PU.1 induces myeloid lineage commitment in multipotent hematopoietic progenitors

Abstract

Little is known about the transcription factors that mediate lineage commitment of multipotent hematopoietic precursors. One candidate is the Ets family transcription factor PU.1, which is expressed in myeloid and B cells and is required for the development of both these lineages. We show here that the factor specifically instructs transformed multipotent hematopoietic progenitors to differentiate along the myeloid lineage. This involves not only the up-regulation of myeloid-specific cell surface antigens and the acquisition of myeloid growth-factor dependence but also the down-regulation of progenitor/thrombocyte-specific cell-surface markers and GATA-1. Both effects require an intact PU.1 transactivation domain. Whereas sustained activation of an inducible form of the factor leads to myeloid lineage commitment, short-term activation leads to the formation of immature eosinophils, indicating the existence of a bilineage intermediate. Our results suggest that PU.1 induces myeloid lineage commitment by the suppression of a master regulator of nonmyeloid genes (such as GATA-1) and the concomitant activation of multiple myeloid genes.

Figure 1

Phenotype of colonies transformed by E26–PU.1 virus. (A) Structure of the E26–WT and E26–PU.1 proviruses. In E26–PU.1, the PU.1 cDNA is translated from a bicistronic genomic RNA via an internal ribosomal entry site (IRES). (LTR) Long terminal repeat. (B) Distribution of cell types in colonies transformed by E26–WT and E26–PU-1. (Light-shaded bars) MEP21; (dark-shaded bars) EOS47; (solid bars) MYL51/2. Transformed colonies were pooled and analyzed by FACS for cell surface antigen expression. The average number of cells positive for each antigen, as determined in two independent experiments, is shown (no value deviated >4% from the mean). In the case of the E26–PU.1-transformed populations ∼15% weakly MYL51/2-positive cells were scored as negative. Staining with MEP26 (another MEP/thrombocyte marker) and anti-MHC II or 1C3 (myeloid lineage markers) antibodies gave similar results as for MEP21 and MYL51/2, respectively; surface markers for the erythroid, B-, and T-cell lineages could not be detected.

Figure 1

Phenotype of colonies transformed by E26–PU.1 virus. (A) Structure of the E26–WT and E26–PU.1 proviruses. In E26–PU.1, the PU.1 cDNA is translated from a bicistronic genomic RNA via an internal ribosomal entry site (IRES). (LTR) Long terminal repeat. (B) Distribution of cell types in colonies transformed by E26–WT and E26–PU-1. (Light-shaded bars) MEP21; (dark-shaded bars) EOS47; (solid bars) MYL51/2. Transformed colonies were pooled and analyzed by FACS for cell surface antigen expression. The average number of cells positive for each antigen, as determined in two independent experiments, is shown (no value deviated >4% from the mean). In the case of the E26–PU.1-transformed populations ∼15% weakly MYL51/2-positive cells were scored as negative. Staining with MEP26 (another MEP/thrombocyte marker) and anti-MHC II or 1C3 (myeloid lineage markers) antibodies gave similar results as for MEP21 and MYL51/2, respectively; surface markers for the erythroid, B-, and T-cell lineages could not be detected.

Figure 2

Induction of myeloid differentiation by an inducible PU.1 allele. (A) Structure of the inducible PU.1 allele and of the PU.1 reporter construct. The hormone-binding domain (HBD) of the human estrogen receptor (hER) was fused to the carboxyl terminus of the PU.1 protein, generating PUER. The reporter pPU3TK–LUC contains three PU.1-binding sites upstream of the HSV thymidine kinase minimal promoter (B) Cotransfection of PU.1/PUER with the reporter pPU3TK–LUC and pRSV–βGal control plasmid in the presence (solid bar) or absence (shaded bar) of βE. The luciferase/β-galactosidase ratios were expressed relative to those obtained with the PU.1 expression vector in the absence of βE. The average values of four determinations are shown; (error bars) standard deviations. (C) Effect of βE on the phenotype of MEPs transformed by E26–PUER. MEP clones transformed by E26–PUER were treated with βE (green graphs) or left untreated (red graphs), and the expression of MEP (MEP21, MEP26), eosinophil (EOS47), and myeloid (1C3, MYL51/2) specific antigens determined at the indicated timepoints. MHC II surface antigen was induced more slowly and only in a subset of the clones (data not shown). βE treatment had no detectable effect on E26–WT MEPs (blue graphs). No decrease in cell viability caused by PUER activation was observed. (D) FACS profiles of βE-treated E26–PUER–MEPs. MEP21 antigen (red graphs) and 1C3 antigen (green graphs) expression was determined at 2, 4, and 6 days after addition of βE. (c-Src) Control antibody (black graphs). Data are displayed as cell number (linear scale) plotted against fluorescence intensity (logarithmic scale).

