The Transcriptional Repressor GFI-1 Antagonizes PU.1 Activity Through Protein-Protein Interaction

Abstract

Mice lacking the zinc finger transcriptional repressor protein Gfi-1 are neutropenic. These mice generate abnormal immature myeloid cells exhibiting characteristics of both macrophages and granulocytes. Furthermore Gfi-1^-/- mice are highly susceptible to bacterial infection. Interestingly Gfi-1^-/- myeloid cells overexpress target genes of the PU.1 transcription factor such as macrophage- colony stimulating factor receptor (M-CSFR) and PU.1 itself. We therefore determined if Gfi-1 modulates the transcriptional activity of PU.1. Our data demonstrates that Gfi-1 physically interacts with PU.1 repressing PU.1- dependent transcription. This repression is functionally significant as Gfi-1 blocked PU.1- induced macrophage differentiation of a multipotential hematopoietic progenitor cell line. Retroviral expression of Gfi-1 in primary murine hematopoietic progenitors increased granulocyte differentiation at the expense of macrophage differentiation. We interbred Gfi-1^+/- and PU.1^+/- mice and observed that heterozygosity at the PU.1 locus partially rescued the Gfi-1^-/- mixed myeloid lineage phenotype but failed to restore granulocyte differentiation. Our data demonstrate that Gfi-1 represses PU.1 activity and that lack of this repression in Gfi-1^-/- myeloid cells contributes to the observed mixed-lineage phenotype.

Gfi-1 is a zinc finger transcriptional repressor containing an amino terminal SNAG domain necessary for transcriptional repression and six carboxyl terminal zinc fingers critical for DNA binding (1). Gfi-1 was first characterized as a T cell oncogene (2-5). It is expressed in the common lymphoid progenitor and developing T and B lymphocytes however during normal development levels decrease as these cells mature (6,7). Gfi-1 is also expressed in non-lymphoid hematopoietic cells including the hematopoietic stem cell (HSC), and the granulocyte macrophage progenitor (GMP) (6). In mature hematopoietic cells, its expression is limited to granulocytes, however Gfi-1 can be induced in mature T cells and macrophages by mitogenic stimuli and LPS respectively (1,8-10).

Gfi-1 is required for granulocyte development. Human GFI-1 mutations were isolated from neutropenic patients, and these mutations hinder Gfi-1's ability to repress transcription (11). Additionally two independent Gfi-1^-/- mouse strains were engineered, which exhibit neutropenia (9,10). An abnormal population of Gr1⁺CD11b⁺ cells sharing characteristics of granulocytes and macrophages develop in Gfi-1^-/- mice (10). These cells lack cytoplasmic granules and do not express RNA for secondary granule proteins. Bone marrow from Gfi-1^-/- mice exhibits higher levels of M-CSFR (macrophage colony stimulating factor receptor) and PU.1 RNA compared to wildtype. Since both of these genes are transcriptional targets of the Ets family transcription factor PU.1, we hypothesized that some of the defects observed in mutants may be due to the lack of antagonism of PU.1 activity in granulocytes by Gfi-1.

Mice lacking PU.1 do not make mature macrophages, granulocytes, or B cells (12,13). Interestingly PU.1 concentration is critical for the specification of cell fates in the hematopoietic system. High levels of PU.1 via retroviral expression direct macrophage development of PU.1^-/- fetal liver hematopoietic progenitors, however low levels promotes B cell differentiation instead (14). We have observed that PU.1 levels are involved in determining whether myeloid progenitors develop into macrophages or granulocytes (15). PU.1 interacts with several important transcription factors involved in the specification of cell fates: GATA-1, C/EBPα and BSAP (16-21). All of these factors inhibit PU.1's ability to activate transcription of specific promoters. For GATA1 and C/EBPα it has been proposed that the ratios of PU.1 to these factors in uncommitted hematopoietic progenitors are important in determining cell fate decisions (15,19,20).

Here we show that Gfi-1 physically interacts with PU.1 and represses its transactivation capability. Gfi-1 specifically blocks PU.1- induced macrophage differentiation of a myeloid cell line and exogenous Gfi-1 expression in primary hematopoietic progenitors increases granulocyte differentiation. Lastly we examined bone marrow from Gfi-1^-/-PU.1^+/- mice, and observed that decreasing PU.1 dosage reduced the mixed-myeloid lineage phenotype of Gfi-1 deficient cells. Our data suggest that Gfi-1 antagonism of PU.1 is critical for downregulating macrophage gene expression in developing granulocytes.

Experimental Procedures

Reporter constructs and expression plasmids

Human M-CSFR promoter was isolated by polymerase chain reaction using K562 DNA as template. The -416 to +124 human M-CSFR promoter was amplified with a previously described primer set (22) and the resultant product was subcloned into pGL3 (Promega). The pcDNA3-PU.1, MigR1-C/EBPα, MigR1-PU.1, (GAL4)₅-luciferase plasmid and GAL4-PU.1 fusion plasmids have been previously described (15,23). The rv-GFP, rv-Gfi-1 and rv-Gfi-1 P to A viral constructs (24) were provided by Dr. Jinfang Zhu (NIH). Plasmid pSport-Gfi-1 was obtained from ATCC, Image clone #5585794.

Transient transfections

U937 cells were electroporated as previously described (25) using 25 ug of luciferase reporter plasmid, 50 ug rv-Gfi-1 plasmid and 5 ug thymidine kinase promoter renilla luciferase (pRL-tk) plasmid. Total amount of plasmid DNA was kept constant with rv-GFP. 293T cells were transfected using the BD CalPhos mammalian transfection kit (BD Biosciences). For M-CSFR promoter assays, cells were co-transfected with 2 ug pGL3-MR, 4ug MigR1-PU.1 or MigR1-C/EBPα, 4 or 8 ug rv-Gfi-1, and 25 ng pRL-tk. For GAL4 regulated promoter assays cells were transfected with 5 ug (GAL4)₅-luciferase, 2.5 ug GAL4DBD, GAL4-full length PU.1 or GAL4-PU.1 transactivation domain, 4 ug rv-Gfi-1 or rv-Gfi-1P/A, and 25 ng pRL-tk. Total amount of plasmid was kept constant with appropriate empty expression plasmids. 48h post-transfection cell lysates were harvested using Promega cell lysis buffer. Firefly and renilla luciferase activity was measured using the Dual-Luciferase Assay System (Promega). All samples were done in triplicate and firefly luciferase values were normalized to renilla luciferase values.

