Coordination of erythropoiesis by the transcription factor c-Myb - PubMed (original) (raw)

Coordination of erythropoiesis by the transcription factor c-Myb

Alexandros Vegiopoulos et al. Blood. 2006.

Abstract

The involvement of the transcription factor c-Myb in promoting the proliferation and inhibition of erythroid cell differentiation has been established in leukemia cell models. The anemia phenotype observed in c-myb knockout and knockdown mice highlights a critical role for c-Myb in erythropoiesis. However, determining the reason for the failure of erythropoiesis in these mice and the precise function of c-Myb in erythroid progenitors remains elusive. We examined erythroid development under conditions of reduced c-Myb protein levels and report an unexpected role for c-Myb in the promotion of commitment to the erythroid lineage and progression to erythroblast stages. c-myb knockdown erythroid colony-forming unit (CFU-E) stage progenitors displayed an immature phenotype and aberrant expression of several hematopoietic regulators. To extend our findings, we analyzed the response of normal enriched erythroid progenitors to inducible disruption of a floxed c-myb allele. In agreement with the c-myb knockdown phenotype, we show that c-Myb is strictly required for expression of the c-Kit receptor in erythroid cells.

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Figures

Figure 1.

Figure 1.

Accumulation of immature progenitor cells in the fetal liver of c-_myb_KD/KD embryos. Presented are fluorescence values (log-scale) from flow cytometric analyses of fetal liver single-cell suspensions from E13 c-myb+/+ and c-_myb_KD/KD embryos stained with monoclonal antibodies. Gated live cells are shown. (A) Cells were stained with α-CD71(FITC) and α-TER119(APC). (B-C) Cells were stained with α-CD71(FITC), α-c-Kit(PE-Cy5), and either α-CD45(APC) (B) or α-CD34(PE) (C). Gated c-Kit+ cells are shown.

Figure 2.

Figure 2.

Progenitors from c-_myb_KD/KD fetal liver display retarded commitment and progression through erythropoiesis. Fetal liver single-cell suspensions from E14 c-myb+/+ and c-_myb_KD/KD embryos were stained with α-CD71(FITC), α-CD41(PE), α-c-Kit(PE-Cy5), and α-TER119(APC). CD41– live cells were sorted based on their CD71 and c-Kit staining. (A) Cells in sorting regions 1-4 (top panel) are displayed in the corresponding color in the middle and bottom panels to indicate their TER119 expression pattern. Note that a minor shift in the TER119 signal of c-_myb_KD/KD cells is attributed to overcompensation caused by reduced c-Kit levels (see panel B). (B) Sorted cells were cultured in SP34 medium containing SCF (100 μg/mL), EPO (2 U/mL), and dexamethasone (1 μM). At the indicated time points, cells were stained with α-CD71 (FITC) and α-TER119 (APC) and analyzed by flow cytometry. Panels Bi to Biv show the development of cells from CD71/c-Kit–sorted regions 1 to 4 (A), respectively. Percentages in Biii and Biv represent proportions of cells at the CFU-E stage. Dead cells were excluded by propidium iodide staining. All plots are in log-scale.

Figure 3.

Figure 3.

Reduced levels of c-Myb are sufficient for intense proliferation of immature erythroid progenitors. Fetal liver cells from E14 c-myb+/+ and c-_myb_KD/KD embryos were plated in SP34 medium containing SCF (100 μg/mL), EPO (2 U/mL), and dexamethasone (1 μM). (A) At day 9 of culture, cells were stained with α-CD71 (FITC) and α-TER119 (APC) and analyzed by flow cytometry. (B) At day 9 of culture, cells were transferred to media favoring terminal erythroid differentiation and cultured for 3 additional days before being analyzed by flow cytometry for CD71 and TER119 expression. (C) At day 9 of culture in SP34, cells were permeabilized with 0.1% NP-40 and stained with 25 μg/mL propidium iodide for flow cytometric analysis of DNA content. Histograms represent linear fluorescence intensities.

Figure 4.

Figure 4.

c-_myb_KD/KD CFU-E stage progenitors display aberrant expression of hematopoietic regulators and abnormal differentiation ex vivo. (A) CD71+c-kit+TER119–CD41– progenitors (Figure 2Biii) corresponding to the CFU-E stage were sorted by FACS from the fetal liver of c-myb+/+ and c-_myb_KD/KD E14 embryos. RT-PCR analysis was performed on RNA extracted from sorted cells. Ethidium bromide–stained agarose gels of PCR products from 2-cycle increments are shown. Normalization by β-actin was confirmed by real time PCR analysis (not shown). (B) Real-time PCR was performed on the cDNA samples described for panel A using SYBR green, primers specific for the indicated genes, and β-actin (for normalization). Reactions were repeated at least 6 times. Average fold change–normalized expression values are shown. Error bars represent the SEM. (C) Fetal liver cells were stained with α-CD71 (FITC), α-CD45 (APC) and α-TER119 (APC) (top panel) or α-CD71 (FITC), α-TER119 (PE), and α-c-Kit (PE-Cy5) (bottom panel). Histograms represent fluorescence intensities (log-scale) of cells gated as CD71+TER119– (CFU-E stage). (D) Fetal liver cells were stained and CFU-E stage cells were sorted as for panel A and were cultured for 48 hours, as described in Figure 2. Cells were stained with α-c-Kit (PE) and were analyzed by flow cytometry. Log-scale of PE-fluorescence intensity is shown. (E) CD71+c-Kit+TER119–CD41– (Figure 2Biii) CFU-E stage progenitors (top panels) and CD71– c-Kit+TER119–CD41– (Figure 2Bi) less mature progenitors (bottom panels) from E14 fetal liver were sorted by FACS and cultured in SP34 for 2 days. Cells were permeabilized with 0.1% NP-40 and were stained with 25 μg/mL propidium iodide for flow cytometric analysis of DNA content. Histograms represent linear fluorescence intensities. (F) Lower c-Myb expression limits the size of CFU-E colonies. Fetal liver cells were stained and CFU-E stage cells were sorted as for panel A and then plated in methylcellulose containing SCF and EPO for 3 days. Colonies were observed using an Olympus CKX41 microscope (Olympus, London, United Kingdom) and a 20 ×/0.40 numeric aperture Php objective under phase contrast. Images were acquired using an Olympus Camedia C3030 camera and were processed using Adobe Photoshop version 4.0 (Adobe Systems, San Jose, CA).

