Conditional activation of PUER and GER in MEL cells restarts myeloid and erythroid program, respectively, and inhibits cell proliferation. MEL^PUER (A) and MEL^GER (C) cells were cultured in the presence or absence of 10⁻⁷ mol/L of 17β-estradiol (E) for the indicated time periods. Total RNA was purified and subjected to quantitative reverse transcription-PCR as described in Materials and Methods. Y-axis, mRNA expression of indicated genes relative to housekeeping gene Gapdh. B. Aliquots of MEL^PUER cells stimulated for the indicated time periods were immunostained using antibodies against Itgam, Ptprc, and Ly6g and control isotypic antibodies, as described in Materials and Methods. Y-axis, percentage of immunostained cells by flow cytometry analysis. Bars, SE calculated for two independent experiments. Proliferation of MEL^PUER (D) and MEL^GER (E) cells unstimulated or stimulated by 17β-estradiol for 96 h. Y-axis, maximum of cell number (%); X-axis, time (days).

Gene expression of MEL cells could be reprogrammed either by PU.1 into mixed non-erythroid or by GATA-1 into erythroid transcriptional programs. MEL^PUER and MEL^GER cells were cultured for 24 h (X-axis) in the presence of 17β-estradiol in a duplicate experiment. Total RNA was purified and the fluorescently labeled cRNA probe was synthesized according to Affymetrix protocol for subsequent hybridization on the expression chip (MG-430A 2.0) containing 22602 probes. High-throughput data analyses were done using GeneSpring (Agilent Technologies) and MeV4 softwares. A. Box plots of gene expression patterns of indicated MEL cell lines stimulated for 24 h (X-axis; N, number of probes) identified by expression arrays (for details, see Materials and Methods and Supplementary Fig. S3A and B). Y-axis, relative expression of mRNAs relative to unstimulated cells. B. Cluster analysis of selected expression patterns (transcription factors and hallmarks of lineage differentiation) from gene expression arrays documenting the involvement of recognized lineage-specific mRNAs (for details, see Materials and Methods and Supplementary Fig. S3).

Colocalization of GATA-1 and PU.1 proteins near Zfpm1 and Nfe2 genes and chromatin H3K9 hyperacetylation induced by ectopic GATA-1 activation. ChIP was carried out on cross-linked chromatin as described in Materials and Methods and in the legend to Fig. 4. Antibodies used were anti-PU.1, anti–GATA-1 (gray columns), anti–acetylated histone H3K9 (black columns), and antirabbit IgG antibody (letter C on the X-axis). A and B. Occupancy of PU.1 and GATA-1 proteins at the indicated positions (relative to transcription start site, in kilo-bases) near Zfpm1 and Nfe2 was determined in unstimulated MEL^GER cells. Levels of H3K9 hyperacetylation in these cells in the presence of 10⁻⁷ mol/L of 17β-estradiol for 24 h (third graphs) was determined relative to acetylation determined in stimulated MEL^PUER cells under the same conditions. Primary binding sites of GATA-1 are functional in reporter assays. A. Right, HeLa cells were transfected with reporter plasmid [Zfpm1, 0.25 μg (R)] and cDNA constructs [pXM-GATA-1, 0.125 μg (G) and pXM-PU.1, 0.125 μg (P); 0.375 μg (3P); 1.125 μg (9P)]. B. Right, MEL^GER cells were lipofected with Nfe2 reporter plasmid (1.7 μg) and either un-stimulated (−E) or further stimulated with 17β-estradiol after 24 h (+E) followed by measurement of luciferase activity at 72 h (for details, see Materials and Methods). C. MEL^PUER cells were either stimulated with 17β-estradiol (+E) or treated with PU.1-inhibiting siRNAs (si) for 48 h, and GATA-1 and PU.1 occupancy was detected by qChIP near Nfe2 (0 kb) and Zfpm1 (+3.5 kb) genes. All ChIP bars in C indicate the fold change (Y-axis) of DNA fragment in specific immunoprecipitates above the immunoprecipitates using control antirabbit IgG antibody. Bars, SE of at least two independent experiments.