In vitro binding assays

Glutathione-S-transferase (GST) fusion proteins were prepared as described (23). Bacterial cells expressing GST-FL Gfi-1 (aa 1-423), GST-NT Gfi-1 (aa 1-258), and GST-CT Gfi-1 (aa 193-423) were obtained from Dr. H.L. Grimes (University of Cincinnati). GST-Znfs3-5 and GST-ΔZnf3-6 were constructed by PCR with specific primers and subcloning of the PCR product into pGEX-4T3. GST-Gfi-1 fusion proteins bound to glutathione agarose were incubated in NETN buffer (20mM Tris-HCl, pH 8.0, 200mM NaCl, 1mM EDTA, and O.5%NP-40) with in vitro translated PU.1 proteins prepared by the TNT reticulocyte lysate system (Promega). Plasmids containing PU.1 deletion mutants have been described and were provided by Dr. M. Atchison (University of Pennsylvania) (26). After 2h incubation bound complexes were washed 4 × in NETN buffer. Bound complexes were eluted in sample buffer and separated by SDS-PAGE. Bound proteins were visualized by autoradiography.

Co-immunoprecipitations

293T cells were transfected with 5 ug pcDNA3-PU.1 and/or pSport-Gfi-1 using lipofectamine 2000 (Invitrogen) according to manufacturers instructions. Forty-eight hours post-transfection whole cell lysates were prepared with cell lysis buffer (50mM Tris-HCl, pH 7.5, 100mM NaCl, 5mM EDTA, 1mM ZnSQ4, 5% glycerol, 1% NP40 and 1% Sodium deoxycholate). Lysates were incubated with 2 ul of monoclonal anti-PU.1 antibody (BD Pharmingen) or 2ul of anti-Gfi-1 (Santa Cruz, N20) and Protein A agarose (Invitrogen) overnight at 4°C. Agarose beads and captured protein complexes were washed 5× in lysis buffer. Protein lysates were eluted in SDS-PAGE sample buffer, separated by SDS-PAGE, and immunoblotted. For U937 immunoprecipitations 1× 10⁷ cells were lysed. Lysates were incubated either with anti-PU.1 or anti-tubulin. Immunoprecipitates were then analyzed as described above for transfected 293T cells.

PUER cells differentiations

The generation of PUER progenitor cells and their differentiation into macrophages and granulocytes is described in detail elsewhere (15,27). Cells were maintained in IMDM, 10% fetal calf serum (Hyclone), 1U/ml penicillin/ streptomycin, 2mM L-glutamine, and 50uM β-mercaptoethanol. Media contained either 5 ng/ml IL-3 or 10 ng/ml G-CSF (R&D systems). Cells were differentiated by adding 100 nM of OHT to the media. For morphological analysis of PUER cells 2.5 ×10⁵ cells were cytocentrifuged onto glass slides, fixed for 30 seconds in methanol, and stained with Wright stain.

Isolation of murine hematopoietic progenitors

The procedure was performed as described previously with minor modifications (14,28). Briefly, bone marrow cells were isolated from tibias and femurs of 8-week old mice. Mature erythroid cells were removed by ammonium chloride lysis. For hematopoietic colony assays 2.5 × 10⁴ nucleated bone marrow cells were plated into methylcellulose medium containing hematopoietic cytokines (Methocult GF 3434, Stem Cell Technologies). Colonies were counted after 7 days of incubation. For retroviral transduction nucleated cells were lineage depleted with StemSep murine hematopoietic progenitor enrichment kit according to manufacturers instructions (Stem Cell). The use of mice in these experiments was approved by the University of New Mexico LACUC (protocol # 0409).

Retroviral transductions

Retroviral vectors, rv-GFP, rv-Gfi-1 or rv-Gfi-1 P to A, were co-transfected into 293T cells together with the retroviral packaging vector pCL-Eco (Imgenex) using calcium phosphate precipitation. Forty-eight hours post-transfection retroviral supernatants were harvested. PUER and primary progenitor cells were infected by resuspending in retroviral supernatant containing polybrene (8 μg/ml) and centrifuging at 2000 × g for 2h at 25°C. PUER cells were re-cultured in fresh media containing IL-3 (R&D systems). Cell lines expressing high levels of GFP were obtained by limiting dilution cloning. Infected primary Hematopoietic progenitors were expanded in the presence of IL-3, IL-6 and SCF for 2 days and subsequently cultured for 4 additional days in G-CSF.

Chromatin immunoprecipitations

Chromatin immunoprecipitations were performed as described (29). Briefly, 2.0 × 10⁷ cells were crosslinked with 0.4% formaldehyde for 10 minutes at room temperature and the reaction was subsequently stopped with 0.125M glycine. Cells were washed in PBS and then lysed in cell lysis buffer. Nuclei were recovered by centrifugation and then lysed in nuclei lysis buffer. Chromatin was sonicated and precleared overnight with 50ul of normal rabbit serum and 30ul of protein A/G agarose (Invitrogen). Precleared lysate was incubated with normal rabbit serum, anti-PU.1 antibody (sc-352, Santa Cruz Biotechnology), or anti-Gfi-1 antibody (sc-8558, Santa Cruz Biotechnology) pre-bound to protein A/G agarose. An aliquot of precleared lysate was saved for input. Immunoprecipitates were washed and then eluted with 100mM NaHCO3, 1%SDS. Crosslinks were reversed at 65°C for 4 h, and protein digested with proteinase K. Isolated DNA was purified by phenol/ chloroform extraction and ethanol precipitation. DNA pellets were resuspended in sterile H2O. The M-CSFR and GlutR2 (negative control) promoters were amplified by PCR.