Figure 5.

Figure 5.

Inducible inactivation of c-myb reveals its requirement for maintenance of c-Kit expression in erythroid progenitors. (A) Schematic representation of c-myb alleles: wild type (wt), targeted floxed (F), and recombined by Cre-recombinase (Δ). Exons are shown as black boxes. Arrowheads represent loxP sites. The probe (P) used in hybridization to Southern blots is indicated by the gray box. H indicates _Hin_dIII. (B) Detection of Cre-mediated recombination of the c-_myb_F allele in cultured erythroid progenitors. Fetal liver cells from E13 c-_myb_–/F/Cre embryos were cultured as described in Figure 3 for 8 days to enrich for erythroid progenitors. IFN–α-A (2000 U/mL) was then added to the cultures and washed off at 24 hours. Genomic DNA was harvested at the indicated time points and digested with _Hin_dIII. Southern blot analysis was performed with probe P, and signals were quantified by phosphorimaging. The deletion rate of the c-_myb_F allele is represented as the ratio of intensities of the c-_myb_Δ signal to the constant c-_myb_– signal (right panel). (C) Flow cytometric analysis of c-Kit surface expression on cultured c-_myb_–/F/Cre and c-_myb_–/F cells in response to IFN treatment. Day 8 fetal liver cultures were treated with IFN–α-A (2000 U/mL) for 24 hours (open histogram) or were left untreated (filled histogram). At 24 or 48 hours, cells were stained with α-c-Kit-PE and analyzed by flow cytometry. (D) Real-time RT-PCR analysis of c-Kit mRNA expression in response to c-myb inactivation. Cells were treated with IFN–α-A, as described, and RNA was harvested at the indicated time points and reverse transcribed. Real-time PCR was performed as described in “Materials and methods.” (Left) Ratio of normalized relative expression levels from samples with IFN to samples without IFN was calculated for c-_myb_–/F/Cre and c-_myb_–/F separately. The average of at least 6 replicates is shown. ANOVA (2 × 2) was performed, and P values are given for the interaction term (cell type × treatment). (Right) Absolute expression (normalized to 8-hour control IFN) of c-Kit RNA determined by real time RT-PCR is shown for the c-_myb_–/F and c-_myb_–/F/Cre samples with or without IFN at 8 and 24 hours. Data for the 24-hour time point from a second independent experiment are illustrated on the right. (E) Real-time RT-PCR analysis of c-Kit mRNA expression in c-myb+/+ and c-_myb_KD/KD CFU-E stage cells sorted from E14 fetal liver. Normalized relative expression levels are shown. Error bars represent SEM. (F) c-myb inactivation does not lead to a general induction of differentiation. Enriched erythroid progenitors from c-_myb_–/F/Cre and c-_myb_–/F fetal livers were treated with IFN–α-A (2000 U/mL) for 24 hours (open histogram) or were left untreated (filled histogram) and subsequently were cultured for another 24 hours. Cells were stained with α-TER119 (PE) and analyzed by flow cytometry. (G) Cells were treated with IFN–α-A, as described in panel F, and were collected on cytospins at the indicated time points. After _o_-dianisidine/hematoxylin staining, 4 fields were counted for the proportion of hemoglobin-positive cells. The ratio of the frequencies of Hb+ cells in samples with IFN to samples without IFN is represented. Error bars represent the SEM.

Figure 6.

Figure 6.

Coordination of erythropoiesis by c-Myb. c-Myb has a dual role as a regulator of erythroid development. Both in uncommitted/early stages (CD71–/low) and in later stages (CFU-E/erythroblast—CD71+TER119–/low), high levels of c-Myb are required for an efficient response of progenitors to erythropoietic stimuli and progression to later stages. c-Myb is indirectly involved in the down-regulation of GATA-2, Runx1, PU.1, Fli-1, and CD45, a process that is a prerequisite for terminal differentiation. At later stages of erythropoiesis, high levels of c-Myb allow the cells to undergo terminal cell divisions. Finally, c-Myb maintains high levels of c-Kit expression on erythroid progenitors, thereby rendering them responsive to external signals regulating proliferation and differentiation.

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