Transgene insertion in proximity to the c-myb gene disrupts erythroid-megakaryocytic lineage bifurcation

Abstract

The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival, and differentiation of hematopoietic cells. We established three lines of erythropoietin receptor-transgenic mice and found that one of them exhibited anemia, thrombocythemia, and splenomegaly. These abnormalities were independent of the function of the transgenic erythropoietin receptor and were observed exclusively in mice harboring the transgene homozygously, suggesting transgenic disruption of a certain gene. The transgene was inserted 77 kb upstream of the c-myb gene, and c-Myb expression was markedly decreased in megakaryocyte/erythrocyte lineage-restricted progenitors (MEPs) of the homozygous mutant mice. In the bone marrows and spleens of the mutant mice, numbers of megakaryocytes were increased and numbers of erythroid progenitors were decreased. These abnormalities were reproducible in vitro in a coculture assay of MEPs with OP9 cells but eliminated by the retroviral expression of c-Myb in MEPs. The erythroid/megakaryocytic abnormalities were reconstituted in mice in vivo by transplantation of mutant mouse bone marrow cells. These results demonstrate that the transgene insertion into the c-myb gene far upstream regulatory region affects the gene expression at the stage of MEPs, leading to an imbalance between erythroid and megakaryocytic cells, and suggest that c-Myb is an essential regulator of the erythroid-megakaryocytic lineage bifurcation.

FIG. 1.

Homozygously transgenic line B mice exhibit hematological abnormalities. (A) Three independent lines of transgenic mice were produced. Levels of transgene-derived mRNA were determined by semiquantitative RT-PCR. Reverse-transcribed cDNAs were subjected to 27, 30, 33, 36, and 39 cycles of amplification. (B) The integration levels of transgene (Tg) were analyzed by Southern blot hybridization analysis and compared with endogenous (endo) DNA levels. Tg/endo ratios are in arbitrary units. Phenotypes were anemia, thrombocythemia, and splenomegaly. (C to H) Wright-Giemsa staining of peripheral blood smears (C to E) and bone marrow smears (F to H). Photomicrographs show samples from wild-type mice (C and F), heterozygously transgenic mice (D and G), and homozygously transgenic mice (E and H). In panel E, large platelets and spindle-shaped platelets are indicated by black and white arrowheads, respectively. Original magnification, ×200.

FIG. 2.

Chromosomal localization and expression analysis of neighboring genes around the transgene integration site. (A) Karyogram of mouse chromosome 10. (B) A3 region. Known genes are shown. (C) Physical and transcript map of the genomic region around the integration site. Arrowheads in the boxes indicate the direction of transcription. (D) Precise map of the region around the integration site, shown with a vertical arrow. The combined thick and thin lines show an EST clone. (E) c-Myb mRNA expression levels determined by RNA blotting analyses. RNA samples were obtained from hematopoietic (bone marrow [BM] and spleen [Sp]) and nonhematopoietic (liver [Li]) tissues of wild-type mice (Wild) or homozygously transgenic mice (Tg homo). (F) c-Myb mRNA expression levels in Ter119⁺, CD41⁺, CD19⁺, and CD3⁺ cells. Semiquantitative RT-PCR was carried out using hypoxanthine phosphoribosyltransferase (HPRT) as a standard. (G) Immunoblotting analyses of BM-MNCs without Mac-1⁺, Gr-1⁺, CD4⁺, or CD8⁺ cells with antibodies against c-Myb and β-actin.

FIG. 3.

Numbers of erythroid-lineage cells decreased in line B homozygously transgenic mice. (A and B) In vitro hematopoietic colony formation using BM-MNCs of wild-type, heterozygously transgenic (Tg hetero), and homozygously transgenic (Tg homo) mice. Note that the production of erythroid progenitor cells, BFU-E (A) and CFU-E (B), is reduced in homozygously transgenic mice. The data are means plus standard errors of the means for experiments performed in triplicate. The colonies of BFU-E cells from homozygously transgenic mice were significantly smaller than those from wild-type mice (inset). (C and D) The expression of erythroid lineage markers was analyzed by FACS using BM-MNCs. The percentage in each quadrant is shown. (C) Numbers of c-Kit⁺ Ter119⁺ immature erythroid cells were decreased in homozygously transgenic mice. (D) R1, R2, R3, and R4 represent Ter119^med CD71^high, Ter119^high CD71^high, Ter119^high CD71^med, and Ter119^high CD71^low populations, respectively. The relative number in each region as a percentage of gated cells is given.