RNA analysis

RNA was isolated from PUER and bone marrow cells using TRIzol (Gibco BRL). One ug of total cellular RNA was used in a 20 ul reverse transcriptase reaction (Superscript III, Invitrogen). Two ul of the reverse transcriptase reaction were used in a PCR reaction with gene specific primers. For real-time PCR 1ul of prepared cDNA was used in a 20ul real-time PCR reaction (Taqman assay) using gene specific probes obtained from Applied Biosystems. The results were normalized to the levels of β actin expression, which was assayed in the same reaction tube. Samples were run in triplicate with a DNA Engine Opticon thermocycler (BioRad). Northern blots were prepared with 15ug of total RNA for each sample. Blots were sequentially probed with random hexamer primed ³²P labeled cDNA encoding murine myeloperoxidase, and β tubulin.

Flow cytometry

Single cell suspensions were prepared and stained with monoclonal antibodies obtained from Pharmingen (Mac3-FITC) and Caltag (Gr1-PE, F4/80-PE, CD11b-biotin). Stained cells were analyzed on a dual laser cell sorter (FACScalibur, Becton Dickinson). Cell preparations were preincubated with antibody to FcγRII/III to reduce non-specific antibody binding and were subjected to propidium iodide uptake to exclude dead cells from the analysis. FACS data was analyzed using FloJo software (Tree-Star).

Results

Gfi-1 inhibits trans activation from the M-CSFR promoter

Because Gfi-1 deficiency results in increased myeloid expression of M-CSFR mRNA, we determined if Gfi-1 represses M-CSFR promoter activity (22). U937 cells were transfected with a 540 base pair M-CSFR promoter luciferase construct in the presence and absence of Gfi-1 expression plasmid. We observed that Gfi-1 repressed M-CSFR promoter activity more than 2-fold in this myeloid cell line (Fig. 1A). To determine if Gfi-1 targeted PU.1 in order to repress the M-CSFR promoter, we repeated the reporter assay in 293T cells. The M-CSFR promoter construct is activated in non-hematopoietic cells by co-transfection of the PU.1 and/or C/EBPα transcription factors (30-32). In transfected 293T cells, PU.1 transactivated the M-CSFR promoter construct 14-fold compared to cells transfected with the reporter plasmid alone. However, if Gfi-1 was co-transfected with PU.1, promoter activity was decreased to approximately 5-fold (Fig. 1B). We did not observe a significant affect on transcription when the M-CSFR promoter construct and Gfi-1 were co-transfected in the absence of PU.1. Additionally Gfi-1 did not significantly repress M-CSFR promoter activity induced by C/EBPα (Fig. 1C). Since there was a slight but reproducible transcriptional synergy between Gfi-1 and C/EBPα, we tested whether Gfi-1 repressed MCSFR promoter activated by both PU.1 and C/EBPα. When PU.1 and C/EBPα activated the MCSFR promoter, transcription was still repressed by Gfi-1 (Fig. 1D). This result indicated that C/EBPα does not abrogate Gfi-1's repression of PU.1.

FIGURE 1. Gfi-1 inhibits M-CSFR promoter activity induced by PU.1.

(A) Transient transfection of U937 cells with a 540bp M-CSFR promoter luciferase construct in the presence or absence of rv-Gfi-1 plasmid. The decrease in transcriptional activity induced by Gfi-1 is statistically significant (p<0.0001). (B) M-CSFR promoter activity induced in 293T cells with co-transfection of MigR1-PU.1 in the presence or absence of increasing amounts of rv-Gfi-1 plasmid. The decrease in transcriptional activity induced by Gfi-1 is statistically significant (p<0.05). The differences in reporter activity between M-CSFR reporter transfected in the presence or absence of Gfi-1 (no PU.1 expression plasmid) was not statistically significant. (C) M-CSFR promoter activity induced in 293T cells by co-transfected MigR1-C/EBPα in the presence or absence of rv-Gfi-1 plasmid. The observed increase in transcriptional activity induced by Gfi-1 was statistically significant (p<0.02). (D) 293T cells co-transfected with MigR1-PU.1, MigR1-C/EBPα, and rv-Gfi-1. Gfi-1 represses transcription mediated by both PU.1 and C/EBPα. This repression was statistically significant (p<0.002). For (A) luciferase activity is reported as relative light units. For (B, C and D) luciferase activity is reported as fold-induction above the activity seen in 293T cells transfected with only the reporter plasmid and rv-GFP. Total DNA content for all transfections was kept constant with rv-GFP plasmid. All transfections also contained the renilla luciferase plasmid pRL-tk. Luciferase values were determined 48h post-transfection, and were normalized to renilla luciferase values. Luciferase values are the mean +/- standard error of the mean of three independent transfections.

The results from the M-CSFR promoter assays suggested that Gfi-1 inhibition is mediated by the repression of PU.1. To confirm these observations, we determined if Gfi-1 affects PU.1 transactivation when PU.1 is tethered to DNA by a heterologous DNA binding domain. Full-length PU.1 was fused to the GAL4 DNA binding domain and co-transfected into 293T cells with a luciferase reporter gene regulated by five upstream GAL4 binding sites. The GAL4-PU.1 protein (G4PU.1) transactivated the reporter over 14-fold above the activity induced by the GAL4 DNA binding domain alone (G4DBD, Fig. 2A). However, similar to results obtained with the M-CSFR promoter, this activity was reduced 7-fold in the presence of Gfi-1. This effect was specific to PU.1, as Gfi-1 had no effect on transcription induced by a fusion protein of GAL4 and the activation domain of the unrelated transcription factor HIF-3α (G4HIF3AD). Interestingly Gfi-1 did not repress a fusion between GAL4 and the PU.1 transactivation domain (G4PUAD, Fig. 2B).