Figure 3

Effect of PUER activation on GATA-1 protein and mRNA expression. (A) E26–PUER transformed MEPs (from a single clone) were treated with βE, and aliquots subjected to Western analysis at different time points with anti-GATA-1 antibody. (B) Pooled MEP clones (four clones) were treated with βE and RNA isolated after 2 and 4 days of induction, as well as from uninduced cells. Sequential Northern blot analysis was performed with chicken GATA-1 and GAPDH cDNAs. The 28S rRNA is shown as a control for total RNA loading.

Figure 3

Effect of PUER activation on GATA-1 protein and mRNA expression. (A) E26–PUER transformed MEPs (from a single clone) were treated with βE, and aliquots subjected to Western analysis at different time points with anti-GATA-1 antibody. (B) Pooled MEP clones (four clones) were treated with βE and RNA isolated after 2 and 4 days of induction, as well as from uninduced cells. Sequential Northern blot analysis was performed with chicken GATA-1 and GAPDH cDNAs. The 28S rRNA is shown as a control for total RNA loading.

Figure 4

Induction of myeloid differentiation by transient PUER activation. (A) Experimental protocol. E26–PUER MEPs were subjected to a βE pulse of 1–4 days, washed extensively, incubated for a total of 8 days, and assayed for the lineage-specific markers described below. (B) Expression of MEP-specific [(•, black) MEP21; (○, blue) MEP26], (C) myeloid-specific [(▪, black) MYL51/2; (▵, blue) MHC 11; (, red) 1C3], and (D) eosinophil-specific (EOS47) antigens. Results are representative of five independent clones. E26–WT MEPs showed no change in antigen expression (data not shown).

Figure 5

Phenotype of E26–PUER-transformed MEPs pulse treated for 3 days with βE. (A) May–Gruenwald–Giemsa staining of E26–WT cells untreated (a) or subjected to a 3-day βE pulse (b), and E26–PUER MEPs untreated (c), or βE treated (d). (B) cMGF dependence of E26–PUER cells βE untreated or treated as indicated. Cells were incubated under low serum conditions with or without recombinant cMGF (rcMGF) for 2 days and the number of cells in S phase determined by short-term [³H]thymidine incorporation. Average values of triplicate determinations are shown; (error bars) standard deviations. (C) E26–WT and E26–PUER MEPs untreated or pulsed with βE for 4 days were expanded in liquid culture for 10 days and subjected to Western blot analyses with anti-chicken GATA-1 (top) and anti-chicken C/EBPβ antisera (bottom).

Figure 5

Phenotype of E26–PUER-transformed MEPs pulse treated for 3 days with βE. (A) May–Gruenwald–Giemsa staining of E26–WT cells untreated (a) or subjected to a 3-day βE pulse (b), and E26–PUER MEPs untreated (c), or βE treated (d). (B) cMGF dependence of E26–PUER cells βE untreated or treated as indicated. Cells were incubated under low serum conditions with or without recombinant cMGF (rcMGF) for 2 days and the number of cells in S phase determined by short-term [³H]thymidine incorporation. Average values of triplicate determinations are shown; (error bars) standard deviations. (C) E26–WT and E26–PUER MEPs untreated or pulsed with βE for 4 days were expanded in liquid culture for 10 days and subjected to Western blot analyses with anti-chicken GATA-1 (top) and anti-chicken C/EBPβ antisera (bottom).

Figure 5

Phenotype of E26–PUER-transformed MEPs pulse treated for 3 days with βE. (A) May–Gruenwald–Giemsa staining of E26–WT cells untreated (a) or subjected to a 3-day βE pulse (b), and E26–PUER MEPs untreated (c), or βE treated (d). (B) cMGF dependence of E26–PUER cells βE untreated or treated as indicated. Cells were incubated under low serum conditions with or without recombinant cMGF (rcMGF) for 2 days and the number of cells in S phase determined by short-term [³H]thymidine incorporation. Average values of triplicate determinations are shown; (error bars) standard deviations. (C) E26–WT and E26–PUER MEPs untreated or pulsed with βE for 4 days were expanded in liquid culture for 10 days and subjected to Western blot analyses with anti-chicken GATA-1 (top) and anti-chicken C/EBPβ antisera (bottom).