Distinct cell types are induced by PUER and GER transgenes from MEL cell line. MEL (A), MEL^PUER (B), and MEL^GER (C) cells were cultured in the presence of 10⁻⁷ mol/L of 17β-estradiol for 72 h, fixed, cyto-spinned, and stained using May-Grünwald-Giemsa. Microscopy (Olympus BX-51 apparatus, E-410 Camera) was done under ×1,000 magnification according to the manufacturer’s recommendations. Black arrow, proerythroblasts; dashed arrows, basophilic erythroblasts; empty arrows, atypical cells. The table displays the distribution of these cell types in MEL, MEL^PUER, MEL^PUER induced with 17β-estradiol for 3 d, MEL^GER, and MEL^GER cells induced with 17β-estradiol for 3 d.

PU.1 and GATA-1 colo-calization near Cbfb and Cebpa genes and chromatin H3K9 hyperacetylation induced by ectopic PUER activation. ChIP was carried out on cross-linked chromatin as described in Materials and Methods using the following antibodies: anti-PU.1, anti–GATA-1 (both gray columns), anti–acetylated histone H3K9 (black columns), and control anti-rabbit IgG antibody (white columns, letter C on the X-axis). A and B. Occupancy of PU.1 and GATA-1 proteins at indicated positions (relative to transcription start site, in kilobases) near Cbfb and Cebpa was determined in unstimulated MEL^PUER cells. Levels of H3K9 hyperacetylation in these cells in the presence of 10⁻⁷ mol/L of 17β-estradiol for 24 h (third graphs in A and B) was determined relative to acetylation determined in stimulated MEL^GER cells under the same conditions. Primary binding sites of PU.1 were functional in reporter assays: 1.4 × 10⁵ MEL^PUER cells were lipofected with Cbfb^(+1531) (A, right) or Cebpa^(−2978) (B, right) reporter plasmids (1.7 μg each). Cells remained unstimulated (−E) or treated with 17β-estradiol at 24 h (+E). Luciferase activity was determined 72 h after trans-fection (for details, see Materials and Methods). C and D. DNA regions (50–60 bp) near Cebpa^(−2978) (no. 1 on X-axis) and Cbfb^(+1531) (no. 2) were cloned together with a Cebpa^(−2978) mutant (no. 3) into the reporter plasmid pGL3 and transfected into MEL^PUER (C) and MEL^GER (D) cells stimulated with 17β-estradiol for 72 h. Transfected qChIP technique using antibodies to PU.1, GATA-1, and antirabbit IgG antibody was done as described in Materials and Methods. PU.1 significantly stimulated luciferase activity of PU.1 binding site nos. 1 and 2, but not in site no. 3 (data not shown). The occupancy of PU.1 near Cebpa (−3.2 kb; E) and Cbfb (+2 kb; F) genes was tested by ChIP in either stimulated (48 h; +E) or unstimulated (−E) MEL^PUER cells and in MEL^PUER cells with knockdown of GATA-1 (si). All ChIP bars in C to F indicate the fold change (Y-axis) of DNA fragment in specific immunoprecipitates above the immunoprecipitates using control antirabbit IgG antibody. Bars, SE of at least two independent experiments.

Inhibition of GATA-1 derepresses PU.1 target genes and inhibition of PU.1 derepresses GATA-1 targets in MEL cells. A. MEL^PUER cells (8 × 10⁴) were transfected either with siRNA oligos inhibiting GATA-1 (si) or with negative control oligo (CO) using Lipofectamine. The cells were cultured either in the absence (CO) or in the presence of 10⁻⁷ mol/L of 17β-estradiol (+E). B. MEL cells (8 × 10⁴) were transfected either with siRNA oligos inhibiting PU.1 (si) or with negative control oligo (CO) using Lipofectamine and cultured for 72 h. Total RNA was purified and subjected to quantitative reverse transcription-PCR as described in Materials and Methods. Y-axis, mRNA expression of indicated genes relative to housekeeping gene Hprt1. Bars, SE of two independent experiments.