FIGURE 2. Gfi-1 inhibits the activity of a fusion protein between full-length PU.1 and the GAL4 DNA binding domain.

Transient transfections of 293T cells with a reporter construct containing five GAL4 sites upstream of a minimal tk promoter driving luciferase expression. (A) Cells were co-transfected with either GAL4 DNA binding domain (G4DBD), GAL4-full length PU.1 (G4PU.1) or GAL4-Hif3 activation domain (G4HIF3AD) expression plasmid in the presence or absence of rv-Gfi-1 plasmid. Gfi-1's repression of transcriptional activity induced by GAL4-PU.1 was statistically significant (p<0.0001). (B) Cells were co-transfected with a GAL4 DNA binding domain or GAL4-PU.1 transactivation domain (G4PUAD) expression plasmid in the presence or absence of rv-Gfi-1. The slight enhancement of transcription by Gfi-1 was statistically significant (p<0.05) (C) GAL4-full-length PU.1 was co-transfected either with either rv-Gfi-1 or rv-Gfi-1 P to A plasmid. Rv-Gfi-1 P to A encodes a form of Gfi-1 that contains an inactivating mutation in the repressive SNAG domain. The difference in activity observed between Gfi-1 and Gfi-1 P to A transfected cells was statistically significant (p<0.001). Total DNA content for all transfections was kept constant with rv-GFP plasmid. All transfections contained the renilla luciferase plasmid pRL-tk. Luciferase values were determined 48h post-transfection, and were normalized to renilla luciferase values. Luciferase activity is the mean +/- standard error of the mean of three independent transfections, and luciferase activity is reported as fold-induction above the activity seen in 293T cells transfected with the GAL4 DNA binding domain expression plasmid.

Since Gfi-1 inhibited PU.1 activity when PU.1 was tethered to DNA via the GAL4 DNA binding domain, we concluded that Gfi-1 did not block PU.1 activity via inhibition of DNA binding (Also see Fig. 4D). Since Gfi-1 contains an amino terminal repression domain (SNAG domain), we tested whether this domain was necessary for antagonizing PU.1. Mutating the second amino acid of Gfi-1 from a proline to alanine abrogates its repression activity (33). When Gfi-1 P to A was analyzed for its ability to inhibit PU.1 activity, a significant decrease in repressive activity compared to wildtype Gfi-1 was observed (Fig. 2C). This result demonstrated that the Gfi-1 SNAG domain is necessary for inhibiting PU.1 transactivation. The results from the GAL4 and M-CSFR reporter assays demonstrate that Gfi-1 inhibits PU.1 activation of two independent promoters in vitro.

FIGURE 4. Gfi-1 blocks macrophage differentiation of PUER myeloid progenitor cells.

(A) Immunoblot of Gfi-1, PU.1 and β actin expression in PUER cells that were superinfected with rv-GFP or rv-Gfi-1 virus. Cell lines were either undifferentiated (no OHT) or differentiated (100nM OHT) for eight days in the presence of IL-3. (B) PUER (GFP and Gfi-1) cell lines were induced to differentiate with 100nM OHT for eight days and subsequently cytocentrifuged. Cells were Wright stained to evaluate morphology. Magnification 40× (C) Immunoblot analysis of M-CSFR expression in whole cell lysates obtained from OHT treated PUER cells infected with either rv-GFP or rv-Gfi-1 retrovirus. Whole cell lysates were prepared from PUER cells at 0,1,2,4, and 8 days after OHT addition. (D) Chromatin immunoprecipitation (ChIP) of the M-CSFR promoter from PUER cells using anti-PU.1 antibody. PUER cell lines were either untreated or treated for 7 days with OHT as indicated. (E) ChIPs as described above except anti-Gfi-1 was used to immunoprecipitate. (F) RT-PCR analysis of RNA obtained from control PUER cells (GFP) and cells expressing Gfi-1. Cells were either untreated or treated with 100nM OHT for 8 days.

Gfi-1 associates with PU.1

Results from the transient transfection assays suggested that Gfi-1 represses transcription by directly binding to PU.1. To determine if Gfi-1's inhibition of PU.1 is mediated by protein-protein interaction, we performed co-immunoprecipitation assays. 293T cells were transfected with Gfi-1 and/or PU.1 expression vector(s). Whole cells extracts were prepared and immunoprecipitated with either anti-PU.1 or anti-Gfi-1 antibody. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with a PU.1 or Gfi-1 antibody to assay for co-immunoprecipitation. PU.1 was detected in anti-Gfi-1 immunoprecipitated complexes (Fig. 3A, upper panel lane 2) obtained from cells co-expressing PU.1 and Gfi-1. Some non-specific precipitation of PU.1 occurred in the absence of PU.1 (lane 3), however significantly more PU.1 was immunoprecipitated in the presence of Gfi-1 indicating that these proteins interact in vivo. Similarly when the same extracts were immunoprecipitated with an anti-PU.1 antibody, Gfi-1 was detected in the immunoprecipitated complexes in cells co-expressing PU.1 and Gfi-1 (Fig. 3A, lower panel).

FIGURE 3. Gfi-1 interacts with PU.1.

(A) 293T cells were transfected with PU.1 and Gfi-1 expression plasmids as indicated. Whole cell lysates were prepared and subjected to immunoprecipitation with either PU.1 or Gfi-1 antibody. Immunoprecipitated complexes were separated by SDS-PAGE. Immunoblots were prepared and probed with anti-PU.1 antibody or anti-Gfi-1 as indicated. (B) U937 cell extracts were prepared and immunoprecipitated with either anti-PU.1 antibody or non-specific antibody (anti-tubulin). 10% input and immunoprecipitates were immunoblotted and probed with anti-Gfi-1 antibody. (C) Full-length ³⁵S-methionine labeled PU.1 was incubated with GST, GST full-length (FL) Gfi-1, GST N-terminal (NT) Gfi-1, GST Gfi-1 lacking the last 4 zinc fingers (deltaZnfs3-6) GST Gfi1 zinc finger 3, 4, and 5 (Znfs3-5) or GST C-terminal (CT) Gfi-1 Binding of PU.1 to different GST-Gfi1 proteins was detected by autoradiography. (D) ³⁵S-methionine labeled in vitro translated PU.1 deletion mutants were incubated with GST-Gfi-1 protein. Protein complexes were washed and separated by SDS-PAGE. Binding was detected as described above.