Figure 6

Phenotype of E26–PUER-transformed MEPs pulse treated for 1 day with β-estradiol. (A) EOS47 antigen expression of 1-day βE pulse-treated E26–PUER MEPs (left) and 3-day pulse-treated control E26–C/EBPβ–ER MEPs (right). The fluorescence intensities of cells stained with EOS47 (top) and anti-c-Src (control) antibodies (bottom) are shown. Fluorescence intensity (y-axis; logarithmic scale) is plotted against forward scatter (x-axis; linear scale). (B) May–Gruenwald–Giemsa (top) and peroxidase staining (bottom) of the above cells.

Figure 7

Requirement of the PU.1 transactivation domain for induction of myeloid differentiation. (A) Maps of full-length PU.1 and amino-terminal deletion mutants (the number indicates the first amino acid still present). The positions of structural and functional domains (Klemsz and Maki 1996) are indicated above. (B) Transactivation potential of PU.1 alleles (see Fig. 2B legend). The activity of PU.1 WT was arbitrarily assigned a value of 1. The averages of three determinations are shown; (error bars) standard deviation. (C) Maps of PU.1 constructs fused to the GAL4 DNA-binding domain. (D) Transactivation potential of the GAL4–PU.1 fusions on the pG5B–Luc reporter. The normalized luciferase activity is expressed as fold activation above the value obtained with control vector (pcDNAI).

Figure 7

Requirement of the PU.1 transactivation domain for induction of myeloid differentiation. (A) Maps of full-length PU.1 and amino-terminal deletion mutants (the number indicates the first amino acid still present). The positions of structural and functional domains (Klemsz and Maki 1996) are indicated above. (B) Transactivation potential of PU.1 alleles (see Fig. 2B legend). The activity of PU.1 WT was arbitrarily assigned a value of 1. The averages of three determinations are shown; (error bars) standard deviation. (C) Maps of PU.1 constructs fused to the GAL4 DNA-binding domain. (D) Transactivation potential of the GAL4–PU.1 fusions on the pG5B–Luc reporter. The normalized luciferase activity is expressed as fold activation above the value obtained with control vector (pcDNAI).

Figure 7

Requirement of the PU.1 transactivation domain for induction of myeloid differentiation. (A) Maps of full-length PU.1 and amino-terminal deletion mutants (the number indicates the first amino acid still present). The positions of structural and functional domains (Klemsz and Maki 1996) are indicated above. (B) Transactivation potential of PU.1 alleles (see Fig. 2B legend). The activity of PU.1 WT was arbitrarily assigned a value of 1. The averages of three determinations are shown; (error bars) standard deviation. (C) Maps of PU.1 constructs fused to the GAL4 DNA-binding domain. (D) Transactivation potential of the GAL4–PU.1 fusions on the pG5B–Luc reporter. The normalized luciferase activity is expressed as fold activation above the value obtained with control vector (pcDNAI).

Figure 7

Requirement of the PU.1 transactivation domain for induction of myeloid differentiation. (A) Maps of full-length PU.1 and amino-terminal deletion mutants (the number indicates the first amino acid still present). The positions of structural and functional domains (Klemsz and Maki 1996) are indicated above. (B) Transactivation potential of PU.1 alleles (see Fig. 2B legend). The activity of PU.1 WT was arbitrarily assigned a value of 1. The averages of three determinations are shown; (error bars) standard deviation. (C) Maps of PU.1 constructs fused to the GAL4 DNA-binding domain. (D) Transactivation potential of the GAL4–PU.1 fusions on the pG5B–Luc reporter. The normalized luciferase activity is expressed as fold activation above the value obtained with control vector (pcDNAI).

Figure 8

(A) Biological activity of PU.1 transactivation domain deletions. E26 derivatives containing PU.1 amino-terminal deletion mutants were constructed, and viruses were produced and used to infect blastoderm cultures, along with E26–WT control virus. Proportions of MEPs, eosinophils and myeloid cells were determined as in Fig. 1. (Light-shaded box) MEP21; (dark-shaded box) EOS47; (solid box) MYL51/2. Average values of three independent transformation assays are shown. (B) Equal amounts of protein from pooled colonies transformed by E26 viruses encoding the indicated PU.1 alleles were subjected to Western blotting with the 9E10 monoclonal antibody (directed against the amino-terminal epitope tag of the PU.1 proteins). The positions of molecular weight markers are shown at left; specific bands representing PU.1 proteins are indicated with asterisks. (C) Correlation between the ratio of MYL51/2-positive and MEP21-positive cells transformed by E26–PUDN viruses and the transactivation potential of the corresponding PUDN constructs. (Black diamonds) Luciferase activity; (shaded circles) MYL/MEP.