FIG. 4.

Supraphysiological production of megakaryocytes in homozygous line B transgenic mice. (A) In vitro megakaryocytic colony formation examined using BM-MNCs. The data are means plus standard errors for triplicate experiments. (B) Serum TPO concentration measured with ELISA. (C) Expression of megakaryocyte lineage markers analyzed by FACS using BM-MNCs. The percentage in each quadrant is given. Numbers of CD41⁺ CD61⁺ megakaryocytes were increased in homozygously transgenic mice. (D) Ploidy analysis of megakaryocytes using FACS. Homozygously transgenic mouse megakaryocytes had a lower ploidy (2N to 8N) than wild-type megakaryocytes (16N and 32N).

FIG. 5.

Hematological analysis of spleens from homozygous line B transgenic mice. (A) Gross appearance of spleens. Note that massive splenomegaly was observed in homozygously transgenic mice. Bar, 1 cm. (B to M) Sections of spleens from wild-type (B, E, H, and K), heterozygously transgenic (C, F, I, and L), and homozygously transgenic (D, G, J, and M) mice, stained with HE (B to G), AchE (H to J), and B220 (K to M). Original magnifications, ×40 (B to D and H to M) and ×200 (E to G). (N) FACS analysis for expression of the erythroid lineage markers Ter119 and CD71 in spleen MNCs. R1 to R4 represent the same regions as in Fig. 3D. The relative number in each region is given as a percentage of gated cells. (O) FACS analysis for the expression of the megakaryocyte lineage markers CD41 and CD61 in spleen MNCs. The percentage in each quadrant is given.

FIG. 6.

MEPs analyses of homozygously transgenic mice. (A) Expression level of c-Myb mRNA in MEPs analyzed by RT-PCR. HPRT, hypoxanthine phosphoribosyltransferase. (B and C) In vitro erythroid (B) and megakaryocyte (C) colony formation by MEPs, examined using a coculture system with OP9. Erythroid colonies were stained with benzidine (B), and megakaryocyte colonies were stained with AchE (C). The numbers of total colonies and benzidine (Be)- and AchE-positive colonies are given. The data are means ± standard errors for experiments done in triplicate. (D and E) FACS analyses of MEPs after coculturing with OP9 cells for 4 days. Results for Ter119⁺ erythroid cells (D) and CD41⁺ megakaryocyte cells (E) are boxed.

FIG. 7.

c-Myb regulates erythroid-megakaryocytic bifurcation at the stage of MEPs. (A) Virus-infected MEPs observed by bright and fluorescent microscopy. GFP expression was detected in c-Myb-infected MEPs but not in uninfected MEPs. (B and C) In vitro erythroid (B) and megakaryocyte (C) colony formation by MEPs supplemented with c-Myb examined by the OP9 coculture system. Columns labeled “wild” and “Tg homo” show numbers of colonies without viral infection, while those labeled “vector” and “c-Myb” show numbers of colonies with viral vector and c-Myb vector infection, respectively. The numbers of total colonies and benzidine (Be)- and AchE-positive colonies are indicated. The data are means ± standard errors for experiments carried out in triplicate. MEPs used in these experiments (A to C) were obtained from homozygously transgenic mice.

FIG. 8.

Phenotype analyses of bone marrow transplant mice. (A) Chimerism of recipient BM-MNCs examined using FACS analysis. Ly5.1 indicates recipient origin, and Ly5.2 indicates donor origin. (B) Expression of erythroid lineage markers in recipient mouse BM-MNCs examined by FACS analyses. R1 to R4 are the same as in Fig. 3D. The relative number in each region is given as a percentage of gated cells. (C) Expression of megakaryocyte lineage markers examined by FACS analyses. The percentage in each quadrant is shown.

FIG. 9.

Hematopoietic abnormalities in homozygously transgenic mice. Broad and narrow lines indicate pathways that are enhanced or repressed, respectively. HSC, hematopoietic stem cell; CMP, common myeloid progenitor.