To determine if endogenous PU.1 and Gfi-1 proteins co-immunoprecipitated, we performed immunoprecipitations on lysates obtained from U937 cells. Both PU.1 and Gfi-1 are detected in extracts of this myeloid cell line (unpublished observation). Lysates were prepared from U937 cells and incubated with either anti-PU.1 antibody or non-specific antibody (anti-tubulin). Protein complexes were isolated, immunoblotted, and probed with anti-Gfi-1 antibody. Immunoreactive Gfi-1 bands were detected in whole cell extract and in the anti-PU.1 immunoprecipitation, but not in the lane precipitated with non-specific antibody (Fig. 3B).

The Gfi-1 domain(s) required for mediating the interaction with PU.1 was identified by incubating in vitro translated (IVT) PU.1 protein with GST fusions to Gfi-1 deletion mutants (Fig. 3C). Consistent with the immunoprecipitation experiments, full-length Gfi-1 associated with PU.1 in vitro. PU.1 also bound a fusion protein containing all six Gfi-1 zinc fingers but not to a fusion containing the N-terminal portion of Gfi-1 lacking all of the zinc fingers. This result suggested that the zinc fingers were necessary and sufficient to mediate Gfi-1 binding to PU.1. To further narrow the region of Gfi-1 required for binding to PU.1 we incubated PU.1 protein with a GST fusion to the region of Gfi-1 containing zinc fingers 3 through 5 (Znfs3-5) and to a fusion containing Gfi-1 truncated after the second zinc finger (ΔZnfs3-6). PU.1 did not bind to a GST-Gfi-1 deleted of its 4-carboxy terminal zinc fingers (ΔZnfs3-6), however it did bind to the GST-Gfi-1 containing only zinc fingers 3-5 (Znfs3-5). Interestingly these are the zinc fingers, which are sufficient to mediate DNA binding(1).

To determine which PU.1 domain(s) interacts with Gfi-1, IVT PU.1 deletion mutants were incubated with GST-Gfi-1. Deletion of the N-terminus (Δ7-30), C-terminus (Δ255-272), or the PEST domain (Δ118-160) did not affect PU.1 binding to Gfi-1. However, deletion of the transactivation domain of PU.1 (Δ33-100) or the PU.1 Ets domain (Δ201-272) greatly diminished binding to Gfi-1 (Fig. 3D). Our results demonstrate that both the transactivation domain and the Ets domain are required for PU.1 to efficiently associate with the zinc finger DNA binding domain of Gfi-1.

PU.1 dependent macrophage differentiation is blocked by Gfi-1

We next determined if Gfi-1 has effects on PU.1's biological activity by expressing Gfi-1 in a PU.1^-/- cell line expressing a conditional version of PU.1 (PUER) (27). When the PUER protein is activated by tamoxifen (OHT), the conditional PU.1 protein induces differentiation of the myeloid progenitor cell line into macrophages and granulocytes depending on the growth conditions (15). To test what effect Gfi-1 has on PU.1- induced differentiation, PUER cells were superinfected with a Gfi-1-IRES-GFP (rv-Gfi-1) retrovirus or a control GFP only (rv-GFP) retrovirus. Clonal ceil lines were generated by limiting dilution.

Cells were grown in IL-3 and 100nM OHT for eight days (macrophage conditions). PUER GFP cells initially expressed endogenous Gfi-1, however upon OHT treatment Gfi-1 expression was extinguished. In contrast Gfi-1 expression was still detected in OHT treated PUER Gfi-1 cells (Fig. 4A). Gfi-1 expression did not affect PUER protein levels as evaluated by western blot. The majority of PUER GFP cells became adherent during the course of OHT treatment, whereas the majority of PUER Gfi-1 cells remained non-adherent. To determine if macrophage development was blocked by Gfi-1, cell morphology was examined by cytocentrifugation and histochemical staining (Fig 4B. OHT-treated PUER GFP cells had the morphology characteristic of macrophages: large cells, vacuolated cytoplasm, and small nucleus. However PUER Gfi-1 cells looked similar to untreated cells. Additionally rv-Gfi-1-infected cells had reduced expression of the macrophage antigen F4/80 compared to rv-GFP-infected cells (Fig. S1A, B).

Since Gfi-1 represses M-CSFR promoter activity in vitro, we further examined expression of M-CSFR protein. In GFP- infected cells, M-CSFR was detected by immunoblot after four days of differentiation. However, M-CSFR was barely detected in Gfi-1- infected cells after eight days of differentiation, consistent with the previous in vitro results (Fig. 4C). We also determined whether Gfi-1 affected PU.1's binding to the endogenous MCSFR promoter. Gfi-1 did not block PU.1 association with the MCSFR promoter as chromatin immunoprecipitation (ChIP) demonstrated that PU.1 associates with the promoter in the presence or absence of Gfi-1 (Fig. 4D). Lastly we examined whether Gfi-1 directly associated with the MCSFR promoter. Using ChIP we detected the MCSFR promoter co-immunoprecipitating with Gfi-1 in PUER Gfi-1 cells induced to differentiate with OHT (Fig. 4E). As expected PUER GFP cell treated with OHT, which do not express detectable levels of Gfi-1 protein, did not precipitate the MCSFR promoter with anti-Gfi-1 antibody. ChIP assays with untreated PUER GFP or PUER Gfi-1 cells did not consistently immunoprecipitate MCSFR above background levels (immunoprecipitations with normal rabbit serum). To show that precipitation of the M-CSFR promoter by anti-PU.1 and anti-Gfi-1 are specific, we tried to amplify glutamate receptor 2 (GlutR2) promoter as a non-specific control from our immunoprecipitations and could not detect it in any of our samples (Fig. S1C).

