B-myb is an essential regulator of hematopoietic stem cell and myeloid progenitor cell development

Abstract

The B-myb (MYBL2) gene is a member of the MYB family of transcription factors and is involved in cell cycle regulation, DNA replication, and maintenance of genomic integrity. However, its function during adult development and hematopoiesis is unknown. We show here that conditional inactivation of B-myb in vivo results in depletion of the hematopoietic stem cell (HSC) pool, leading to profound reductions in mature lymphoid, erythroid, and myeloid cells. This defect is autonomous to the bone marrow and is first evident in stem cells, which accumulate in the S and G2/M phases. B-myb inactivation also causes defects in the myeloid progenitor compartment, consisting of depletion of common myeloid progenitors but relative sparing of granulocyte-macrophage progenitors. Microarray studies indicate that B-myb-null LSK(+) cells differentially express genes that direct myeloid lineage development and commitment, suggesting that B-myb is a key player in controlling cell fate. Collectively, these studies demonstrate that B-myb is essential for HSC and progenitor maintenance and survival during hematopoiesis.

Keywords: myelodisplastic syndrome; myeloipiesis.

Fig. 1.

Murine B-myb genomic structure and target production. (A) Expression of B-myb in HSCs and progenitor cells. (All mRNA levels shown are normalized to that of β-actin.) All values represent mean ± SEM. (B) Schematic representation of the mouse B-myb genomic clone. (Note: not drawn to scale.) LoxP sites are depicted as black arrowheads. Homologous recombination will generate the B-myb^F(neo) allele that will be used for subsequent recombination by Cre recombinase. ES cells with either a type I or II deletion (B-myb^ΔEx6 and B-myb^F) will be produced following Cre expression. The probe that can be used to determine homologous recombination is indicated. Restriction enzyme sites: B, BamHI; Bg, BglII: H, HindIII; S, SmaI; Xb, XbaI; Xh, XhoI. Deletion of exon 6 produces a B-MYB protein without a DNA-binding domain and a shift in reading frame. (C) Representative semiquantitative PCR analysis of genomic DNA isolated from pIpC-treated control and B-myb floxed mice showing the presence of floxed and deleted alleles in the BM, spleen (SP) and thymus (THY). The percentage of deletion is shown. (D) Western blot analysis of B-MYB protein (arrow) in sorted Lin⁻ BM cells derived from pIpC-treated control (F/F) and floxed/Mx1Cre⁺ (F/Fcre) mice. β-Actin is shown as a loading control.

Fig. 2.

B-myb is required for hematopoiesis. B-myb–deficient (F/Fcre; Mx1-cre-B-myb^F/F) and control (B-myb^+/F or B-myb^F/F) mice were treated with pIpC every other day over a 5-d period. Tissues were harvested on day 21 posttreatment. Total number of cells isolated from BM (A) and spleen (B) of control and B-myb–deficient mice. Analysis of mature lineage cells present in the BM (C) and spleen (D) of control and B-myb–deficient mice. Erythroid, B-, T-, and myeloid lineage cells are defined as TER119⁺, B220⁺, CD3⁺, and CD11b⁺Gr1⁺, respectively. Total number of thymocytes (E) and mature populations (F) in the thymuses of control and B-myb F/Fcre mice. (G) Total number of HSCs in the BM of control and B-myb–deficient mice. (H) Total number of MPPs, myeloid progenitors (CMPs, GMPs, and MEPs), and lymphoid progenitors (CLPs) in the BM of control and B-myb–deficient mice. (I) Colony-forming assay showing the frequency of whole BM cells that formed megakaryocyte (GEMM), erythroid (E), granulocyte–monocyte (GM), monocyte (M), or granulocyte (G) colonies in culture. All values represent mean ± SEM. n ≥ 3 per population for each genotype. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005.

Fig. 3.

Cell-autonomous role for B-myb in hematopoiesis. Wild-type mice (CD45.1) were transplanted with control or B-mybF/Fcre (CD45.2) whole BM (WBM). (A) Animals transplanted with BM of both genotypes showed nearly equal donor cell contribution at 8–10 wk posttransplant, before pIpC administration. (B) Total number of control and B-myb–deficient CD45.2⁺ cells in the WBM of recipient animals following treatment with pIpC. (C) Total number of erythroid (TER119⁺), B- (B220⁺), T- (CD3⁺), and myeloid lineage (CD11b⁺Gr1⁺) CD45.2⁺ cells in the BM of recipients transplanted with control and B-myb–deficient BM following pIpC treatment. Total number of control and B-myb–deficient CD45.2⁺ HSCs (D) and myeloid progenitors (CMPs, GMPs and MEPs) (E) in the BM of recipient animals following treatment with pIpC. All values represent mean ± SEM. n ≥ 3 per population for each genotype. ***P ≤ 0.0005.

Fig. 4.

Loss of B-myb results in aberrant cell cycle progression in HSCs and increased cell death of myeloid progenitors. (A) BM was harvested from pIpC-treated control and B-myb F/Fcre mice 2 h following injection with BrdU. The cells were then subjected to flow cytometric analysis to determine the percentage of cells in the indicated populations that are in the G₀/G₁ (lower left gate), S (upper gate), and G₂/M (lower right gate) phases of the cell cycle. (B) Frequency of Annexin V⁺DAPI⁺ HSCs and myeloid progenitors of pIpC-treated control and B-myb–deficient mice. (C) Frequencies of myeloid progenitors in the BM of pIpC-treated control and B-myb–deficient mice. Representative plots are shown. All numerical values represent mean ± SEM for three independent experiments. n ≥ 3 per population for each genotype. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005.

Fig. 5.

Computational analysis of the B-myb LSK⁺ transcriptiome. (A) Clustering analysis of altered gene expression in microarrays of control and B-myb–depleted LSK⁺ cells. (B) Quantitative mRNA expression (qPCR) of differentially expressed genes in control and B-myb KO LSK⁺ cells. All results were normalized to β-actin expression and are graphed as the average fold-change (±SEM). PCR reactions were performed in triplicate using cDNAs isolated from at least two independent pools of sorted cells per genotype.