex vivo differentiated ICSBP-/- granulocytes or monocytes with SLV peptide abolished the difference in Fas-induced apoptosis between these cells and WT cells. In fact, Fas antibody induced more apoptosis in SLV peptide-treated, monocyte-differentiated ICSBP-/- cells than in similarly treated WT cells. This implied that other components in the Fas pathway might be up-regulated in cells with constitutive ICSBP deficiency. This would be consistent with studies of apoptosis resistance of chronic phase CML cells (35). These results also suggested that interference with Fas-Fap-1 interaction might be a profitable approach to the problem of persistent CML clones.

Our studies indicated that regulation of PTPN13 transcription is dependent upon both abundance and tyrosine phosphorylation of ICSBP. PTPN13 repression by tyrosine-phosphorylated ICSBP would permit signal-dependent regulation of apoptosis during myelopoiesis. However, abnormal PTPN13 transcription in myeloid malignancies is likely to be related to decreased ICSBP expression levels. These results suggested that leukemia-associated mutations that impaired ICSBP-tyrosine phosphorylation might synergize with decreased ICSBP expression for disease progression in myeloid malignancy.

Fas plays other roles in addition to apoptosis regulation during myelopoiesis. Fas may be involved in expansion of hematopoietic stem cells (36). A role for Fap-1 in this process would be of interest. Fas is also involved in phagocyte recruitment to sites of inflammation (37). If Fap-1 antagonizes this activity, one would anticipate decreased recruitment in ICSBP-deficient malignancies such as CML and therapy-related myelodysplasia. This might provide a mechanism for the impaired innate immune response in these diseases, despite normal or elevated numbers of mature appearing neutrophils. Such an association might also provide a mechanism for abnormal cell migration in CML. Therefore, ICSBP deficiency may impact multiple pathways involved in the innate immune response in myeloid malignancies such as CML and myelodysplasia.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Medicine, The Feinberg School at Northwestern University, 710 N. Fairbanks Ct., Olson 8524, Chicago, IL 60611. Tel.: 312-503-4625; E-mail: e-eklund{at}northwestern.edu.

2 The abbreviations used are: ICSBP, interferon consensus sequence-binding protein; ISRE, interferon-stimulated response element; AML, acute myeloid leukemia; CML, chronic myeloid leukemia; GM-CSF, granulocyte-macrophage colony-stimulating factor; M-CSF, macrophage colony-stimulating factor; EMSA, electrophoretic mobility shift assay; INF, interferon; TSS, transcription start site; shRNA, short hairpin RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type; IRF, interferon regulatory factor. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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