The expression of several myeloid genes was examined in PUER GFP and Gfi-1 cells by RT-PCR (Fig. 4F). These genes are direct transcriptional targets of PU.1: CD64, M-CSFR, CD11b and c-fes (22,25,34,35). Gfi-1 efficiently repressed the expression of CD64 and M-CSFR, which are expressed preferentially in macrophages. In contrast, Gfi-1 barely repressed the expression of CD11b or c-fes, which are transcribed in both granulocytes and macrophages. The expression levels of CD11b and M-CSFR were confirmed by real-time RT-PCR analysis (Fig. S1D). These results suggest that Gfi-1 preferentially represses PU.1 macrophage target genes, but not PU.1 targets expressed in both macrophages and granulocytes. Interestingly the PU.1 target genes c-fes (25) and CD11b (35) were abundantly expressed in the absence of OHT. CD11b has been observed previously in untreated PUER cells (-OHT) due to slight leakiness of the PUER protein (27). When CD11b and c-fes expression was examined 24 hrs post-OHT treatment, we observed a slight induction of both genes above levels detected in untreated cells (data not shown). However, the induction was not greatly affected by Gfi-1 expression (data not shown).

Gfi-1 does not block PUER induced granulocyte commitment

When PUER cells are pre-treated with G-CSF before OHT activation of PU.1, they differentiate into granulocytes instead of macrophages upon activation of the PUER protein (15). PUER cells do not fully differentiate into mature granulocytes as their nuclei do not completely segment and although they express the secondary granule protein neutrophil gelatinase we have never detected lactoferrin (Dahl et al. unpublished). PUER GFP and Gfi-1 cells were differentiated under granulocyte conditions to determine if enforced Gfi-1 expression blocked only macrophage development or all myeloid development. Differentiation was evaluated by examining the morphology of cells after cytocentrifugation and histochemical staining. Both GFP- and Gfi-1- infected PUER cells appeared by gross morphology to be neutrophillic granulocytes with characteristic segmented nuclei and were indistinguishable from one another (Fig 5A). RNA was prepared from GFP- and Gfi-1-infected granulocyte cultures and RT-PCR was performed for the granulocyte genes myeloperoxidase (MPO), neutrophil gelatinase and gp91. Results obtained from representative GFP and Gfi-1 expressing cell lines are shown. Neutrophil gelatinase and gp91 expression were detected in PUER GFP differentiated cultures, but not in undifferentiated cells (Fig. 5B). Similar results were obtained in Gfi-1 expressing cells, indicating that Gfi-1 expression is permissive for PU.1 induced granulocyte differentiation. MPO is a primary granule protein whose expression is greatly enhanced in Gfi-1^-/- hematopoietic cells along with another primary granule protein, neutrophil elastase (ELA2) (10). Consistent with Gfi-1 knockout data, MPO expression was reduced in PUER cells by exogenous Gfi-1 as determined by northern blot (Fig. 5C).

FIGURE 5. Gfi-1 does not block neutrophil differentiation induced by PU.1.

(A) rv-GFP and rv-Gfi-1 infected PUER cells were grown under neutrophil differentiating conditions. Cells were subsequently cytocentrifuged and stained with May-Grunwald-Giemsa. (B) RT-PCR and (C) northern blot analysis were performed on RNA prepared from rv-GFP and rv-Gfi-1 infected PUER cells. IL-3 indicates cells grown in the cytokine IL-3. A + mark indicates that cell were treated with 100nm OHT. G indicates cells Grown in the cytokine G-CSF.

Infection of primary murine bone marrow progenitors with a Gfi-1 retrovirus promotes granulocyte differentiation

Bone marrow was harvested from 8-10 week old wildtype mice. Nucleated bone marrow cells were depleted of lineage positive cells (CD5, CD11b, Gr-1, B220, and Terrl 19) and infected for 2 days with the rv-Gfi-1 retrovirus in the presence of the cytokines IL-3, IL-6, and SCF. Cells were grown an additional 4 days in G-CSF before being analyzed for differentiation by flow cytometry (Fig. 6). We have previously shown that cultured bone marrow cells expressing cell surface CD11b and F4/80 are macrophages and cells expressing CD11b and Gr-1 are granulocytes (15).

FIGURE 6. Gfi-1 increases granulocyte differentiation of primary progenitors.

Nucleated bone marrow cells were lineage depleted to enrich for hematopoietic progenitors and infected with (A) rv-Gfi-1 or (B) rv-Gfi-1 P to A retrovirus for 2 days in the presence of SCF, IL-6, and IL-3. Cells were cultured 4 additional days in G-CSF and analyzed by FACS for expression of GFP, CD11b, Gr-1 and F4/80. Cells were gated on two populations: GFP^low and GFP^high. Upper panels show expression of CD11b and Gr-1, and the bottom panels show expression of CD11b and F4/80. Sorting of populations has shown previously that CD11b⁺Gr-1⁺ cell are granulocytes and CD11b⁺F4/80⁺ cells are macrophages (Dahl et al., 2003).

When we gated on cells expressing no or low levels of GFP (corresponding to low levels of Gfi-1 since the two protein are expressed from the same mRNA), approximately 60% of the cells were granulocytes and 40% of the cells were macrophages as determined by CD11b and Gr-1 staining (Fig. 6A, upper panels). However if we gated on the GFP high population (high Gfi-1 levels), 80% of the cells were granulocytic and only 20% were macrophages. Similar results were obtained by evaluating differentiation with CD11b and F4/80 staining (Fig. 6A, lower panels). Since we observe an increased percentage of the CD11b⁺ cell population becoming Gr-1⁺ in the GFP^high fraction, we conclude that Gfi-1 is increasing granulocyte differentiation at the expense of the macrophage population. Results shown are representative of three independent infections.

This increase in granulocytic differentiation requires an active SNAG repressor domain, as the Gfi-1 P to A inactivating mutant (33) did not increase granulocytic differentiation. Interestingly the Gfi-1 P to A mutant induced a mixed lineage phenotype with approximately 90% of infected cells (high GFP) co-expressing Gr1 and F4/80 (Fig 6B). This result is consistent with knock-in studies that show that when the P to A mutant is knocked into the Gfi-1 locus it phenocopies the null mutant animals (36). Lastly no differences in myeloid differentiation were observed between GFP^low and GFP^high populations of bone marrow progenitors infected with rv-GFP control virus (data not shown).

PU.1 heterozygosity decreases the mixed lineage phenotype of Gfi-1^-/- ' hematopoietic cells

To determine if antagonism of PU.1 activity by Gfi-1 had functional consequences in vivo we interbred PU.1 and Gfi-1 mutant mice. We performed hematopoietic colony assays with bone marrow obtained from wildtype, Gfi-1^-/-, and Gfi-1^-/-PU.1^+/- mice. Compared to wildtype we observed a significant increase in CFU-M (P<0.05) and CFU-G (P<0.02) colonies obtained from Gfi-1^-/- bone marrow. The Gfi-1^-/- and Gfi-1^-/-PU.1^+/- CFU-G colonies did not contain mature cells, as RT-PCR from colony RNA did not detect expression of either neutrophil gelatinase or lactoferrin (data not shown). Similar to our previous studies with CSF3 deficient mice PU.1 heterozygosity resulted in increased CFU-G colonies compared to CFU-M colonies when expressed on a neutropenic background (CSF3^-/- or Gfi-1^-/-, Fig 7A)(15).

FIGURE 7. PU.1 heterozygosity reduces the mixed lineage phenotype of Gfi-1^-/- hematopoietic cells.

(A) Hematopoietic colony assays were performed with nucleated bone marrow harvested from 8-week old mice. 25,000 cells from each animal were plated into methylcellulose media containing SCF, IL-3, IL-6, and Epo. Duplicate cultures were prepared and after 7 days of culture hematopoietic colonies were scored for number and types of colonies. Average number of colonies and the standard error are shown for each genotype assayed. 6 wildtype, 3 Gfi-1^-/-, and 6 Gfi-1^-/-PU.1^+/- animals were examined in this assay. (B) Realtime RT-PCR analysis of M-CSFR, CD11b, and ELA2 expression in nucleated bone marrow obtained from WT, PU.1^+/-, Gfi-1^-/-, and Gfi-1^-/-PU.1^+/- mice. Expression was normalized to β-actin and show as fold difference from levels detected in wildtype mice. 3 mice for each genotype were examined. * indicates a statistical difference between the levels of M-CSFR detected in Gfi-1^-/- and Gf-1^-/-PU.1^+/- samples (P<0.05) (C) Flow cytometry analysis of nucleated bone marrow isolated from 8 week old mice. Representative FACs plots obtained from Mac3 and Gr-1 analysis of wildtype, Gfi-1^-/- and Gfi-1^-/-PU.1^+/- cells is shown.

The expression of MCSFR, CD11b and ELA2 were examined by real-time RT-PCR from wildtype, PU.1^+/-, Gfi-1^-/- and Gfi-1^-/-PU.1^+/- bone marrow. As previously observed MCSFR and ELA2 were overexpressed in Gfi-1 mutant marrow (Fig. 7B)(10). Interestingly levels of MCSFR but not the pan-myeloid gene CD11b were significantly reduced in the compound mutant (Gfi-r^-/-PU.1^+/-) marrow compared to the Gfi-1^-/- marrow (P<0.05). The granulocyte gene ELA2 was slightly increased in the compound mutant animals compared to the Gfi-1 mutants but this was not statistically significant. Three mice of each genotype were examined. This decrease in the compound mutant does not appear to be due to a general reduction in PU.1 targets by PU.1 heterozygosity since we did not observe a significant decrease in PU.1^+/- mice analyzed. Although PU.1 heterozygosity rescues the overexpression of the macrophage targets it did not rescue the expression of the granulocyte genes neutrophil gelatinase or lactoferrin (Supplementary data, Fig S2B).

Lastly Hock et al. observed that Gfi-1 mice exhibit increased numbers of mixed lineage Mac3⁺Gr-1⁺ cells in their bone marrow (10). Using flow cytometry we examined wildtype, Gfi-1^-/- and Gfi-1^-/-PU.1^+/- marrow for the presence of these cells. In 4 independent experiments performed we always observed a decrease in Mac3⁺Gr-1⁺ cells in the Gfi-1^-/-PU.1^+/- marrow compared to marrow isolated from Gfi-1 mutants. Representative FACS plots from one experiment are shown (Fig. 7C). These results demonstrate that decreased PU.1 gene dosage lowers the overexpression of macrophage genes in the Gfi-1 mutant and reduces the number of mixed lineage cells. This supports the conclusion that an important function for Gfi-1 in myelopoiesis is to antagonize PU.1.

Discussion

Since PU.1 target genes are overexpressed in myeloid cells obtained from Gfi-1^-/- mice (9,10), we examined whether the essential granulocyte transcription factor Gfi-1 antagonizes PU.1 activity. We observed that Gfi-1 inhibits PU.1's transactivation activity and physically interacts with PU.1. Inhibition required an intact amino terminal SNAG repression domain. The interaction between these two factors is functionally significant as Gfi-1 expression blocked PU.1-induced macrophage differentiation of a myeloid cell line. Although Gfi-1 blocked PU.1-induced macrophage differentiation of the PUER myeloid progenitor cell line, it did not block granulocyte commitment. We also observed that exogenous Gfi-1 expressed in primary murine hematopoietic progenitors, increased granulocyte differentiation at the expense of macrophage development. Previously the related protein Gfi-1b was shown to block differentiation of the myelomonocytic M1 cell line (37). However we report for the first time that Gfi-1 inhibits macrophage differentiation of a myeloid cell line and primary hematopoietic cells.

These results suggest that Gfi-1 may be important in the GMP decision to become a macrophage or granulocyte by antagonizing PU.1 activity similar to what we previously hypothesized for C/EBPα (15). However in the absence of Gfi-1, hematopoietic cells commit to the granulocyte cell fate as CFU-G progenitors are generated in the bone marrow as shown by our hematopoietic colony assays (Fig. 7A). Importantly we do not observe a significant change in the ratio of CFU-G to CFU-M progenitors between wildtype and Gfi-1 mutant animals. We observed an increase in the total number of both CFU-G and CFU-M colonies, which is likely due to the increased percentage of GMP cells in the bone marrow of Gfi-1^-/- mice (6). If Gfi-1 were playing a role in the granulocyte/macrophage cell fate decision one would expect that its deletion would result in an increase in CFU-M progenitors relative to the CFU-G. Retroviral expression results may be due to an effect of Gfi-1 being misexpressed in common myeloid progenitors (CMPs). Annexin V staining did not show that Gfi-1 expression induced apoptosis of macrophage progenitors (Data not shown).

We did not demonstrate a role for Gfi-1 in directing cell fate decisions by repressing PU.1. However we suggest that Gfi-1 “locks in” the granulocyte cell fate by repressing macrophage genes regulated by PU.1. This is demonstrated by the reduction of MCSFR expression when the PU.1 gene dosage was reduced in a Gfi-1 null background but the expression of the pan-myeloid PU.1 target gene CD11b was unaffected. Additionally it was previously shown that Mac3⁺Gr-1⁺ mixed lineage cells are increased in Gfi-1 mutant bone marrow and we showed that there is a reduction in this cell population in Gfi-1^-/-PU.1^+/- animals (Fig 7C) (10).

Interestingly Gfi-1 efficiently repressed PU.1 macrophage specific target genes (MCSFR and CD64) but not targets that are expressed in both macrophages and granulocytes (CD11b, c-fes). It is unclear how Gfi-1 discriminates between repressing PU.1 specific macrophage target genes and PU.1 targets expressed in all myeloid cells (Fig. 4F). However one difference we observed in the expression of these two sets of genes is that PU.1 targets expressed in both macrophages and granulocytes require only low levels of PU.1 for their expression. Low PU.1 activity present in the PUER cell line in the absence of OHT is enough to induce their expression whereas it is not enough to activate the expression of macrophage specific targets M-CSFR and CD64. This suggests that PU.1 regulation of pan-myeloid and granulocyte-specific genes is mechanistically distinct from PU.1 regulation of macrophage-specific genes. Optimal regulation of the macrophage-specific genes M-CSFR and macrosialin require the transcription factor, c-Jun as a co-factor for PU.1 (38,39). In granulocytes c-Jun expression decreases during granulocyte differentiation suggesting that its role as a PU.1 co-factor is less important in granulocytes (40). We are currently investigating whether the requirement for c-Jun as a PU.1 co-activator confers specificity for Gfi-1 repression of PU.1 target gene subsets.

Targeted mutations in mice and naturally occurring mutations isolated from human neutropenic patients demonstrate that Gfi-1 is essential for granulocyte development (9-11). Therefore, an understanding of how Gfi-1 functions in granulocyte differentiation is important for the design of future targeted therapies for granulocyte disorders. Here we have shown that Gfi-1 binds PU.1 repressing its ability to activate transcription. Genetic data from interbreeding PU.1 and Gfi-1 mutants demonstrates that this interaction is important in vivo. The data supports a conclusion that a critical function of Gfi-1 is to repress PU.1 regulated macrophage specific genes for proper granulocyte development.

Supplementary Material

SD 1. FIGURE S1. Gfi-1 blocks macrophage differentiation of the PUER cell line.

(A) For F4/80 immunohistochemistry PUER cells were plated onto chamber slides (Nunc) and cultured for 8 days in IL-3 and 100nM OHT. Adherent cells were then fixed and stained with rat anti-mouse F4/80 (Caltag) using a Vectastain ABC-alkaline phosphatase kit (Vector Laboratories Inc.). (B) PUER cells were infected with either rv-GFP or rv-Gfi-1 retroviruses and differentiated for 8 days in 100nm OHT. F4/80 expression was analyzed by flow cytometry gating on the GFP- and GFP+ cell fractions. (C) Control for non-specific immunoprecipitation of genomic DNA in the chromatin immunoprecipitation (ChIP) assays. ChIP products from fig. 4D and E were used in a PCR amplification of the glutamate receptor 2 (GlutR2) promoter. (D) Realtime RT-PCR assay for CD11b and M-CSFR expression in undifferentiated and differentiated PUER GFP and Gfi-1 cell lines. The expression levels were normalized to β actin expression. CT values were obtained for M-CSFR and β actin. dCT values were obtained by subtracting the β actin CT value from the M-CSFR CT value. Lower dCT value correlates with higher expression.

SD 2. FIGURE S2: Expression of lactoferrin and neutrophil gelatinase in bone marrow RNA samples.

RT-PCR analysis from RNA prepared from nucleated bone marrow cells isolated from 8 week old mice. Representative results from two animals of each genotype are shown. At least three animals of each genotype were analyzed.

The abbreviations used are

Gfi-1

Growth factor independence-1

C/EBPα

CCAAT/enhancer binding protein alpha

GST

glutathione S-transferase

M-CSFR

macrophage-colony stimulating factor receptor

Hif3α

hypoxia inducible factor 3 alpha

ChIP

chromatin immunoprecipitation

HPRT

hypoxanthine guanine phosphoribosyl transferase

CFU-M

colony forming unit-macrophage

CFU-G

colony forming unit-granulocyte

OHT

hydroxy tamoxifen

CMP

common myeloid progenitor

CLP

common lymphoid progenitor

GMP

granulocyte-macrophage progenitor

IRES

internal ribosome entry site

GFP

green fluorescent protein